Methods: In this study, retinyl acetate, a derivative of vitamin A, was successfully incorporated into electrospun polycaprolactone fibres. Discussion: These results suggest that incorporation of retinyl acetate into electrospun fibres has may mediate the damaged microenvironment post cancer radiation therapy. Volume 11 - 2023 | https://doi.org/10.3389/fbioe.2023.1233801 Background: Increased cancer rates denote that one in two people will be diagnosed with cancer in their lifetime Over 60% of cancer patients receive radiotherapy either as a stand-alone treatment or in combination with other treatments such as chemotherapy and surgery Whilst radiotherapy is effective in destroying cancer cells it also causes subsequent damage to healthy cells and surrounding tissue due to alterations in the tumor microenvironment and an increase in reactive oxygen species (ROS) This can cause extensive damage that impairs tissue function and the likelihood of tissue regeneration and restoration of function is significantly reduced as new healthy cells cannot survive in the damaged environment radiotherapy can cause salivary gland dysfunction This significantly impairs the patient’s quality of life and there is currently no cure Tissue engineering approaches are used to mimic the microenvironment of the tissue and can mediate the damaged microenvironment via bioactive compounds healthy cells into the damaged environment was successfully incorporated into electrospun polycaprolactone fibres Results: SEM images and characterization analyses showed that all scaffolds produced had similar characteristics including fiber morphology and scaffold wettability The vitamin scaffolds were shown to exert an antioxidant effect through scavenging activity of both DPPH and hydroxyl radicals in vitro the antioxidant scaffolds supported the growth of human submandibular gland cells and significantly upregulated the expression of GPx1 when cultured under both normal conditions and under a simulated oxidative stress environment Discussion: These results suggest that incorporation of retinyl acetate into electrospun fibres has may mediate the damaged microenvironment post cancer radiation therapy the design of a delivery carrier is highly important in the translation of antioxidant therapies electrospun polycaprolactone (PCL) scaffolds with different retinyl acetate (RA) concentrations were fabricated and their functional properties characterized In vitro testing with human submandibular gland (HSG) cells a stable and functional salivary gland cell line under both normal and simulated oxidative stress environments was performed This was used to determine cell viability and compatibility to ensure that the scaffolds cause no further or any new damage to the radiation damaged site PCL (Mn = 80,000 Da) (Sigma) was dissolved in hexafluoroisopropanol (HFIP) (Manchester Organics) with different amounts of retinyl acetate (RA) (Sigma) in a glass vial Each solution was left on a tube roller overnight at room temperature to create a homogenous solution the appropriate polymer solution was loaded into a 20 mL syringe attached to a syringe pump and electrospun using an IME technologies EC-DIG electrospinning system A 0.3 mm inner diameter brass needle was used at a 15 cm distance from an aluminum foil covered rotating mandrel (8 cm diameter A total volume of 4.0 mL polymer solution was spun at a rate of 0.8 mL/h across a distance of 100 mm The scaffold mat was left to air-dry in a fume hood overnight to allow any residual solvent to evaporate The morphology and diameters of the electrospun fibres was characterized using scanning electron microscopy (SEM) analysis 10 mm scaffolds (n = 3) were punched and gold sputter coated for 30 s (Emscope SC500A) prior to imaging SEM images were obtained using 15 kV BSE accelerating voltage (Hitachi TM4000Plus SEM) Average fiber diameter size was measured using ImageJ software (150 fiber diameters for each sample) Tensile testing of the scaffolds was performed using an Instron material testing machine (Instron Model 3,367 50 N load cell) with Bluehill 3 software Scaffolds were cut into strips (80 mm × 10 mm) and mounted 20 mm at each end to give a gauge length of 40 mm The samples (n = 5) were then stretched at a rate of 50% strain per minute until failure Scaffold thickness was measured using handheld digital vernier calipers The incremental Young’s Modulus was calculated for each sample in 5% increments to a total of 20% strain A 5 μL water droplet was pipetted onto the top of each scaffold (n = 5) Images were taken every 0.2 s using a DMK 41AU02 (ImagingSource) camera at 5 Hz The contact angle was then measured using the ImageJ Contact Angle plugin where AB and AS are the absorbances of the blank and sample Equation 1 Percentage of DPPH Scavenging Activity 100 and 25 μM hydrogen peroxide solutions were prepared by adding 0.3 μL hydrogen peroxide (9.8 M) (Sigma) to 30 mL MEM (non-supplemented) to prepare the 100 μM solution and then 5 mL of this was added to 20 mL MEM non-supplemented media (Gibco) EtOH (70%) was used to lift the scaffolds from the foil before they were added to the appropriate wells 10 mm scaffolds were sterilized in 0.5 mL EtOH (70%) for 10 min Spent solution was aspirated to waste and scaffolds were subsequently rinsed with 1 mL PBS for 10 min in triplicate 1 mL of the appropriate media (supplemented MEM 100 μM hydrogen peroxide) was added to each well and the plates incubated at 37°C 1 mL samples of the three different medias were taken for the Day 0 samples and frozen at −80°C 70 μL samples were collected and frozen The hydrogen peroxide assay was performed using a fluorescent hydrogen peroxide assay kit (Sigma-Aldrich) samples were incubated with red peroxidase and horseradish peroxidase in assay buffer for 30 min at RT The fluorescence intensity of the samples was then measured using a Clariostar® Plus microplate reader (BMG LABTECH) at λex = 540 nm and λem = 590 nm 10 mm scaffolds were punched and EtOH (70%) was used to lift the scaffolds from the foil backing before the scaffold mats were transferred to 48 well plates Scaffolds were then incubated in 1 mL EtOH (70%) at RT for 30 min and rinsed with PBS (10 min) in triplicate Scaffolds were then incubated in 0.5 mL minimum essential media (MEM) cell culture media (37°C scaffolds were left in the biohood to air dry for a minimum of 30 min This change in media was noted as day 0 for the purpose of the cell study An additional 500 μL media was added to all remaining samples using normal media on day 3 Cell viability was determined using CellTitre-Blue® assay (Promega) conducted as per the manufacturer’s instructions at 24 h cell seeded scaffolds (N = 5) were transferred to a fresh suspension plate with 400 µL fresh media and 100 µL CellTitre-Blue and incubated at 37°C 100 μL of each sample was transferred to a 96 well plate and fluorescence measured at 560/590 nm using a Clariostar® Plus microplate reader (BMG LABTECH) Seeded scaffolds were dissolved via papain digest in a solution containing papain (2.5 U/mL) cysteine (5 mM) and EDTA (5 mM) (all Sigma) at 24 h Scaffolds were incubated in 0.5 mL solution at 60°C and left overnight to fully dissolve Samples (N = 5) were then used in the Quant-iT™ Picogreen® assay (Thermo-Scientific) as per the manufacturer’s instructions to determine the amount of dsDNA present in each scaffold The fluorescence of the samples was read at 490 nm (λex 480 nm λem 520 nm) using a Clariostar® Plus microplate reader (BMG LABTECH) Each sample was rinsed in triplicate with PBS before being fixed with formalin (10% 500 μL) (Sigma) in the incubator (37°C scaffolds were rinsed twice with PBS (10 min) The stain was prepared by creating working stocks of both dyes 0.5 μL calcein (Invitrogen) was added to 1 mL PBS; and 1.0 μL ethidium (EthD) (Invitrogen) was added to 1 mL PBS and protected from light 20 μL EthD and 5 μL Calcein working solutions were added to 10 mL PBS 150 μL of stain was added to each scaffold and incubated at RT Images were taken using a Zeiss AxxioImager epifluorescence microscope at ×25 magnification Samples were incubated in 0.2% Triton X-100 (Sigma) at RT for 10 min 1% v/v Phalloidin conjugate in PBS with 0.5% bovine serum albumin (BSA) was prepared by combining 1% BSA (Sigma) in 20 mL PBS and 0.2 μL Phalloidin 514 (Sigma) 100 μL was added to each scaffold and incubated at RT Phalloidin solution was aspirated to waste and samples were rinsed in triplicate with PBS (10 min) 4’6-diamino-2-phenylindole (DAPI) solution was prepared by adding 20 μL DAPI stock (Thermofisher) to 20 mL PBS scaffolds were rinsed twice with PBS (10 min) before images were taken using a Zeiss AxxioImager epifluorescence microscope at ×40 magnification Images were then processed using ICY software (n = 5) RNA was extracted from the samples using Tri-Reagent (Invitrogen) and purified using an RNA extraction kit (Promega) Gene expression was determined using reverse transcriptase polymerase chain reaction (RT-PCR) Forward and reverse primer sequences were designed using PromoBlast and custom made by Merck (Table 1) and glutathione peroxidase 1 (GPx-1) in the HSG cells grown in both normal and hydrogen peroxide medias was measured The levels of gene expression were measured using the 2−ΔΔCT method and are given relative to the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at D3 in the respective media type Forward and reverse primer sequences used in PCR All data was subjected to statistical analysis conducted by one-way ANOVA with Tukey’s post hoc test (OriginLab) Statistical differences considered were as follows: *p < 0.05 and ***p < 0.001 Data is reported as mean ± standard deviation (SD) RA was successfully incorporated into electrospun PCL polymer fibres. SEM image analysis confirmed that the fibres obtained all had similar morphology (Figure 1). The fibres were smooth and randomly aligned. A summary of characteristics for each scaffold is shown in Table 2 Minimal change was observed between the groups indicating that the inclusion of RA in the PCL fibres did not have any deleterious effect on the scaffold morphology SEM images of each scaffold group taken at ×4,000 magnification Scaffold Properties for each scaffold group There was significant difference at the p < 0.01 level between the fiber diameters of each group (n = 150) and p < 0.001 between both PCL and 0.1% RA with the 0.5% RA scaffold; with diameters ranging from 1.60 to 1.71 µm there was only a 2.3% and 6.4% reduction in fiber diameter size for the 0.1% RA and 0.5% RA scaffolds Water contact angle (WCA) confirmed the hydrophobic properties of the scaffold. PCL is known to be hydrophobic (Deng et al., 2019) WCA measurements were taken to determine if the inclusion of RA into the PCL fibres had any impact at all on the hydrophobicity of the fibres as this could potentially impact cell adherence when culturing the cells on the scaffold which would therefore influence the rest of the study No significant difference was found between the WCA of the three groups When comparing the ultimate tensile strength (UTS) of the scaffolds there was statistical significance of p < 0.01 between the two vitamin-containing groups and statistical significance at the p < 0.05 level between the PCL and 0.5% RA scaffolds Figure 2 displays the Young’s modulus (YM) for each scaffold group measured over four increasing 5% strain increments the incorporation of RA in the fibres led to a decrease in YM with the PCL only scaffolds being the stiffest at each range This trend was observed in all strain increments up to 20% and overall the material stiffness statistically reduced as the strain applied increased Incremental Young’s modulus measured from 0% to 20% strain (A) Results of the DPPH scavenging assay (B) results of the fluorometric H2O2 assay The DPPH assay (Figure 3A) showed that at 30 min both the PCL control and the RA containing scaffolds showed similar absorption with approximately 27% scavenging activity The scavenging activity was slightly higher in the RA scaffolds however this was not statistically significant As there was little difference between the groups the scaffolds were incubated again for a total period of 24 h and the analysis performed again scavenging activity increased to between 35% and 45% and there was a more notable difference between the RA scaffolds and the PCL control Over the duration of the test, the concentration of hydrogen peroxide was reduced when the RA containing scaffolds were incubated in H2O2 media (Figure 3B) It can be seen that there was a significant reduction between each timepoint taken from the 0.1% and 0.5% RA scaffold samples the 0.1% RA scaffold was shown to be the most effective in reducing the level of H2O2 This result aligns with the DPPH assay to show that the RA scaffolds are capable of producing an antioxidant effect A series of assays were performed on cells grown on each of the scaffolds to confirm that RA and the scaffolds themselves were not cytotoxic to the cells. As shown in Figures 4A, B cell viability in both normal and H2O2-doped media increased over the duration of the study (A) Cell viability in normal media (B) cell viability in H2O2 media (C) DNA quantitation in normal media (D) DNA quantitation in H2O2 media and there was no statistical significance seen between any of the groups at any timepoint Both Live/Dead and DAPI/Phalloidin staining (Figures 5A, B, respectively) confirm that the cells were adhered to and successfully proliferated on the surface of the scaffold. In the Live/Dead staining of seeded scaffolds incubated in H2O2 media (Figure 5A) it can clearly be seen that whilst cells are still able to proliferate in the H2O2 media There are two dead cells that were observed in the seeded PCL scaffold incubated in H2O2 sample showing that the scaffolds are not toxic to the HSG cells There were no dead cells observed on the seeded scaffolds containing RA indicating that the inclusion of the vitamin had no significant detrimental effect Representative images of (A) Live/Dead staining at D7 Green represents live cells; red represents dead cells (B) DAPI/Phalloidin staining at D7 Cyan represents nuclei (DAPI); green represents actin filaments (Phal.) Expression of genes associated with salivary glands and their function, as well as one for antioxidant activity, were determined using RT-PCR assays (Figure 6) Results were normalized to tissue culture plastic (TCP) at D3 for each gene This is represented by the horizontal dashed line in the PCR graphs Relative expression of (A) AQP5 in normal media (B) AQP5 in H2O2 media (C) BCL2 in normal media (D) BCL2 in H2O2 media (E) GPx1 in normal media (F) GPx1 in H2O2 media normalized to D3 TCP (represented by the horizontal dashed line in each graph) AQP5 is a water channel, expressed by the secreting acinar cells and is an essential component of functional salivary gland epithelium; it is expression is routinely used to assess salivary gland function and regeneration (Matsuzaki et al., 2012; Emmerson et al., 2018). AQP5 expression was notably reduced in the cells grown in hydrogen peroxide media (Figure 6B), in comparison to those grown in normal media (Figure 6A) Statistical significance (p ≤ 0.001) was found between the 0.5% RA scaffolds in the two different media types at 24 h Expression of the apoptosis marker BCL2 increased in the cells grown in hydrogen peroxide (Figure 6D), when compared to cells grown in normal media (Figure 6C) There was no significant difference seen within groups At 24 h statistical difference (p ≤ 0.001) was seen between 0.1% RA scaffolds in normal and H2O2 media while expression of BCL2 dropped with time in the 0.1% RA scaffolds Across all groups, expression of GPx1, an antioxidant marker, was highest at D7 (Figure 6E), increasing as the amount of antioxidant in the scaffold increased. There was still some expression, particularly at D3, in the hydrogen peroxide media, with PCL and 0.1% RA being upregulated in comparison to the same groups in the normal media (Figure 6F) Statistical difference at the p ≤ 0.001 level was observed between normal and H2O2 conditions with both the 0.1% and 0.5% RA scaffolds at 24 h the statistics showed that p ≤ 0.05 this is the first application of an electrospun antioxidant scaffold designed to regulate ROS levels in the salivary gland with the potential to support survival and proliferation of SG cells Significantly, the DPPH and hydrogen peroxide fluorometric assays produced results which confirmed that the scaffolds produced were capable of exerting an antioxidant effect. Previously published literature has shown that the scavenging activity of tissue engineered scaffolds can be increased by incorporating antioxidant compounds. Kheradvar et al. fabricated nanofibers containing vitamin E (VE) to be used for promoting repair and wound healing (Kheradvar et al., 2018) They found that increasing VE concentration lead to an approximate 48% difference in antioxidant activity between 1% and 5% VE within the first 4 h A similar trend was observed in our study where results showed an 18% increase between 0.1% and 0.5% RA however this was within the first 24 h there is a three-fold difference between these sets of results there are several factors that can be attributed to these differences PCL is insoluble in methanol (used to prepare the DPPH solution in the assay) whereas poly (vinyl alcohol) (PVA) is readily soluble This means that PVA fibres from the Kheradvar et al releasing and exposing more vitamin from within the fibres to the DPPH The fiber sizes and morphology will also influence their function as will the differences between the two vitamins Whilst the DAPI/Phalloidin staining indicates that there is a reduction in cell numbers when exposed to H2O2 this is not observed in the Live/Dead images. Based on the arbitrary Live/Dead imaging (Figure 5A) it seems that RA reacts with calcein (green) in the stain Upon closer inspection of the images with the antioxidant scaffolds what initially appears to be background noise actually shows that the calcein from the live/dead stain has adhered to the scaffold fibres The visibility of the fibres stained with calcein increases with increasing concentration of RA implying that a reaction is occurring between the RA on the surface of the fibres with the dye The green fibres cannot be seen in the PCL control images confirming that the fluorescence is due to the retinyl acetate this finding has not been stated in any previous literature Whilst these images are representative and live cells are still observed on the scaffold as expected this apparent reaction between the RA and calcein should be taken into consideration and taken as an artefact of using calcein as a live stain in the presence of RA Gene analysis was performed on a range of genes found within the SG (Figure 6) Genes were selected based on function that was most applicable to the function of the salivary gland and that would reflect the impact that the harsh ROS-mimicking environment has on the growth of the HSG cells and GPx1 genes code for proteins associated with water-channels RT-PCR was performed to determine gene expression of cells grown in both normal media and H2O2-doped media and normalized to tissue culture plastic (TCP) at D3 for each condition The upregulation of BCL2 observed in the H2O2 media is logical in the sense that the cells are trying to protect themselves by blocking the apoptosis pathway and this is intensified by the harsh environment In normal conditions, oxidative stress causes upregulation of GPx1, an enzyme that oxidizes GSH to GSSG to scavenge ROS and maintain cellular redox (Dequanter et al., 2017; Zhao et al., 2022) this may not be the case for damaged environments GPx1 expression in normal media showed a similar increasing trend between timepoints in each group with the highest expression seen in the 0.5% RA scaffold at each timepoint expression is significantly reduced in H2O2 media both conditions see an increase for GPx1 compared to 24 h which may indicate that the RA has a positive effect on these cells the important finding here is that the gene is maintained and the presence of RA does not disrupt the production of the gene Another notable observation is that expression of GPx1 increases even though cell viability decreases; there are fewer live cells on the scaffold however they are producing and exponentially increasing their antioxidant effect Whilst there was limited trends/statistical significance observed during RT-PCR all genes were still functionally active under both conditions and at all RA concentrations (as well as in PCL controls) highlighting the positive ability of HSG cells to maintain gene expression The scaffold did not have any negative impact on the genes expressed by HSG cells and the results also suggest that the presence of RA promotes cell survival Whist there are many other factors that can influence regulation of a gene the amount present on the surface of the scaffold there is an overall general trend that suggests that the inclusion of the antioxidant in the scaffold fibres has a positive benefit on HSG cells cultured under oxidative stress conditions Scaffolds were fabricated by electrospinning PCL polymer solution where PCL pellets and RA were dissolved in HFIP RA is insoluble in water therefore an alternative solvent had to be used Whilst water is preferable in terms of safety for use in vivo the solvent is allowed to evaporate and is also rinsed in ethanol followed by multiple PBS washes to ensure that the scaffold is completely sterile and void of any residual solvent This method gives polymer fibres with the RA incorporated throughout the scaffold While it would be interesting to measure the exact quantity of RA within the scaffolds when it comes to the function of these scaffolds we are mainly interested in how much of the RA is released from the fibres to produce the desired antioxidant effect We predominantly focused on the external functionality of the scaffolds (through the DPPH assay) as the amount of RA in the scaffold may not be representative of the amount present on the surface of and released by the fibres The cells used in this study are HSG epithelial cells, which are known to be contaminated with HeLa cells (Lin et al., 2018) This may influence the results and outcome in terms of testing the suitability of this process; however HSG cells still provide a good representative model for salivary gland cells for determining the effect that an antioxidant may have on their survival and proliferation however HSG cells are functioning SG cells that are robust enough to withstand the test conditions subjected to in this study and to ensure that the scaffold does not further damage or induce new damage upon gland cells that have survived radiation treatment could be investigated to further prove function and provide a more representative model system Whilst the cell viability assay measures the metabolic activity of the cells DNA quantitation results confirm the proliferation of the cells Both should be considered as metabolic activity within cells varies at different stages of the cell cycle and therefore can only be used as an indicator of viability the cells can be alive but depending on their state will exert different metabolic activity the combined results provide confirmation that the HSG cells are proliferating on the scaffolds and that this is not hindered by the inclusion of RA within the scaffold fibres There is evidence to suggest that the material needs time to activate the cells, as we see in the RT-PCR results of GPx1 after 3 days exposure to H2O2 and then again at D7 (Figures 6E, F) the upregulation of the gene suggests that there is an activation of antioxidant activity within the HSG cells themselves It could be that these cells require pre-exposure to the material prior to being placed into the ROS-mimicking condition before producing an antioxidant effect Introducing a pre-exposure time would allow an initiation period for the activation of the gene which would be more beneficial when employed as a therapeutic approach The positive results seen within this study provide motivation to investigate this further in future studies As previously mentioned, vitamin A (and its derivatives) have been shown to produce an anticancer effect (Blumenthal et al., 2000; Doldo et al., 2015) An important study in the future would entail determining if the RA scaffolds were capable of producing such an effect This could be done in vitro using organoids/spheroids A dual-action scaffold that is able to not only reduce the amount of ROS within the damaged environment but also provide an anticancer effect would be extremely beneficial this study has successfully proven that retinyl acetate can be incorporated into an electrospun fiber scaffold which can successfully promote cell survival and proliferation in both normal and mimicked ROS environments whilst producing an antioxidant effect This provides encouraging support for the potential use of such tissue engineering approaches to restoring the damaged microenvironment post-radiation to a state that can support the survival and proliferation of re-introduced cells with the ultimate aim of tissue repair and ultimately restoration of salivary gland function The work within this study emphasizes the potential of antioxidant tissue engineered scaffolds as a viable therapeutic approach and provides sufficient evidence to warrant further studies into such applications The original contributions presented in the study are included in the article/Supplementary Material further inquiries can be directed to the corresponding author LW performed all experimental work and processing of results and wrote the first draft of the manuscript All authors contributed to the article and approved the submitted version This work is funded by EPSRC grant number EP/T517884/1 and MRC grant MR/L012766/1 EE is funded by UKRI/MRC grant MR/S005544/1 and by a Chancellor’s Fellowship from the University of Edinburgh Alistair Elfick for use of lab facilities within the Institute of Bioengineering The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1233801/full#supplementary-material 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2023 Westwood, Emmerson and Callanan. 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Generally, data from Sunday’s games will be available early Tuesday afternoon. For an immediate look at the games from the previous weekend, please check out our Monday Review tool which includes a collection of preliminary stats Why would a review make the point of saying someone’s not a genius You think I’m especially not a genius [Pause] You didn’t even have to think about it Bears’ leaders met with Shane Waldron and he responded with D’Andre Swift Unleashed But on a week where Rashee Rice was lost for the season it’s imperative to focus on the positives despite playing half of his games with Malik Willis A couple other RotoViz favorites showed out in Week 4 as well. You have to select Jayden Daniels in every draft That was our dynasty drumbeat and a recurrent theme of the Best Ball Banana Stand with Peter Overzet And Chase Brown was one of my top RB recommendations. The Brown developments were beyond frustrating through three weeks, but he again demonstrated both elite talent and a perfect team fit in scorching the Panthers. You want the list of RBs who have both scored more fantasy points and averaged more EPA/game than Brown Brown looks like the most explosive RB in football and we’ve seen what he can do with a second-level crevice as his 54-yard receiving score in 2023 was the second-fastest ball carrier time in the NFL last year I’d say hopefully the Bengals have learned their lesson but I strongly doubt that’s the case The more realistic hope — or at least a parallel wish — is that Cincinnati could morph into an offense with a large enough rushing pie to support both Brown and Zack Moss. If you can get a Brown team through the midseason gauntlet, the Strength of Schedule Streamer really likes the Bengals backs for the fantasy stretch run If you’re starting to participate in resurrection-style drafts Brown is a compelling selection as a dynamic back in what should be a rising offense Jordan Addison was my other top WR play this season and it was brutal watching the first half of Week 1 and wondering what might have been if he’d stayed healthy turning five opportunities into 79 yards and two scores Addison has been a TD-machine at the NFL level and his 24 yards per reception on Sunday offered a reminder of the vertical ability that tends to be a bit undersold Addison’s return put my OT guillotine squad in position to overcome Breece Hall’s nightmare performance where he got benched for Braelon Allen Two weeks ago, I discussed a conservative bidding approach early in guillotine contests but the injury to DeVonta Smith put me in serious jeopardy heading into Week 4 especially after I ended up as the second-highest bidder on several of the top players chopped after Week 3 It’s a horrible feeling to rely on Trevor Lawrence who has been worse than Daniel Jones and Gardner Minshew from a reality Expected Points Added perspective The Jaguars signed Lawrence to a five-year and the signal caller has responded by failing as a viable starter Lawrence did manage to target his stars in Week 4 throwing 21 of 33 passes in the direction of Brian Thomas and Christian Kirk and the remaining 12 passes notched only 22 yards total Lawrence missed a wide-open Thomas — who looks like an instant star and fantastic first-round reality pick — and Kirk on what could have been long TDs Speaking of resurrection, Colm and I will be hosting a new guillotine league for the RotoViz community soon, maybe even this Wednesday evening. To get involved, sign up at guillotineleagues.com and contact Colm (@OvertimeIreland) We may have a fun RV-related prize for the winner Membership Required You must be a member to access this content View Membership Levels What’s included in your subscription? Shawn Siegele updates his 2025 dynasty superflex rookie rankings after 60 frantic hours of drafting in the FFPC He discusses what these high-stakes leagues can teach us about all dynasty rookie drafts this spring and summer Beginning Saturday morning I’ve spent almost every waking moment living and breathing fantasy rookie drafts With nine FFPC squads and seven different co-managers Shawn Siegele discusses rankings and tiers for 2025 dynasty superflex rookie drafts and proposes scenarios for maximizing your total haul The RotoViz TriFlex leagues and regular FFPC dynasty leagues begin their rookie drafts on Saturday morning In this exercise I work through projected rookie draft slots and help subscribers navigate individual player tiers to create the most total value and best address team needs Shawn Siegele updates his top-60 dynasty rookie rankings live on draft weekend Earlier this week I published my final pre-draft rookie rankings As a reference piece that included a) my most recent thoughts b) results and visualizations from the combine c) intel on peripherals from Sports Info Solutions and d) intel from various articles and RotoViz tools Sign-up today for our free Premium Email subscription Terms of Service Volume 3 - 2016 | https://doi.org/10.3389/frobt.2016.00064 Middlewares are fundamental tools for progress in research and applications in robotics They enable the integration of multiple heterogeneous sensing and actuation devices as well as providing general purpose modules for key robotics functions (kinematics no existing middleware yet provides a complete set of functionalities for all robotics applications and many robots may need to rely on more than one framework This paper focuses on the interoperability between two of the most prevalent middleware in robotics: YARP and ROS Interoperability between middlewares should ideally allow users to execute existing software without the necessity of (i) changing the existing code and (ii) writing hand-coded “bridges” for each use case We propose a framework enabling the communication between existing YARP modules and ROS nodes for robotics applications in an automated way Our approach generates the “bridging gap” code from a configuration file connecting YARP ports and ROS topics through code-generated YARP bottles We support YARP/ROS and ROS/YARP sender/receiver configurations which are demonstrated in a humanoid on wheels robot that uses YARP for upper body motor control and visual perception and ROS for mobile base control and navigation algorithms which supports the run-time conversion of YARP bottles to ROS messages Our framework provides both YARP/ROS and ROS/YARP as sender/receiver use cases The YARP/ROS use cases illustrate how to visualize in Rviz (a ROS application) (i) the current state of the robot joints controlled with YARP and (ii) the current gaze point of a robotic head The ROS/YARP use case shows how to send target gaze points from a ROS topic to a robotic head controller implemented in YARP Naur and Randell (1969) define middleware as a piece of software that gives an extra level of abstraction to the developer through a layer between the operating system and the applications The majority of the communication middlewares are based on an Interface Definition Language (IDL) approach 2 and have several implementations.3 Although the IDL is an agnostic standard the implementations require libraries for all the supported languages/operating systems The dependency on specific libraries has shifted the problem from the IDL to the adoption of a particular implementation Vendors and designers encourage the users to adhere to a particular middleware while not considering other options so the interoperability between middlewares is not usually addressed In the robotics context, Ceseracciu et al. (2013) describe the middleware as the entity which provides the glue that holds all the software modules together. Furthermore, as noted by Mohamed et al. (2008) a middleware helps collaborative development since each developer may orient its efforts to a specific module a high effort is put on the messaging system in terms of efficiency and coordination mechanisms we focus on the communication services that allow to send and to receive information between software components in the YARP and ROS middlewares Metta et al. (2006) introduced YARP an open-source middleware initially designed to provide an abstraction layer to the communications between modules It has evolved into a multipurpose middleware that provides libraries and utilities that act as the control system of a robot which uses YARP as its “circulatory system” the robots Coman and Vizzy have adopted it adding them to the more than 20 labs that use the iCub and YARP for research The basic communication objects in YARP are Ports that send Bottles over the network The Bottles may be constructed by hand or using the Thirft Interface Definition Language (IDL) that allows to define the bottle from a struct (i.e. The most recent developments by the YARP team, described in Fitzpatrick (2016) allow to communicate bidirectionally (read/write from/to ROS topics) for all the basic types and some of the non-basic types such as images There are two options to translate types between YARP and ROS: (i) generate ROS-compatible types at compilation time using Thrift ILD or (ii) generate ROS-compatible types at run-time using Bottles both options require custom code written purposefully for each particular set of messages which is an effortful process in systems with many communication links Aspects such as conversion between data types packaging/unpackaging of messages (many-to-one or one-to-many conversions) all have to be hand coded by the developer we propose a semi-automated way to generate the interface code the developer describes just the minimal set of information required to perform the conversion between message formats we describe the automatic code generator that allows to exchange information between existing YARP modules and ROS nodes Our framework is based on a tool that generates C++ code from a configuration text file that describes the set of inputs (i.e. data conversions that may be applied to the inputs and a detailed specification of the output (i.e. We denote this tool as the yarp bottle generator which after parsing the configuration file creates a C++ file that after compilation and subsequent execution acts as a bridge between YARP and ROS From the point of view of Software Patterns that transforms data between a source and a destination while lowering the coupling level The generated code allows to communicate between YARP port objects and ROS publisher/subscriber objects developed previously Our generator abstracts YARP and ROS developers from dealing directly with interoperability issues The main concepts of the bottle generator are: the hub which are explained in the following subsections Figure 1A illustrates the main concepts of the generated code and their interaction for creating the output The output message is assembled from the information provided by the converters (units and data types conversion between the different systems) constants (additional data required by ROS) and counters (time and sequence information required by ROS and YARP) which collects information from several YARP ports/ROS topics and merges/splits/reorders data elements according to the requirements of the different platforms Figure 1. Top image shows the architecture of the generated code. Bottom table shows the overview of the configuration parameters. (A) Generated code detailed architecture for the main use cases. The dashed boxes represent the previously implemented YARP with ROS Fitzpatrick (2016) interoperability tools (B) Overview of configuration file parameters If the message section has a hierarchical structure additional sections must be added and the instructions on how to do this are available online (see text footnote 8) A hub reads data coming from several ports/topics and stores the data in a YARP bottle The data are ordered in the bottle sequentially according to the list of port names indicated in the configuration file The user can define as many hubs as needed in the configuration file according to the needs The role of hubs is mostly related to (i) the memory layout of the output and (ii) the converter functions to be applied a hub reads the data coming from several YARP ports that contain the motor encoder values of a robot head and arms This hub reads the ports and stores the values sequentially in a bottle The hub idea is similar to the YARP Port Arbitrator of Paikan et al. (2014) because the arbitrator and the hub read data from several ports The main difference is that the hub stores the data in a bottle while the arbitrator selects one of the ports to be sent according to a rule The converter receives a bottle from the hub and then applies a function to every element of the bottle The conversion function was designed to perform tasks such as unit conversion (e.g. The current approach is to have a converter for each hub so in the configuration file each hub has the field “function” where its corresponding converter function is defined The conversion result is stored in the same bottle received by the converter which provides this functionality between YARP ports in run-time The output builder constructs a YARP bottle using a hierarchical structure of data types, defined in the configuration file. The hierarchy of allowed types includes the hubs, constants, timestamps, and counters, as illustrated in Figure 1A The structure of the hierarchy is comprised by the root’s data type and its children The hierarchical data type is denoted as msg (borrowing from the ROS message definition) which means that the definition of this type is a list of data types The remaining data types can be divided into (i) containers (list The containers have several values wrapped in one bottle and the basic types are just single elements added to the output while the single_value is a constant (i.e. The examples in Section 4 will show the application of the components explained in this section Top part shows the YARP–ROS configuration file and bottom part shows the ROS–YARP configuration file there are two sensors: (i) a fake motor control board interface that sends the motor encoder values to a YARP port (mux1) and (ii) a fake inertial sensor that sends readings to a YARP port (mux2) Readings from both sensors are sent to a ROS topic that reads the MotorsInertial message file (right side of the figure) The MotorsInertial file indicates that the data are composed of two floating point arrays (B) ROS–YARP configuration file example whose motors are controlled by a ROS program The ROS message contains the joint number and the position value to be commanded which is shown on the left side of the figure The right side of the figure shows the configuration file for the ROS–YARP bridge where there the position values for a motor are read from a ROS topic configured in mux1 (/fakebot_motor_control ros) The read values are sent to a YARP port (/fakebot/motor/rpc:i) configured in the section message Figure 3. Example of the generated C++ code by running the yarp-bottle-generator executable with the configuration file yarp_ros_tutorial example in Figure 2A Figure 4. Example of the generated C++ code by running the yarp-bottle-generator executable with the configuration file ros_yarp_tutorial example in Figure 2B the middle table shows the computational overload of the generated code and the bottom table shows the network overload of the generated code The results show the additional execution time of reading the inputs and writing the output Bottle The critical issue for managing the source/destination is that connections between ports/topic are done differently on each middleware YARP does not assume either read or write status of a port so the sender–receiver connection is done by the user ROS topics have to be declared as either publishers or subscribers so the roscore server does the connection sender–receiver according to the topic definition connecting to YARP ports when sending/receiving data to/from YARP ports and letting the roscore server manage the connections when sending/receiving data to/from ROS topics The concepts explained in Section 3.1 correspond to sections or parameters of the configuration file. Figure 1B summarizes the sections (in square brackets) and their parameters, taking into account the source–destination middleware configuration. More details on how to write your own configuration file are available online.8 Section 4.3 analyses the additional computational resources required by our approach followed by known issues and limitations section Figure 2A shows the configuration file for reading two YARP ports and converting them into a MotorsInertial ROS message (right side of Figure 2A). The generated program shown in Figure 3 reads from the YARP ports /dummy_head/state:o and /inertial and creates a MotorsInertial message which is published on the ROS topic /motors_inertial_port the program converts the angles to radians and fills up the arrays inertial and encoders of the MotorsInertial message with the converted values The configuration file contains four sections: [general] • The output entity name (string output_name) • The source/destination middlewares flags (from_ros_topics = false,to_ros = true) • The number of hubs (integer num_mux) • The rate of execution of the generated code (number rate) • The ROS message name (string ros_msg_name) The subsequent sections of the configuration file describe each of the hubs which in this example are two (mux1 and mux2) • Number of ports (integer num_ports = 1 for both hubs) • Name of the ports/topics (string ports = /dummy_head/state:o for mux1 and ports =/inertial for mux2) • Converter function function = deg_to_rad for mux1 which converts the read encoder angles from degrees to radians function = non-e_double parses the coming data as a floating point number • Verbose flag (string verbose = false) in case the user wants to print on the screen the values red by the hub The rest of the file describes how the output’s structure is built The section message needs to know the number of types (integer num_fields) which is this case is the number of fields of MotorsInertial each field corresponds to the data stored by the hub • The first type 1_type = mux corresponds to the data stored in a hub In order to know which hub corresponds to this field the line 1_mux = mux2 assigns the data from the inertial sensor • The second type 2_type = mux and 2_mux = mux1 assigns the data from the motors to the second field of the message Figure 3 shows the code generated by this configuration file example The code guarantees that there is successful connection to all the YARP input ports waiting until all the YARP ports are connected to the bridge ports moves to a loop that (i) reads the input data It is important to remark that for the output port is not necessary to check the connection status because ROS automatically connects the publisher to the subscriber as soon as they are available The right side of Figure 2B shows the configuration file for reading the ROS message MotorControl (on the left side of Figure 2B) from a ROS topic converting the message into a YARP Bottle that commands a motor of the fakebot robot The generated program reads the joint and angle values from the topic /fakebot_motor_control_ros then sending the command “set pos joint angle” to the RPC YARP port /fakebot/motor_rpc:i The main differences with respect to the previous example are • The general section has the flags to_ros = false and from_ros_topics = true • No converter function applied to the hub (function = none) • The [message] generated has two string values that are constant (1_msg and 2_msg) if the user in the configuration file associates a wrong type between the middlewares the code is generated without any warnings/errors the user has to verify the association between types Another limitation of our approach is the applicability area the main goal of the middleware is the correct streaming of sensors and actuators data for control systems so abstractions such as meta-messages that will work for several middlewares are difficult to implement because of the low-level streaming approach (e.g. different data code/decode for the same type of sensor) direct and intellectual contribution to the work This work was supported by FCT (UID/EEA/50009/2013) programme RBCog and FCT project AHA (CMUP-ERI/HCI/0046/2013) CrossRef Full Text | Google Scholar Google Scholar Architectures for enterprise integration and interoperability: past Fitzpatrick, P. (2016). Using YARP with ROS. Available at: http://www.yarp.it/yarp_with_ros.html Google Scholar CrossRef Full Text | Google Scholar CrossRef Full Text | Google Scholar “Middleware for robotics: a survey,” in IEEE International Conference on Robotics Google Scholar “Vizzy: a humanoid on wheels for assistive robotics,” in Robot 2015: Second Iberian Robotics Conference: Advances in Robotics Google Scholar Software Engineering: Report of a Conference Sponsored by the NATO Science Committee Google Scholar Google Scholar Ros: an open-source robot operating system Google Scholar Sucan, I. A., and Chitta, S. (2016). MoveIt! Available at: http://moveit.ros.org Google Scholar Moreno P and Bernardino A (2016) Middleware Interoperability for Robotics: A ROS–YARP Framework Received: 03 February 2016; Accepted: 06 October 2016; Published: 28 October 2016 Copyright: © 2016 Aragão, Moreno and Bernardino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Plinio Moreno, cGxpbmlvQGlzci50ZWNuaWNvLnVsaXNib2EucHQ= Metrics details Hypoxia is known to be detrimental in cancer and contributes to its development we present an approach to fate-map hypoxic cells in vivo in order to determine their cellular response to physiological O2 gradients as well as to quantify their contribution to metastatic spread We demonstrate the ability of the system to fate-map hypoxic cells in 2D We identify distinct gene expression patterns in cells that experienced intratumoral hypoxia in vivo compared to cells exposed to hypoxia in vitro The intratumoral hypoxia gene-signature is a better prognostic indicator for distant metastasis-free survival Post-hypoxic tumor cells have an ROS-resistant phenotype that provides a survival advantage in the bloodstream and promotes their ability to establish overt metastasis Post-hypoxic cells retain an increase in the expression of a subset of hypoxia-inducible genes at the metastatic site suggesting the possibility of a ‘hypoxic memory.’ The vast majority of studies on hypoxia-regulated gene expression have been conducted in an in vitro setting by exposing cells to 1% O2 for short periods of time The 4xHRE-CRE-ODD transgene integrated at chromosome 3 position 61,062,240 c Triple-transgenic mice were sacrificed at different time points over a 4-month period in order to detect hypoxic cells during breast cancer progression from hyperplasia to early carcinoma and invasive late-stage carcinoma H&E-stained sections of paraffin-embedded tissue (left) or fluorescent imaging of frozen tissue sections (right) The second inset at the DCIS stage highlights early detection of hypoxia h O2 measurements performed by using OXNANO nanoprobes dispersed in 3D Matrigel surrounding embedded organoids (mean ± SEM n > 200); ****P < 0.0001 versus day 1 (matched one-way ANOVA with Bonferroni posttest) the results suggest that (1) organoids cultured in 3D develop severe O2 gradients that range from 14% to 1.7%; (2) the change in O2 levels over time may be an important environmental cue that dictates the fate of individual or clusters of cells in organoids grown in 3D cultures Ratios (%) are displayed in the graph at different time points of tumor progression (mean ± SEM n = 48); ****P < 0.0001 versus day 15 (Two-way ANOVA with Bonferroni posttest) The box extends from the 25th to 75th percentiles the median is marked by the vertical line inside the box and the whiskers represent the minimum and maximum points f MCF7 hypoxia fate-mapping cells were injected into the nipple and delivered to a single ductal tree of multiparous NSG mice A whole mount of the mammary fat pad was imaged by fluorescent microscopy 60 days after injection g Tissue sections were stained with DAPI to detect cell nuclei and imaged for DsRed and GFP h Tissue sections were stained with DAPI to detect cell nuclei and labeled with a HIF-1α antibody these data show that cells that experienced hypoxia in the primary tumor (GFP+) can migrate toward a more oxygenated invasive front of tumor regions in orthotopic MDA-MB-231 and 4T1 models and form invasive structures in the intraductal MCF7 model we compared the gene expression changes that occur in vivo with standard hypoxic exposure in vitro b–d Relative expression of mRNA measured by qPCR in tumor-derived cells (TG or TR) or MDA-MB-231 cells exposed to 20% or 1% O2 in vitro c genes co-regulated by intratumoral and in vitro hypoxia (CA9 and d genes exclusive to upregulation upon intratumoral hypoxia (ITGA10 and CP) (mean ± SEM n = 3); ****P < 0.0001 TG versus TR and 1% versus 20% (two-tailed t-test) e Heat map of the 41-gene signature derived from the overlap of intratumoral and in vitro hypoxia The distribution of the relative fold change of each gene in the 41-gene signature is displayed for GFP+ versus DsRed+ sorted tumor cells (T) or cells (C) exposed to 1% versus 20% O2 conditions Genes with fold change higher than 75% of the fold change of genes in the set are red and genes with fold change lower than 25% of the fold change of genes in the set are blue (Pearson correlation factor r = 0.84 and P = 9.6 × 10−7) f–h Microarray expression data from 664 breast cancer patients were used to perform multigene survival analysis (n = 664; HGU133 plus 2.0 arrays Kaplan–Meier analysis of distant metastasis-free survival (DMFS) of breast cancer patients stratified by high or low expression by using f the 40 most induced genes by intratumoral hypoxia g the 41-gene signature derived from the overlap of intratumoral and in vitro hypoxia or h the 40 most induced genes by in vitro hypoxia these data demonstrate that (1) the response to in vitro hypoxic culture conditions (24 h) does not recapitulate the response to physiologic levels of intratumoral hypoxia; (2) intratumoral hypoxia-induced gene expression is not entirely reversible even after standard tissue culture whereas the in vitro hypoxic effect is reversed within 2 days of culture under 20% O2 Both in vitro and intratumoral hypoxia showed significant dow-regulation of pathways associated with proliferation Post-hypoxic cells have enhanced metastatic potential a Orthotopic tumors derived from MDA-MB-231 hypoxia fate-mapping cells and tumor were harvested at 2 weeks for half of the mouse cohort Surgical tumor resection was performed on the second half of the cohort and lungs were harvested 2 weeks after tumor removal to assess late-stage metastasis Pie charts represent the ratio of DsRed+ to GFP+ cells in each site b The probability of a GFP+ (or DsRed+) CTC in the blood was obtained by dividing the percentage of GFP+ (or DsRed+) CTCs detected in the bloodstream with the percentage of GFP+ (or DsRed+) cells in the matched primary tumor (N = 3 n = 17) by using flow cytometry (FC); ****P < 0.0001 GFP versus DsRed (two-tailed t-test) c Cross-sectional imaging of DsRed and GFP expression and DAPI labeling of lung micrometastasis at week 2 The inset shows magnified GFP+ micrometastasis e The probability of a GFP+ metastatic event in the lung is obtained by dividing the percentage of GFP+ micrometastases by the percentage of GFP+ cells in the matched primary tumor by using d image analysis or e flow cytometry (N = 3 IA: n = 23; FC: n = 22); ****P < 0.0001 GFP versus DsRed (two-tailed t-test) median is marked by the line inside the box and whiskers represent the minimum and maximum f Cross-sectional imaging of DsRed and GFP expression and DAPI labeling of lung macrometastasis 2 weeks after tumor resection h The probability of a GFP+ metastatic event in the lung is obtained by dividing the percentage of individual GFP+ macrometastatic colonies by the percentage of GFP+ cells in the matched primary tumor by using g image analysis or h flow cytometry (N = 3 IA: n = 16; FC: n = 22); ****P < 0.0001 GFP versus DsRed (two-tailed t-test) j Tumor and lungs from triple-transgenic mice were harvested at 4 months of age Probability of lung metastatic events was obtained as described in g (mean ± SEM N = 8); ****P < 0.0001 GFP versus tdTom (two-tailed t-test) k MDA-MB-231 hypoxia fate-mapping cells were injected into the tail vein of NSG mice and harvested 5 weeks later the findings demonstrate that (1) a portion of hypoxic cells in regions adjacent to the perinecrotic region of the tumor can overcome O2 deprivation and metastasize; (2) cells that leave the hypoxic region of the primary tumor and enter the bloodstream will be exposed to higher O2 levels (reoxygenation); (3) GFP+ cells detected in the blood and lung became hypoxic in the primary tumor; (4) post-hypoxic cells have 4–5× higher chance at being found in the blood and lung Exposure to intratumoral hypoxia promotes invasion a Schematic representation of the early and late steps of the metastatic cascade b Tumor-derived cells were incorporated with 90% non-labeled wild-type MDA-MB-231 cells in a 3D spheroid fully embedded in collagen spheroids were subjected to time-lapse imaging every 5 min for 16 h Phase-contrast and fluorescent images taken on days 0 and 4 of the same spheroid The white dashed ring marks the invasive front of the spheroid d Total diffusivity (mm/min) (c) and persistent time (min) (d) of DsRed+ and GFP+ (mean ± SEM n = 80–92 cells); ****P < 0.0001 GFP versus DsRed (two-tailed t-test) e Projection of DsRed+ (red) or GFP+ (green) cell trajectories f Number of DsRed+ and GFP+ cells at the invasive front of the spheroid (beyond the dashed white line in (b)) (mean ± SEM n = 44 spheroids); ****P < 0.0001 GFP versus DsRed (two-tailed t-test) Post-hypoxic CTCs have ROS-resistant phenotype a Schematic representation of the proposed mechanism Post-hypoxic cells (GFP+) are resistant to ROS and have a survival advantage over DsRed+ cells in the bloodstream b Mitochondrial ROS levels were measured by using MitoROS by flow cytometry in matched tumor and blood samples freshly harvested from mice (N = 2 n = 8); ****P < 0.0001 GFP versus DsRed and ####P < 0.0001 tumor versus blood (Two-way ANOVA with Bonferroni posttest) the median is the vertical line inside the box and the whiskers represent the minimum and maximum c Freshly isolated CTCs were treated with the ROS inhibitor N-acetylcysteine (NAC) for 1 h and ROS levels were measured using MitoROS by flow cytometry (N = 1 n = 7); ****P < 0.0001 GFP versus DsRed and ####P < 0.0001 NAC– versus NAC+ (Two-way ANOVA with Bonferroni posttest) d Cell viability was measured by using Sytox Blue by flow cytometry (N = 1 n = 9); ****P < 0.0001 GFP versus DsRed (two-tailed t-test) f Tumor-derived cells sorted for DsRed+ or GFP+ expression were treated ex vivo with H2O2 for 1 h and f were quantified 48 h later by image analysis by using nuclei segmentation to determine cell confluence (e) (N = 4 n = 16); ****P < 0.0001 GFP versus DsRed (Two-way ANOVA with Bonferroni posttest) g Tumor-derived hypoxia fate-mapping cells (1 × 105 cells) were injected directly into the tail vein of NSG mice Organs were harvested either 48 h or 25 days after injection i Cryo-sections of the lung were stained with DAPI and imaged for DsRed and GFP to detect micrometastasis after 48 h (h) or macrometastasis after 25 days (i) j Quantification of the number of metastatic events by image analysis of mounted lung sections after 48 h or 25 days (N = 1 n = 9–10); ****P < 0.0001 GFP versus DsRed (Two-way ANOVA with Bonferroni posttest) k Quantification of metastatic burden by flow cytometry analysis of lungs after 48 h or 25 days (N = 1 n = 10); ****P < 0.0001 GFP versus DsRed (Two-way ANOVA with Bonferroni posttest) the findings demonstrate that GFP+ cells are more resistant to oxidative stress and maintain lower levels of mitochondrial ROS in the bloodstream than DsRed+ cells DsRed+ cells are also more susceptible to ROS-induced apoptosis Two times more GFP+ than DsRed+ cells that successfully extravasate are able to initiate metastatic colonization our results suggest that post-hypoxic cells acquire a ROS-resistant phenotype in the primary tumor as well as the ability to migrate and intravasate into the bloodstream The ROS-resistant phenotype allows them to survive high ROS levels in the bloodstream to promote their metastasis-initiating capability in the lung c Heat map of the 19-gene signature derived from the overlap of tumor and lung GFP+ induction The distribution of the relative fold change of each gene in the 19-gene signature is displayed for in vitro hypoxic exposure (in vitro) GFP+ versus DsRed+ sorted tumor cells (TG/TR) and GFP+ versus DsRed+ sorted lung metastatic cells (LG/LR) Genes with fold change higher than 75% of the fold change of genes in the in vitro set are blue and genes with fold change lower than 25% of the fold change of genes are white Genes with fold change higher than 75% of the fold change of genes in the in vivo sets are dark green and genes with fold change lower than 10% of the fold change of genes are white (Pearson correlation factor TG/TR vs d Overview of the role of post-hypoxic cells in the metastatic cascade Post-hypoxic cells (GFP+) have enhanced metastatic potential associated with enhanced invasion and a ROS-resistant phenotype that improves survival in the bloodstream A global gene expression analysis demonstrated that the intratumoral exposure versus in vitro exposure to hypoxia results in distinctly different gene expression profiles oxygenation is finely tuned across time and length scales reflecting important parameters such as the dilation and constriction of nearby blood vessels our result is not entirely surprising since nearly all of these processes including interactions with the tumor microenvironment We found that the 41-gene signature derived from the overlap of intratumoral and in vitro hypoxia serves as a prognostic indicator of DMFS This is also consistent with the <30-min half-life of HIF-1α upon reoxygenation nine of the nineteen RNA transcripts that are regulated in common in the GFP+ versus DsRed+ cells in the primary tumor and metastatic lung lesions are hypoxia-regulated and HIF-inducible This result suggests that a limited number of hypoxia-inducible genes maintain the same magnitude of increased expression at the metastatic site Further work is warranted to determine the mechanistic underpinnings of this finding Future work is needed to determine whether post-hypoxic cells at the metastatic site are more resistant to current chemotherapy regimens and whether targeting post-hypoxic cells could have a therapeutic benefit for patients with metastatic disease and 4T1 (ATCC® CRL-2539™) cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM (Sigma-Aldrich) or RPMI-1640 (4T1; Sigma-Aldrich) with 10% FBS (Corning) and 1% penicillin/streptomycin (Invitrogen) Hypoxic conditions were achieved by using an InvivO2 workstation (Baker) with an ICONIC (Baker) electronically controlled gas-mixing system maintained at 37 °C and 75% humidity The loxp-DsRed-loxp-eGFP sequence was PCR amplified from the plasmid pMSCV-loxp-DsRed-loxp-eGFP-Puro-WPRE (#32702 Addgene) by using primers containing Sal1 and Not1 restriction enzyme sites The amplified PCR fragment was digested with Sal1 and Not1 treated with Calf intestinal phosphatase (New England BioLabs) The fragmented DNA was ligated into the pENTRA1 vector cut with Sal1 and Not1 The Gateway SystemTM (Invitrogen) was used to recombine the pENTR1A shuttle vector with pLenti CMV/TO Zeo DEST (644-1) (#17294 Lentiviral vector 1 encoding CMV-loxp-DsRed-loxp-eGFP or lentiviral vector 2 encoding 4xHRE-MinTK-CRE-ODD were co-transfected with plasmid psPAX2 (#12260 Addgene) into 293T cells by using Polyjet (SL10088 Filtered viral supernatant generated from lentiviral vector 1 was collected 48 h post transfection and added to MDA-MB-231 or 4T1 cells with 8 μg/mL polybrene (Sigma-Aldrich) overnight zeocin (Invitrogen) was added to the medium of cells for selection (100–250 μg/mL) cells were transduced with lentivirus encoding 4xHRE-MinTK-CRE-ODD The cell lines were exposed to 5% O2 and flow sorted to remove GFP+ cells The flow-sorted cells were single-cell cloned and screened by image analysis and flow cytometry to identify clones that switched from DsRed expressing to GFP expressing under 0.5% but not at 3% O2 conditions Aliquots of whole-cell lysates were prepared in NP-40 buffer (150 mM NaCl pH 8.0) and fractionated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) Proteins were transferred from the SDS gel to a nitrocellulose membrane for 10 min by using a Trans-blot Turbo (Bio-Rad) The nitrocellulose membrane was blocked in 5% milk (% w/v) in Tris-buffered saline and 0.1% Tween-20 (TBS-T) for 30 min and incubated overnight with primary antibodies (1:1000) against HIF-1α (#610959 the nitrocellulose membrane was incubated with the corresponding anti-mouse (1:2500) (#AC2115 Azure Biosystems) or anti-chicken (1:5000) (AP162P Sigma-Aldrich) HRP-secondary antibody for 1.5 h at room temperature with orbital shaking β-actin was detected with a β-actin HRP-conjugated antibody (1:10,000) (#HRP-60008 ECL (Enhanced Chemiluminescent Substrate) (Perkin Elmer) was utilized as the substrate for HRP-catalyzed detection The chemiluminescent signal was imaged by using a c300 imager (Azure Biosystems) DsRed-only and GFP-only cell lines were used as control for the GFP antibody injected with 1.25 mg of pimonidazole in saline (12.5 mg/ml) (Hypoxyprobe™-1) 1 h prior to sacrificing Mammary fat pads located on the left side of the mouse were formalin-fixed MFPs located on the right side of the mouse were fixed in 4% PFA at 4 °C for 4 h saturated in 30% sucrose (Sigma-Aldrich) at 4 °C overnight and mounted onto Superfrost Plus Microscope Slides (Fisher Scientific) To evaluate the GFP and tdTom distribution tumor tissue was stained with DAPI (1:1000 for 15 min Bright-field images of H&E-stained sections were taken with an Olympus (UPLFLN 10XPh) phase objective 10 × (BioTek Instruments) GFP and tdTom expression were evaluated by using frozen tissue mounted on slides and imaged by using a 40×/1.30 PlanNeofluar oil objective with DIC0X/0.45 PlanApo (dry no DIC) and a 63×/1.4 PlanApo oil objective with DIC in a Zeiss LSM780-FCS microscope were identified by using a proprietary Cergentis script Integration sites were detected based on coverage peaks in the genome and the identification of fusion reads between the TG sequence and host genome A mixture of 80–90% wild-type (non-labeled) cells and 10–20% hypoxia fate-mapping cells were plated per well in spheroid formation media (2 × 104)—DMEM (Sigma-Aldrich) and Methocult H4100 (STEMCELL Technologies) spheroids were embedded into 2 mg/ml collagen containing DMEM and soluble rat tail type I collagen (Corning) each spheroid was transferred to a Petri dish where it was individually isolated with a collagen solution mix and quickly transferred to the center of a semi-cross-linked collagen gel in a 96-well plate at 37 °C Spheroids were imaged in an environmentally controlled microscope every 2 days by using an Olympus (UPLFLN 4 × ) objective in Cytation 5 (BioTek Instruments) Multiple images were captured in order to display the entire spheroid (up to a 3 × 3-tile size) Confocal microscopy was performed to obtain z projections of spheroids by using a 10 × /0.45 PlanApo (dry no DIC) objective in a Zeiss LSM780-FCS microscope Z stacks spaced at 6.3-μm intervals of 4 × 4 tiles were processed into a 3D image via Imaris version 9.2 (Bitplane) and 3D surface rendering was used to visualize the color distribution in 3D the same circular region of interest (ROI) was aligned with the center of each spheroid and cells outside the ROI were counted as cells at the invasive front of the spheroid Animal research complied with all relevant ethical regulations according to protocols approved by the Johns Hopkins University Animal Care and Use Committee mice were anesthetized with isoflurane; 5 × 104 MCF7 hypoxia fate-mapping cells were injected directly into the mammary duct of 8- to 12-month-old multiparous (NSG) female mice injected with 1.25 mg of pimonidazole in saline (12.5 mg/ml) (Hypoxyprobe™-1 kit) 1 h prior to sacrificing Tumors were excised at various time points formalin-fixed (Sigma-Aldrich) for 1 h and saturated in 30% sucrose (Sigma-Aldrich) at 4 °C overnight and embedded in OCT media (Fisher Scientific) To evaluate the GFP and DsRed/tdTom distribution To assess the entire cross section of the tumor each slide was imaged with an Olympus (UPLFLN 4×) objective by using Cytation 5 microscope (BioTek Instruments) Multiple image tiles were linearly stitched with Gen5 Software (BioTek Instruments) Tumor removal surgery was performed 12 days (4T1 model) or 2 weeks (MDA-MB-231 model) after tumor implantation Mice were anesthetized and transferred to a heating pad and the tumor was carefully detached from adjacent skin adjacent loose tissue and visible lymph nodes were also removed The wound was closed by using 9-mm autoclips (Braintree Scientific and ophthalmic ointment was used to prevent eye desiccation An average of 500 μl of blood was collected by cardiac puncture into an EDTA tube by using a 26-G syringe needle Red blood cells were lysed by using cold ACK Lysing Buffer (Quality Biological) Cells were incubated with 5 ml of the lysis buffer on ice for 10 min followed by cold centrifugation at 1700 g for 5 min and the remaining cells were washed and resuspended in FACS buffer lungs were inflated with an OCT:PBS solution and excised for both image and flow cytometry analysis Full lung-slide sections were imaged as described for the primary tumor Image analysis was performed by using NIS-Elements software Individual metastatic events were carefully annotated metastatic events were marked as ROIs by using the auto-detect tool available in NIS-Elements lungs were chopped and digested (collagenase 2 mg/mL (Sigma-Aldrich) BSA 2 mg/mL (Gemini Bioproducts)) for 1 h at 37 °C at 160 RPM cells were washed with PBS and resuspended in FACS buffer Animals were warmed for 5–10 min with an overhead heat lamp to dilate the veins and mice were sacrificed at the time points indicated The cell suspension was filtered (70-μm strainer) and enriched by magnetic-activated cell separation (MACS) by using a Mouse Cell Depletion Kit (Miltenyi Biotec) following the manufacturer’s instructions The resultant cell suspension was then washed with PBS and suspended in sorting buffer Cells were sorted by using a SH800 cell sorter (Sony Biotechnology) directly into Tris Reagent (Zymo Research) for RNA extraction followed by RNA sequencing or directly into cell culture media to establish cell lines fixed with 4% PFA (Fisher Scientific) for 5 min Samples were analyzed by using a FACSCalibur (BD Biosciences) flow cytometer and DsRed was detected in the FL-2 channel DsRed-only and GFP-only cell lines were used as controls for compensation Data were analyzed via FlowJo V10 software (Tree Star Needle probes were carefully washed and calibrated to atmospheric O2 before each measurement Paraffin-embedded tissue sections of an MDA-MB-231 tumor were dewaxed with Clarification new-clean® (EMD) and hydrated with a series of decreasing ethanol baths (100% Citrate-EDTA buffer was used for antigen retrieval at 85 °C for 40 min LSAB+ System (Dako) was used for Hypoxyprobe™-1 staining according to the manufacturer’s instructions and counterstained with hematoxylin (Sigma-Aldrich) Immunofluorescence staining was performed in tissue cryo-sections after permeabilization with 0.1% Triton-X (Sigma-Aldrich) for 10 min for the detection HIF-1α All slides were blocked with 2% BSA for 30 min Slides were incubated overnight at 4 °C with primary antibodies against HIF-1α (#SC-10790 Santa Cruz Biotechnology) and/or Hypoxyprobe™-1 (dilutions at 1:50) The slides were then incubated with the corresponding mouse (#A21237) or rabbit (#A21245) secondary Alexa Fluor 647™ (Invitrogen) antibodies at 1:1000 dilution for 1.5 h at RT followed by DAPI staining (1:1000 for 15 min In order to obtain two immunofluorescence stains in the same tissue slide Apoptotic cells were stained with Tunel labeling Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions All slides were mounted with anti-fade solution (90% glycerol Slides were imaged by using a Cytation 5 (BioTek Instruments) or by using a LSM780-FCS laser-scanning confocal microscope (Zeiss) with a 40×/1.30 PlanNeofluar oil objective with DIC0X/0.45 PlanApo (dry Total RNA was extracted from cells by using TRIzol (Invitrogen) and purified by using Direct-zol RNA mini kit (Zymo Research) with DNase I treatment samples were confirmed to have a RIN value > 9.0 when measured on an Agilent Bioanalyzer Libraries for RNA sequencing were prepared with KAPA Stranded RNA-Sequencing Kit Unique adapters were used for each sample in order to multiplex samples into several lanes Sequencing was performed on Illumina Hiseq 3000/4000 with a 150-bp pair-end run by Quick Biology (Pasadena Normalized (TMM) and raw data were uploaded to GEO Matched gene lists were ranked based on fold change upon intratumoral or in vitro hypoxia The fold change of multiple samples was averaged before ranking the gene set Default settings were used (enrichment statistic: classic; normalization mode: meandiv) and the number of permutations was set to 1000 Mitochondrial ROS levels and viability was measured by using Elite Mitochondrial ROS Activity Kit—Deep Red Fluorescence (MitoROS) (e-Enzyme) and Sytox Blue (Invitrogen) according to the manufacturer’s instructions Fluorescence intensity was measured by using a SH800 cytometer (Sony) Tumor-derived cells sorted for DsRed+ or GFP+ were plated in 96-well plates (5000 cells/well) fresh media with hydrogen peroxide (H2O2) (Sigma) was added Whole wells were imaged with an Olympus (UPLFLN 4XPh) phase objective 4× in a 4 × 3 montage (BioTek Instruments) in the RFP and DAPI area was quantified by thresholding to determine well confluency using Gen5 (BioTek Instruments) Tumor-derived cells (1 × 105) were resuspended in 1 ml of fresh media in a 1.5-ml centrifuge tube Confluency was assessed as described for the H2O2 treatment This assay was adapted from published work77 Tumor-derived cells were resuspended in a cell suspension (1 × 105/ml) that was slowly collected into a syringe without a needle attached The suspension was then expelled through a 26-G needle at a constant flow rate of 0.25 ml/s cells were counted by using Trypan Blue to asses viability and statistical analysis was performed by using GraphPad Prism 6 and statistical tests appropriate for each experimental setup All comparisons between DsRed+ and GFP+ populations from the same tumor or organoid were tested by using a matched Two-way ANOVA with Bonferroni multi-comparison tests A comparison of two variables was performed by using an unmatched Two-way ANOVA with Bonferroni multi-comparison tests The comparison of a single variable across multiple experimental conditions was performed by using a one-way ANOVA with Bonferroni multi-comparison test A comparison of a single variable measured in a sample at two different locations was performed via paired two-tailed Student’s t-test Significance levels are reported as ****P < 0.0001 Further information on research design is available in the Nature Research Reporting Summary linked to this article The RNA-sequencing data have been deposited in the GEO database under the accession codes GSE111653 (in vitro hypoxia), GSE126609 (tumor), and GSE136372 (lung) The survival data referenced during the study are available in a public repository from the kmplot.com website All the other data 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N. in Bioinformatics: The Impact of Accurate Quantification on Proteomic and Genetic Analysis and Research 41–74, https://doi.org/10.1201/b16589 (2014) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data The molecular signatures database hallmark gene set collection VennPlex-a novel venn diagram program for comparing and visualizing datasets with differentially regulated datapoints Resistance to fluid shear stress is a conserved biophysical property of malignant cells Download references and Josh DiGiacomo for their feedback over the course of this study Sukumar for guidance with the intraductal mouse model Hanhvy Bui (IIC Flow Cytometry Core) and Dr Jessica Gucwa (SKCCC Flow Cytometry Core) assisted with flow cytometry and Shyanne Salen assisted with tail-vein injections Work in the Gilkes lab is supported by U54-CA210173 (NCI) The Jayne Koskinas Ted Giovanis Foundation for Health and Policy Cindy Rosencrans Fund for Metastatic Triple-Negative Breast Cancer and the SKCCC Core Grant (P50CA006973 (NCI)) Cartoons included in the display items of this work were adapted from Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (smart.servier.com) The Sidney Kimmel Comprehensive Cancer Center The Johns Hopkins University School of Medicine Department of Chemical and Biomolecular Engineering were responsible for the triple-transgenic mice screening; J.A.J assisted on animal work and immune blotting; I.Y were responsible for the transgenic mice genotyping; I.Y assisted on the preparation of the RNAseq samples; G.W performed the intraductal injections; D.M.G was responsible for concept and research design The authors declare no competing interests Peer Review Information Nature Communications thanks Nicola Valeri and other reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-019-12412-1 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research Metrics details Heightened aerobic glycolysis and glutaminolysis are characteristic metabolic phenotypes in cancer cells is featured by frequent genomic amplification of MYCN a member of the Myc oncogene family that is primarily expressed in the early stage of embryonic development and required for neural crest development Here we report that an enriched glutaminolysis gene signature is associated with MYCN amplification in children with NBL The partial knockdown of MYCN suppresses glutaminolysis in NBL cells forced overexpression of MYCN in neural crest progenitor cells enhances glutaminolysis glutaminolysis induces oxidative stress by producing reactive oxygen species (ROS) rendering NBL cells sensitive to ROS augmentation Through a small-scale metabolic-modulator screening we have found that dimethyl fumarate (DMF) a Food and Drug Administration-approved drug for multiple sclerosis suppresses NBL cell proliferation in vitro and tumor growth in vivo DMF suppresses NBL cell proliferation through inducing ROS and subsequently suppressing MYCN expression Our findings suggest that the metabolic modulation and ROS augmentation could be used as novel strategies in treating NBL and other MYC-driven cancers we envision that a persistent metabolic rewiring renders cancer cells highly dependent on certain metabolic pathways in a way that other cells are not (metabolic addiction) hence modulation of this process holds the promise of novel metabolic interventions (metabolic vulnerability) Children with high-risk NBL are treated with aggressive multimodal therapy <50% of patients with high-risk NBL will survive long term with current therapies and survivors are at risk for serious treatment-related late toxicities novel treatments must be developed to enhance therapy efficacy with minimal toxicity the impact of metabolic reprogramming of cancer cells by oncogenes is not entirely clear How to harness the impact of metabolic reprogramming to develop novel therapies is also very important for cancer treatment A better understanding of how genetic alterations (MYCN amplification) impact NBL metabolic reprogramming will enable us to identify key oncogenic events and metabolic characters we report a role of MYCN in regulating NBL metabolic reprogramming and reactive oxygen species (ROS) induction The short hairpin RNA (shRNA)-mediated partial knockdown of MYCN suppresses the expression of metabolic genes and the activity of glutaminolysis in NBL cell lines Heightened glutaminolysis in NBL cells by MYCN provides bioenergetic support and induces ROS as a by-product in mitochondria conferring metabolic vulnerability of NBL cells to ROS-producing agent as cancer cells are more sensitive to agents that cause further accumulation of ROS a Food and Drug Administration (FDA)-approved drug for inflammation and autoimmunity as a novel therapeutic agent that suppresses NBL cell growth through inducing ROS and subsequently suppressing MYCN expression Our studies suggest that metabolic modulation of glutaminolysis and ROS augmentation may represent effective strategies in treating NBL and other MYC-driven cancers a Genes in glutaminolysis pathway were upregulated in MYCN-amplified tumors (93 cases) compared with non-MYCN-amplified tumors (550 cases) (ANOVA These data were derived from Kocak dataset (GSE45547) Age (red > 18 months; green < 18 months); MYCN (red = amplification; green = non-amplification); stage (red = stage 4; blue = stage 4S; brown = stage 3; dark green = stage 2 b MYCN protein and mRNA expression levels were determined by immunoblot (left panel) and qPCR (right panel) NB-1643 control-shRNA-inducible and MYCN-shRNA-inducible cells were collected after 3 days in the absence or presence of 1 μg/ml doxycycline for MYCN level analysis mRNA levels in cells expressing control-shRNA were set to 1 Error bars represent standard deviation from the mean of triplicate samples Data are representative of two independent experiments c Glycolysis and glutaminolysis as determined by the generation of 3H2O from [5-3H]glucose and the generation of 14CO2 from [U-14C]glutamine NB-1643 control-shRNA and MYCN-shRNA cells (collected after incubation with 1 μg/ml doxycycline for the expression of specific RNA in 2% FBS media for 3 days followed by overnight 10% FBS media recovery) were used for measuring the metabolic flux d mRNA levels of genes essential for glutaminolysis in NB-1643 cells were determined by qPCR NB-1643 control-shRNA-inducible and MYCN-shRNA-inducible cell lines were treated with 1 μg/ml doxycycline for 3 days before harvesting the samples for RNA extraction followed by qPCR analyses of metabolic genes in the glutaminolytic pathway mRNA levels in NB-1643 control-shRNA cells were set to 1 e Chromatin immunoprecipitation assessment of the binding of MYCN on indicated genes after 24-h MYCN induction with 500 ng/ml doxycycline in MycN3 cells MYCN was immunoprecipitated from cross-linked DNA and then DNA was extracted for PCR with primers that amplify predicted binding site and non-binding site for each gene a NB-EBC1 cell proliferation curves under indicated culture conditions were determined by live-cell imaging analysis (IncuCyte ZOOM™) Cell confluences of complete media at 96 h were set to 100% Error bars represent standard deviation from mean of quadruplicate samples b JoMa cells were transduced with lentiviral vector carrying mouse MYCN gene or blank control plasmid and cultured for 3 days The ROS level (left) was determined by flow cytometry in cells followed with 30 min incubation with 5 μM CM-H2DCFDA at 37 °C The ROS level in control JoMa cells were set to 1 in the bar graph MCYN protein levels (right) were determined by immunoblot c JoMa cells were transduced with lentiviral vector carrying mouse MYCN gene or blank control plasmid for 3 days and the growth curve of cells treated with indicated doses of H2O2 was determined by live-cell imaging analysis (IncuCyte ZOOM™) d NB-EBC1 cell growth upon treatment of H2O2 in different concentrations was determined by live-cell imaging analysis (IncuCyte ZOOM™) a–c NB-EBC1 cell growth (a and c) and cell death (b) upon indicated treatment were determined by live-cell imaging analysis (IncuCyte ZOOM™) (right panel) The cell confluences or death percentage of control group were set to 100% or 1 in the bar graph (left panel) Error bars represent standard deviation from the mean of quadruplicate samples e Tumor xenograft was established by using NB975A2 cells and mice were treated daily with 300 mg/kg DMF or vehicle controls The tumor volumes of control group and DMF-treated group were monitored daily The tumor volumes of vehicle control group were set to 100% Error bars represent SEM from the mean of 10 tumors Data are representative of three independent experiments a NB-1643 cells were incubated with 100 μM DMF for 12 h and harvested for RNA extraction for microarray analysis followed with gene set enrichment analysis b The expression of indicated proteins in NB-1643 and NB-EBC1 cells was determined by immunoblot c MYCN mRNA levels in NB-1643 and NB-EBC1 cells upon indicated treatments were determined by qPCR MYCN mRNA levels in control cells were set to 1 d NB-1643 and NB-EBC1 cells were incubated with 100 μM DMF for 1 h The ROS level was determined by flow cytometry in cells followed by 30 min incubation with 5 μM CM-H2DCFDA at 37 °C e NB-1643 and NB-EBC1 cells were treated with 100 μM DMF in the absence and presence of 5 mM ROS scavenger NAC and collected at designated time points The expression of indicated proteins was determined by immunoblot f NB-1643 and NB-EBC1 cells were treated with 100 μM DMF in the absence and presence of 5 mM ROS scavenger NAC Cell proliferation was determined by live-cell imaging analysis (IncuCyte ZOOM™) Cell confluences of control groups at the end of day 6 were set to 100% in the bar graph a The expression of indicated proteins in NB-1643 and NB-EBC1 cells following DMF treatment was determined by immunoblot b The mRNA levels of genes in Txn pathway were determined by qPCR mRNA levels of genes in control cells were set to 1 c NB-EBC1 cells were transfected with control or Nrf2 siRNA for 3 days and then treated with DMF Cell proliferation was determined by live-cell imaging analysis (IncuCyte ZOOM™) (left panel) Cell confluences of NB-EBC1 cells transfected with control siRNA without DMF at 144 h were set to 100% (right panel) d NB-EBC1 cells were transfected with control or Nrf2 siRNA for 2 days and then treated with 50 μM DMF for 1 h The ROS level (left panel) was determined by flow cytometry in cells after 30 min incubation with 5 μM CM-H2DCFDA at 37 °C The ROS levels in control siRNA-transfected cells were set to 1 in the bar graph (right panel) we have revealed a tight association between MYCN overexpression and the transcriptional upregulation of metabolic genes in glutaminolysis the partial knockdown of MYCN suppresses glutaminolysis This long record of accomplishment in clinical use demonstrates that DMF is a safe drug with generally mild toxicities and may represent an optimal drug for future development Cell culture materials and Matrigel were purchased from Fisher Scientific Inc Glucose-free and glutamine-free Dulbecco's modified Eagle's media and GSH reduced ethyl ester were purchased from Sigma-Aldrich Inc Chicken embryo extract was from Gemini Biological Products (West Sacramento B-27 supplement and N-2 supplement were obtained from Gibco (Waltham and anti-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz The siRNA oligonucleotides corresponding to human MYCN and Nrf2 were purchased from Fisher Scientific Inc siRNA oligonucleotides (20 nM) were transfected into cells using Lipofectamine RNAiMAX reagent (Invitrogen) IBs were carried out to examine the knockdown of targeted proteins encoding for inducible MYCN-shRNA expression or mouse MYCN expression were directly added to the cell culture medium and incubated for 48–72 h before further analysis and sonicated at 4 °C in a lysis buffer (50 mM Tris-HCl protease and phosphatase inhibitor tablet) Cell lysates were centrifuged at 13,000 × g for 15 min The protein concentrations were determined by using the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific) After 5 min boiling in 4 × NuPAGE® LDS Sample Buffer with 10× Reducing solution (Thermo Fisher Scientific) the proteins were separated by NuPAGE 4–12% Protein Gels (Thermo Fisher Scientific) transferred to PVDF membranes by using the iBlot Gel Transfer Device (Thermo Fisher Scientific) and probed with the appropriate primary antibodies Membrane-bound primary antibodies were detected using secondary antibodies conjugated with horseradish peroxidase IBs were developed on films using the enhanced chemiluminescence technique one million cells were suspended in 0.5 ml fresh media cells were dispensed into 7 ml glass vials (TS-13028 Thermo) with a PCR tube containing 50 μl of 0.2 M KOH glued on the sidewall the vials were capped using a screw cap with rubber septum (TS-12713 The assay was stopped 2 h later by injection of 100 μl of 5 N HCl and the vials were kept at room temperate overnight to trap the 14CO2 The 50 μl of KOH in the PCR tube was then transferred to scintillation vials containing 10 ml scintillation solution for counting A cell-free sample containing 0.5 μci [U-14C]glutamine was included as a background control NB-1643 cells were incubated with 100 μM DMF and harvested at designated time points for RNA extraction RNA samples were then performed microarray by St Microarray data were submitted with assigned GEO number as GSE98241 ROS levels were detected using the CM-H2DCFDA probe purchased from Thermo Fisher Scientific Cells were incubated with 5 µM CM-H2DCFDA for 30 min in serum-free media at 37 °C and analyzed by flow cytometry immediately C57BL/6NHsd mice were purchased from Envigo All mice were kept in specific pathogen-free conditions within the research institute at Nationwide Children’s Hospital Animal protocols were approved by the Institutional Animal Care and Use Committee of the research institute at Nationwide Children’s Hospital NB975A2 cells mixed with 70% Matrigel were injected subcutaneously to mice for the establishment of NBL xenograft model Mice were randomized into different groups when tumors were 100 to 200 mm3 a vehicle control (0.8% hydroxyethyl cellulose) was given by oral gavage every day DMF was administered orally at a dose of 300 mg/kg every day till the endpoint of 2,000 mm3 Tumor volumes were determined with the method of tumor length times tumor width times tumor width times 0.52 All animal experiments were conducted in accordance with institutional animal care and use committee of the Research Institute at Nationwide Children’s Hospital approved protocols designed to minimize the numbers of mice used and to minimize any pain or distress The named institutional review board or ethics committee specifically approved this study P values were calculated with Student’s t test P values < 0.05 were considered significant The biology of cancer: metabolic reprogramming fuels cell growth and proliferation Understanding the Warburg effect: the metabolic requirements of cell proliferation The intercellular metabolic interplay between tumor and immune cells HIF-1: upstream and downstream of cancer metabolism Oncogene addiction versus oncogene amnesia: perhaps more than just a bad habit Neuroblastoma tumour genetics: clinical and biological aspects TheInternational Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report Combinatorial regulation of neuroblastoma tumor progression by N-Myc and hypoxia inducible factor HIF-1alpha Myc promotes glutaminolysis in human neuroblastoma through direct activation of glutaminase 2 ATF4 regulates MYC-mediated neuroblastoma cell death upon glutamine deprivation ATF4 and N-Myc coordinate glutamine metabolism in MYCN-amplified neuroblastoma cells through ASCT2 activation From Krebs to clinic: glutamine metabolism to cancer therapy Reprogramming glucose metabolism in cancer: can it be exploited for cancer therapy Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis MYCN and ALKF1174L are sufficient to drive neuroblastoma development from neural crest progenitor cells The p53 regulatory gene MDM2 is a direct transcriptional target of MYCN in neuroblastoma Mitochondrial reactive oxygen species and cancer The proto-oncometabolite fumarate binds glutathione to amplify ROS-dependent signaling Fumarate induces redox-dependent senescence by modifying glutathione metabolism Activation of endogenous antioxidant defenses in neuronal cells prevents free radical-mediated damage Dimethyl fumarate modulation of immune and antioxidant responses: application to HIV therapy A transplantable TH-MYCN transgenic tumor model in C57Bl/6 mice for preclinical immunological studies in neuroblastoma Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation Embryonic lethality resulting from disruption of both N-myc alleles in mouse zygotes N-myc is essential during neurogenesis for the rapid expansion of progenitor cell populations and the inhibition of neuronal differentiation Targeting mitochondria metabolism for cancer therapy 408 (2016) (doi: 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for psoriasis: dosing Placebo-controlled phase 3 study of oral BG-12 or glatiramer in multiple sclerosis Placebo-controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis The mTORC2 complex regulates terminal differentiation of C2C12 myoblasts PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification Difference in glucose sensitivity of liver glycolysis and glycogen synthesis Relationship between lactate production and fructose 2,6-bisphosphate concentration Glucose and glutamine metabolism in rat thymocytes The role of histone demethylase KDM4B in Myc signaling in neuroblastoma Download references and Doris Phelps for valuable discussions and technical assistance Chris Lauo for providing valuable reagents to this study This work was supported by the Davis Bremer Research Pilot Award from The Ohio State University Center for Clinical and Translational Science (to N.S.) R21AI117547 and 1R01AI114581 from National Institute of Health 128436-RSG-15-180-01-LIB from the American Cancer Society and a research grant from CancerFree KIDs Foundation (to R.W.) American Cancer Society-Research Scholar (to J.Y. the American Lebanese Syrian Associated Charities (ALSAC) the US Public Health Service Childhood Solid Tumor Program Project Grant No Center for Childhood Cancer and Blood Diseases The Research Institute at Nationwide Children’s Hospital The authors declare that they have no conflict of interest Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41419-018-0295-5 Sorry, a shareable link is not currently available for this article. Volume 2 - 2012 | https://doi.org/10.3389/fonc.2012.00112 MAP17 is a small 17 kDa non-glycosylated membrane protein previously identified as being overexpressed in carcinomas Breast tumor cells that overexpress MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in the presence or the absence of contact inhibition MAP17-expressing clones also grow better in nude mice The increased malignant cell behavior induced by MAP17 is associated with an increase in reactive oxygen species (ROS) production and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells The MAP17-dependent increase in ROS and tumorigenesis relies on its PDZ-binding domain because disruption of this sequence by point mutations abolishes the ability of MAP17 to enhance ROS production and tumorigenesis MAP17 is overexpressed in a great variety of human carcinomas Immunohistochemical analysis of MAP17 during cancer progression demonstrates that overexpression of the protein strongly correlates with tumoral progression Generalized MAP17 overexpression in human carcinomas indicates that MAP17 can be a good marker for tumorigenesis and (A) Schematic representation of MAP17 protein domains (B) Schematic representation of MAP17 disposition in the membrane Multiple oncogenes that activate signaling pathways directly involved in cell survival or proliferation have been discovered in previous decades Other genes may provide an advantage to the tumoral cells making them insensitive to physiological signals or altering their normal physiology Although activated macrophages destroy cancer cells more effectively than normal cells the ability to escape activated macrophages is a characteristic of tumor cells One of the mechanisms responsible for the specific killing of tumor cells by macrophages is the production of the cytokine tumor necrosis factor-alpha (TNF-α) resistance to TNF may provide cancer cells with a selective advantage against host elimination Ectopic expression of MAP17 in tumor cells prevents TNF-induced G1 arrest by impairing p21waf1 induction expression of MAP17 does not inhibit TNF-induced apoptosis in Me180-sensitive tumor cells The inhibition of TNF is specific because MAP17 does not alter the response to other cytokines such as IFN-α MAP17 increases the uptake of glucose in some cells but this effect is not responsible for TNF bypass MAP17 overexpression in carcinomas occurs mostly through mRNA amplification, but promoter activation has also been observed by some oncogenes (Kocher et al., 1995; Guijarro et al., 2007c) Immunohistochemical analysis of MAP17 during cancer progression shows that overexpression of the protein strongly correlates with tumoral progression Generalized MAP17 overexpression in human carcinomas indicates that MAP17 can be a good marker for tumorigenesis and especially for malignant progression These expression patterns provide a mechanistic insight and a possible target for future therapies (AKT inhibition) (A) Representative picture of human breast tumors overexpressing MAP17 (B) Same tumor sample showing activated AKT (phosphorylated at S473) (C) Correlation between MAP17 expression and AKT activation in breast tumor samples analyzed which suggests that this effect is dependent upon MAP17 protein expression Schematic representation of the intracellular pathways activated by MAP17 through ROS The AKT pathway is represented in more detail NHeRF1 present also mutations at the PDZ domains in breast tumors which abolishes binding to these suppressor proteins Primary breast tumors with LoH at the NHeRF1 locus show higher aggressiveness the relation of these mutations with MAP17 or other physiological alterations such as ROS of glucose uptake is at present unknown Possible mechanisms involved in MAP17-dependent increase of ROS (A) Direct pH alteration by membrane transports (B) increase in glucose metabolism through mitochondrial respiration (C) increase in aerobic glycolysis (Warburg’s effect) which is allowed by acidic detoxification carried out by membrane transports bund to MAP17–NHeRFs complexes MAP17 overexpression in human breast carcinomas indicates that MAP17 can be a good marker for tumorigenesis and for malignant progression Our results indicate that this protein is likely to play an important role in carcinogenesis The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest This work was supported by grants from the Spanish Ministry of Science and Innovation and Feder Funds (SAF2009-08605) Consejeria de Innovacion y Ciencia (CTS-6844) and Consejeria de Salud (PI-0142) Amancio Carnero’s Lab is also funded by a fellowship from Fundacion Oncologica FERO supported by Fundació Josep Botet The funding source has not had a role in the study design nor in the writing and decision to submit the manuscript Reactive oxygen species activate p90 ribosomal S6 kinase via Fyn and Ras Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Hydrogen peroxide activates p70(S6k) signaling pathway Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Rethinking glycolysis: on the biochemical logic of metabolic pathways Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Regulation of PTP1B via glutathionylation of the active site cysteine 215 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Rat kidney MAP17 induces cotransport of Na-mannose and Na-glucose in Xenopus laevis oocytes Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Control of cell proliferation by reactive oxygen species Pubmed Abstract | Pubmed Full Text The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Tumour hypoxia induces a metabolic shift causing acidosis: a common feature in cancer Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Increased manganese superoxide dismutase expression suppresses the malignant phenotype of human melanoma cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text NHE3 inhibition by cAMP and Ca2+ is abolished in PDZ-domain protein PDZK1-deficient murine enterocytes Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Roles of the scaffolding proteins NHERF in liver biology Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Role of NHERF and scaffolding proteins in proximal tubule transport Pubmed Abstract | Pubmed Full Text | CrossRef Full Text NHERF (Na+/H+ exchanger regulatory factor) gene mutations in human breast cancer Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Transcriptional regulation of the SCL locus: identification of an enhancer that targets the primitive erythroid lineage in vivo Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Free radicals in the physiological control of cell function Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Correlation between the loss of the transformed phenotype and an increase in superoxide dismutase activity in a revertant subclone of sarcoma virus-infected mammalian cells Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Sodium-coupled monocarboxylate transporters in normal tissues and in cancer Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The PDZ-binding chloride channel ClC-3B localizes to the Golgi and associates with cystic fibrosis transmembrane conductance regulator-interacting PDZ proteins Pubmed Abstract | Pubmed Full Text | CrossRef Full Text A major scaffolder in brush borders of proximal tubular cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Transcriptional regulation of the stem cell leukemia gene (SCL) – comparative analysis of five vertebrate SCL loci Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Large scale genetic screen identifies MAP17 as protein bypassing TNF-induced growth arrest Pubmed Abstract | Pubmed Full Text | CrossRef Full Text MAP17 enhances the malignant behavior of tumor cells through ROS increase Pubmed Abstract | Pubmed Full Text | CrossRef Full Text MAP17 overexpression is a common characteristic of carcinomas Pubmed Abstract | Pubmed Full Text | CrossRef Full Text MAP17 inhibits Myc-induced apoptosis through PI3K/AKT pathway activation Pubmed Abstract | Pubmed Full Text | CrossRef Full Text p38α limits the contribution of MAP17 to cancer progression in breast tumors doi: 10.1038/onc.2011.619 [Epub ahead of print] Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Redox modulation of p53 conformation and sequence-specific DNA binding in vitro Pubmed Abstract | Pubmed Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Pubmed Abstract | Pubmed Full Text | CrossRef Full Text MaRX: an approach to genetics in mammalian cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Down regulation of small intestinal ion transport in PDZK1- (CAP70/NHERF3) deficient mice Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Overexpression of PDZK1 within the 1q12-q22 amplicon is likely to be associated with drug-resistance phenotype in multiple myeloma Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The membrane-associated protein pKe#192/MAP17 in human keratinocytes Pubmed Abstract | Pubmed Full Text | CrossRef Full Text PDZ domain-containing 1 (PDZK1) protein regulates phospholipase C-beta3 (PLC-beta3)-specific activation of somatostatin by forming a ternary complex with PLC-beta3 and somatostatin receptors The role of oxidative stress in chemical carcinogenesis Pubmed Abstract | Pubmed Full Text selectively up-regulated in human carcinomas Pubmed Abstract | Pubmed Full Text Identification and partial characterization of a novel mem-brane-associated protein (MAP17) up-regulated in human carcinomas and modulating cell replication and tumor growth Pubmed Abstract | Pubmed Full Text Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Regulatory mechanisms of Na+/glucose cotransporters in renal proximal tubule cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide Pubmed Abstract | Pubmed Full Text pH control mechanisms of tumor survival and growth Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Regulation of ion transport by the NHERF family of PDZ proteins Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Identification of small PDZK1-associated protein as a regulator of PDZK1 and plasma high density lipoprotein levels Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Independent activation of Akt and NF-kappaB pathways and their role in resistance to TNF-alpha mediated cytotoxicity in gliomas Pubmed Abstract | Pubmed Full Text | CrossRef Full Text PDZK1 regulates two intestinal solute carriers (Slc15a1 and Slc22a5) in mice Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Requirement for generation of H2O2 for platelet-derived growth factor signal transduction Pubmed Abstract | Pubmed Full Text | CrossRef Full Text NHERF links the N-cadherin/catenin complex to the platelet-derived growth factor receptor to modulate the actin cytoskeleton and regulate cell motility Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Bypassing cellular senescence by genetic screening tools Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Tumor suppressor genes and ROS: complex networks of interactions Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Sodium-hydrogen exchanger regulatory factor 1 (NHERF-1) transduces signals that mediate dopamine inhibition of sodium-phosphate co-transport in mouse kidney Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated induction of matrix-degrading metalloprotease-1 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Bradykinin prevents the apoptosis of NIT-1 cells induced by TNF-alpha via the PI3K/Akt and MAPK signaling pathways Pubmed Abstract | Pubmed Full Text Manganese-containing superoxide dismutase overexpression causes phenotypic reversion in SV40-transformed human lung fibroblasts Pubmed Abstract | Pubmed Full Text Citation: Carnero A (2012) MAP17, a ROS-dependent oncogene. Front. Oncol. 2:112. doi: 10.3389/fonc.2012.00112 Copyright: © 2012 Carnero. This is an open-access article distributed under the terms of the Creative Commons Attribution License distribution and reproduction in other forums provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc *Correspondence: Amancio Carnero, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío, Consejo Superior de Investigaciones Cientificas, Universidad de Sevilla, Campus Hospital Universitario Virgen del Rocío, Edificio IBIS, Avda. Manuel Siurot s/n, 41013 Sevilla, Spain. e-mail:YWNhcm5lcm8taWJpc0B1cy5lcw== Metrics details Cellular stresses trigger autophagy to remove damaged macromolecules and organelles Lysosomes ‘host’ multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes which regulates autophagy and lysosome biogenesis is activated following the inhibition of mTOR Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca2+-dependent mechanism independent of mTOR Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels This activation triggers calcineurin-dependent TFEB-nuclear translocation autophagy induction and lysosome biogenesis When TRPML1 is genetically inactivated or pharmacologically inhibited clearance of damaged mitochondria and removal of excess ROS are blocked TRPML1’s ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell ROS and autophagy may constitute a negative feedback mechanism that mitigates oxidative stress and promotes cell survival It is not yet known whether the mTOR–TFEB pathway regulates lysosome function in response to other cellular stresses it is unclear whether and how TRPML1 is activated by specific autophagy-inducing conditions we hypothesize that lysosomal conductances particularly through lysosomal Ca2+ channels such as TRPML1 may mediate ROS regulation of lysosomal function we demonstrate a direct and specific activation of lysosomal TRPML1 channels by both exogenous oxidants and mitochondria-derived ROS in the endolysosomal patch-clamp recordings This ROS-induced TRPML1 activation leads to lysosomal Ca2+ release calcineurin-dependent TFEB-nuclear translocation and increases of LC3-II expression and autophagy Genetic inactivation or pharmacological inhibition of TRPML1 impairs ROS-induced autophagy and blocks the clearance of damaged mitochondria and removal of excess ROS Other tested oxidants included cysteine-specific oxidants (DTNB and DTNP The effects of oxidants were normalized to that of ML-SA1 (20 μM) Numbers of patches tested for each oxidant are shown in brackets (e) Representative traces of DTNP-insensitive (red) (f) ChT induced single-channel openings in an inside-out patch isolated from TRPML1-4A-expressing cells (g) The single-channel conductance of ChT- and ML-SA1-activated ITRPML1 Numbers of patches tested for each constructs are shown in brackets (j) Insensitivity of whole-endolysosome TRPML1Va currents to ChT (k,l) ChT activated endogenous whole-endolysosome ITRPML1 in WT but not TRPML1 KO mouse macrophages suggesting that TRPML1 is the primary ROS-regulated conductance in the lysosome (a) Upper panels: application of CCCP increased the fluorescence intensity of CM-H2DCFDA (green) versus the DMSO-treated control (CTL) group The increase was inhibited by co-application of NAC (5 mM) Lower panels: representative traces of basal whole-endolysosomal currents under each condition (CTL CCCP+NAC) in TRPML1-expressing COS-1 cells (b) Summary of CCCP pretreatment effects on basal ITRPML1 and IzTRPML1.1 from at least five patches for each experimental condition Numbers of patches for each experimental condition are shown in brackets (c) Effects of CCCP pretreatment on endogenous ITRPML1 in HEK293 cells (mean±s.e.m. n=4–5 patches for each treatment).*P<0.05 (d) Pretreatment of CCCP (10 μM) for 1 h increased ML-SA1-induced Ca2+ release measured by Fura-2 imaging in TRPML1-expressing HEK293 cells (e) CCCP pretreatment reduced GPN-induced lysosomal Ca2+ release which is presumed to reflect the lysosomal Ca2+ store Mean values (±s.e.m.) are shown for >30 cells per coverslip (f) Quantification of results shown in d,e from at least three independent experiments (mean±s.e.m.) these results suggest that lysosomal TRPML1 is activated or sensitized by mitochondria-generated ROS in the cell these results are mostly consistent with an interpretation that in CCCP-treated cells there is increased TRPML1-mediated Ca2+ release from lysosomes caused by high basal TRPML1 activity resulting from ROS elevation (a) In HeLa cells stably expressing mRFP–GFP–LC3 CCCP treatment (5 μM for 3 h) increased the formation of autophagosomes (b) Quantification of various treatment conditions on CCCP-induced autophagosome formation (mean±s.e.m. n≥30 randomly-selected cells for each treatment) (c) NAC did not affect ML-SA5-induced autophagosome formation (n≥40 randomly-selected cells for each treatment) (d) Western blot analysis of LC3-I and -II (arrows) protein expression in CCCP (10 μM 3 h) -treated WT and ML-IV human fibroblasts Torin 1 (1 μM) was used as a positive control to induce autophagy 0.5 μM) was used to inhibit lysosomal degradation (e) Quantitative analysis of LC3-II levels under various experimental conditions shown in d (from at least three independent experiments); *P<0.05 these results suggest that CCCP treatment may increase mitochondrial ROS thereby activating TRPML1 to induce autophagy (a) Effects of ML-SI3 (10 μM) co-administration on the accumulation of PARKIN-positive puncta (red) induced by CCCP treatment (10 μM for 3 h) followed by 1 h recovery (without CCCP) in PARKIN stable cells (b) Quantitative analysis of ML-SI3 and ML-SI4 effects on the clearance of PARKIN puncta (c) Effects of CCCP treatment on mitochondrial membrane potential monitored by JC-1 fluorescent dyes in WT and ML-IV fibroblasts removal of CCCP for 1 h led to repolarization (re-energization) of mitochondrial membrane potential (green J-aggregates; energized) in WT but not ML-IV cells (d) The ratio of red to green fluorescence of JC-1 was quantified for >30 randomly-selected cells (f) Effect of ML-SI4 (10 μM) on ROS levels measured by CM-H2DCFDA (green) imaging in HeLa cells low levels of ROS may be sufficient to induce mitophagy even though not detected experimentally by PARKIN recruitment these results suggest that TRPML1 may have a role in preventing the accumulation of damaged mitochondria these results suggest that TRPML1 activation is required for ROS-induced ROS removal a negative feedback mechanism that is used by cells to circumvent oxidative stress (a) Differential effects of BAPTA-AM and NAC on CCCP- and Torin-1-induced TFEB-nuclear translocation in HEK293 cells stably expressing mCherry–TFEB Cells were treated with CCCP (5 μM) and Torin 1 (1 μM) for 1 h to induce TFEB-nuclear translocation Nuclei were counterstained with DAPI (pseudo-colored in green) (b) Ratio of nuclear versus cytosolic TFEB (>100 cells per experimental condition) (c) Western blot analysis of cytosolic versus nuclear pools of TFEB proteins with and without CCCP treatment and in the presence and absence of NAC and BAPTA-AM Tubulin and Lamin are proteins abundant in the cytosolic and nuclear fractions (d) Averaged effects of NAC or BAPTA-AM on CCCP-induced TFEB-nuclear translocation based on multiple repeated experiments as shown in c (e) Differential effects of ML-SI3 on CCCP- and Torin-1-induced TFEB-nuclear translocation (f) The quantitative effects of ML-SI3 on CCCP-induced TFEB-nuclear translocation based on the multiple repeated experiments as shown in e (g) CCCP (10 μM for 1 h) induced accumulation of TFEB Torin-1 induced TFEB-nuclear translocation in both WT and ML-IV cells Nuclei were labelled with DAPI (pseudo-colored in red) (h) Average ratios of nuclear versus cytosolic TFEB immuoreactivity (>100 randomly-selected cells per experiment) ML-SI4 (10 μM) inhibited TFEB-nuclear translocation induced by CCCP (10 μM) treatment for 1 h (i) The effects of ML-SA5 (1 μM for 1 h) on TFEB-nuclear translocation in the presence and absence of ML-SI4 (10 μM) in WT and ML-IV cells (j) Average ratios of nuclear versus cytosolic TFEB immuoreactivity (>50 randomly-selected cells per experiment) All quantification data are presented as mean±s.e.m.; *P<0.05 paired t-test for western blots and ANOVA for all other comparisons these results suggest that whereas ROS activation of TRPML1 is sufficient to activate TFEB additional TRPML1-independent mechanisms may be responsible for starvation-induced TFEB activation these results suggest that TRPML1-dependent activation of TFEB plays a crucial role in ROS-induced autophagy and mitophagy autophagy is promoted to facilitate clearance of damaged mitochondria and removal of excessive ROS ML-SA5 induced robust TFEB-nuclear translocation in both mTRPML1- and zTRPML1.1-expressing ML-IV cells redox regulation of TRPML1 is specifically required for ROS-induced TFEB activation and autophagy The subsequent increase in autophagic flux may facilitate removal of damaged mitochondria and restoration of redox homeostasis Hence TRPML1 may serve as a ROS sensor in the lysosome that regulates an autophagy-dependent negative-feedback mechanism essential for cellular redox homeostasis there may be multiple ROS sensors involved in regulating autophagy in which endogenous mitochondrial ROS are generated to induce autophagy autophagy induction is almost completely blocked by Ca2+ chelators and TRPML1 inhibitors The robustness of these results suggests that TRPML1 may play a pivotal role in autophagy regulation our identification of TRPML1 as a lysosomal ROS sensor has revealed a unique ROS-regulated paradigm in which both initiation and maturation of autophagy are coordinated Hence TRPML1 appears to be uniquely positioned to respond to certain stress pathways the TRPML1–TFEB pathway may represent a potential therapeutic target by which preemptive modulation of oxidative stress may alleviate symptoms in patients with lysosomal storage diseases and neurodegenerative diseases characterized by excess ROS All constructs were confirmed by DNA sequencing COS-1 and HEK-293T were cultured in a 1:1 mixture of DMEM and Ham’s F12 (DF12) media with 10% fetal bovine serum (FBS) HeLa and HAP1 cells were maintained in DMEM and IMDM Lipofectamine 2000 (Invitrogen) was used for the transfection of above cells Human skin fibroblast cell lines from a mucolipidosis IV (TRPML1 KO) patient (clone GM02048) and a healthy control (clone GM05659) were obtained from the Coriell Institute for Medical Research (NJ Fibroblasts were transfected with a Neon electroporation kit (Invitrogen) Culture media were refreshed 18–24 h post-transfection and cells were imaged 48 h post-transfection to allow sufficient recovery time following transfection all cell lines were maintained in DMEM medium supplemented with 10% Tet-free FBS at 37 °C in a humidified 5% CO2 incubator TFEB CRISPR KO cells were generated in HeLa cells using the CRISPR/Cas9 system43 The TFEB sequence in the second exon 5′-GACGGGGGTATTGATGGCCG-3′ (TFEB–sgRNA) was targeted with pSpCas9 (BB)-2A-puro vector (Addgene) HeLa cells were then transfected with TFEB–sgRNA-expressing vector using Lipofectamine 2000 and selected in the presence of 5 μg ml−1 puromycin for 48 h their genomic DNAs were sequenced to confirm the intended genetic disruptions For TFEB and TFE3 immunofluorescence detection cells were grown on glass coverslips and then fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 after treatments The cells were then blocked with 1% bovine serum albumin in phosphate buffered saline (PBS) Endogenous TFEB and TFE3 were recognized by incubating cells with anti-TFEB (1:200; Cell Signaling Technology) or anti-TFE3 antibody (1:1,000 Sigma) at 4 °C overnight Cells were then washed four to five times with PBS and incubated with anti-rabbit secondary antibodies conjugated to Alexa Fluor 568 or 488 (Invitrogen) for 1 h coverslips were mounted on the slides with Fluoromount-G (Southern Biotech) Images were acquired with an Olympus Spinning-Disk Confocal microscope The fluorescence intensity at 488 nm (F488) was recorded at 37 °C with the spinning-disk confocal live-imaging system which included an Olympus IX81 inverted microscope an iXon EM-CCD camera (Andor) and MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices) ROS levels were detected with a CM-H2DCFDA dye assay (Invitrogen) cells were incubated with 2.5–5 μM CM-H2DCFDA in the culture media without FBS at 37 °C for 30 min and then recovered in the complete media for 10 min before imaging For non-quantitative estimation of lysosomal luminal pH cells were incubated with 50 nM LysoTracker Red DND-99 (Invitrogen) in complete culture medium for 15 min before imaging The fluorescence was visualized with a DP71 camera (Olympus) mounted on an Olympus IX-71 inverted microscope Images were captured at 20 × magnification with DPController software The fluorescence intensity was quantified with the ImageJ software (NIH) Human fibroblasts were incubated with 1 μM JC-1 (Invitrogen) in complete culture medium at 37 °C for 30 min before imaging The fluorescence was detected at 520 nm for J-monomer and 600 nm for J-aggregates (excitation wavelength=488 nm) by a Leica confocal microscope All bath solutions were applied via a fast perfusion system that produced a complete solution exchange within a few seconds Data were collected via an Axopatch 2A patch-clamp amplifier Digidata 1440 and processed with pClamp 10.0 software (Axon Instruments) Whole-endolysosome currents were digitized at 10 kHz and filtered at 2 kHz All experiments were conducted at room temperature (21–23 °C) and all recordings were analysed in pCLAMP10 (Axon Instruments) and Origin 8.0 (OriginLab) Whole-cell recordings were performed with pipette electrodes (resistance 3–5 MΩ) filled with (in mM): (1) 133 Cs methanesulfonate adjusted with CsOH; free [Ca2+]i<10 nM); or (2) 140 K-gluconate 0.39 CaCl2 and 20 HEPES (pH 7.2; free [Ca2+]i∼100 nM) The standard extracellular bath solution (Tyrode’s solution) contained (in mM): 153 NaCl pipette electrodes with 1–2 MΩ resistance were used The bath solution and pipette solution were the same as those used for whole-endolysosome recordings The following reagents were purchased: ML-SA1 (Princeton BioMolecular Research Inc) BAPTA-AM (Invitrogen) and vacuolin-1 (Calbiochem) ML-SA and ML-SI compounds were identified from a Ca2+-imaging-based high-throughput screening conducted at NIH/NCATS Chemical Genomics Center (NCGC; see https://pubchem.ncbi.nlm.nih.gov/bioassay/624414#section=Top) ML-SA and ML-SI compounds are available upon request Statistical comparisons of confocal images were performed with analyses of variance (ANOVA) Protein expression levels were compared using paired t-test A P-value<0.05 was considered statistically significant The data that support the findings of this study are available from the corresponding author upon request MCOLN1 is a ROS sensor in lysosomes that regulates autophagy Regulation of autophagy by ROS: physiology and pathology Neurodegenerative diseases and oxidative stress Autophagy as a stress-response and quality-control mechanism: implications for cell injury and human disease Parkin is recruited selectively to impaired mitochondria and promotes their autophagy ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy At the end of the autophagic road: an emerging understanding of lysosomal functions in autophagy ROS and autophagy: interactions and molecular regulatory mechanisms Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation Signals from the lysosome: a control centre for cellular clearance and energy metabolism MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis A gene network regulating lysosomal biogenesis and function A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB A dynamic interface between vacuoles and mitochondria in yeast TPC proteins are phosphoinositide- activated sodium-selective ion channels in endosomes and lysosomes PI(3,5)P(2) controls membrane trafficking by direct activation of mucolipin Ca(2+) release channels in the endolysosome Reducing and oxidizing agents sensitize heat-activated vanilloid receptor (TRPV1) current Oxidative regulation of large conductance calcium-activated potassium channels Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release Oxidative challenges sensitize the capsaicin receptor by covalent cysteine modification Redox regulation of transient receptor potential channels Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes Phosphoinositide isoforms determine compartment-specific ion channel activity Mitochondrial bioenergetics and structural network organization Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage Antioxidants can inhibit basal autophagy and enhance neurodegeneration in models of polyglutamine disease Imaging approaches to measuring lysosomal calcium Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1 TFEB links autophagy to lysosomal biogenesis Improved vectors and genome-wide libraries for CRISPR screening Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets Ca2+ in quality control: an unresolved riddle critical to autophagy and mitophagy Lysosomes and oxidative stress in aging and apoptosis The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel Regulation of the transcription factor EB-PGC1 α axis by beclin-1 controls mitochondrial quality and cardiomyocyte death under stress Lysosomal adaptation: how the lysosome responds to external cues Mucolipins: Intracellular TRPML1-3 channels The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing Download references This work was supported by NIH grants (NS062792 We are grateful to Dr David Rubinsztein for GFP–mRFP–LC3 stable cell line Ferguson for GFP–TFEB stable cell line and Dr Susan Slaugenhaupt (Harvard Medical School) for the TRPML1 KO mice we also thank Dr Xinran Li and Mr Stephen Ireland for generating Gcamp7-TRPML1 construct We appreciate the encouragement and helpful comments provided by other Xu lab members Xiping Cheng and Lu Yu: These authors contributed equally to this work Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals National Center for Advancing Translational Sciences performed the laboratory experiments; R.C. wrote the paper with inputs and final approval from all authors The authors declare no competing financial interests Reprints and permissions Download citation Experimental & Molecular Medicine (2024) Molecular and Cellular Biochemistry (2024) Sign up for the Nature Briefing newsletter — what matters in science Metrics details a natural phytochemical isolated from tumeric roots represents a candidate for prevention and therapy of colorectal cancer/CRC the exact mechanism of action and the downstream mediators of curcumin’s tumor suppressive effects have remained largely unknown Here we used a genetic approach to determine the role of the p53/miR-34 pathway as mediator of the effects of curcumin Three isogenic CRC cell lines rendered deficient for the p53 miR-34a and/or miR-34b/c genes were exposed to curcumin and subjected to cell biological analyses siRNA-mediated inhibition and ectopic expression of NRF2 qPCR and qChIP analyses of its target genes were performed injected into NOD/SCID mice and lung-metastases formation was determined by longitudinal In CRC cells curcumin induced apoptosis and senescence and suppressed migration and invasion in a p53-independent manner Curcumin activated the KEAP1/NRF2/ARE pathway by inducing ROS curcumin induced miR-34a and miR-34b/c expression in a ROS/NRF2-dependent and p53-independent manner NRF2 directly induced miR-34a and miR-34b/c via occupying multiple ARE motifs in their promoter regions Curcumin reverted repression of miR-34a and miR-34b/c induced by IL6 and hypoxia Deletion of miR-34a and miR-34b/c significantly reduced curcumin-induced apoptosis and senescence and prevented the inhibition of migration and invasion by curcumin or ectopic NRF2 In CRC cells curcumin induced MET and prevented the formation of lung-metastases in mice in a miR-34a-dependent manner we found that curcumin may enhance the therapeutic effects of 5-FU on CRC cells deficient for p53 and miR-34a/b/c Activation of the KEAP1/NRF2/miR-34a/b/c axis mediates the tumor suppressive activity of curcumin and suggests a new approach for activating miR-34 genes in tumors for therapeutic purposes new approaches and compounds for improved prevention and therapy of CRC are clearly needed As the clinical studies only included small numbers of patients targeted and prospective clinical trials are required to establish curcumin in clinical practice down-regulation of miR-34a/b/c in CRCs does not only commonly occur during intestinal carcinogenesis but is also causally involved in CRC formation these observations suggest that miR-34a and miR-34b/c have tumor suppressive properties and would therefore represent attractive mediators of tumor prevention and/or suppression elicited by curcumin Here we show that curcumin induces the expression of miR-34a and miR-34b/c independent of p53 by activating the KEAP1/NRF2/ARE pathway via the generation of reactive oxygen species/ROS we demonstrate that the miR-34a/b/c genes are important mediators of the pro-apoptotic senescence-inducing and metastasis-inhibiting effects of curcumin these effects of curcumin are independent of p53 and dominant over signals from the micro-environment that repress miR-34 expression the identified mechanisms provide an opportunity to activate miR-34a and miR-34b/c genes in tumor cells that display inactivation/down-regulation of the p53/miR-34 pathway A Detection of p53 protein by Western blot analysis after treatment with curcumin B Cell viability of HCT116 cells exposed to different concentrations of curcumin for 48 hours was determined by MTT assays C Impedance of HCT116 cells treated with curcumin D Determination of cell number at the final time point of the experiment shown in (C) E Cell cycle analysis using propidium iodide (PI) staining F Analysis of apoptosis in HCT116 cells treated with curcumin determined by Annexin V FITC and propidium iodide staining and cleaved caspase-3 was analyzed by Western blot analysis after being treated with curcumin for the indicated periods in HCT116 cells H Detection of senescent cells after exposure to curcumin for 48 hours determined by pH 6 β-gal staining I Wound healing assay of HCT116 cells treated with curcumin for 24 hours (left panel) Results represent the mean (%) of wound closure (right panel) J Determination of invasion in a modified Boyden-chamber assay Relative invasion of HCT116 cells treated with curcumin for 48 hours and (D) (n = 4) mean values ± SD are shown whereas it induced apoptosis and senescence of CRC cells in a p53-independent manner A Analysis of ROS formation in HCT116 cells treated as indicated Right panel: Quantification of fluorescence intensity B Cell viability of HCT116 cells after treatment with curcumin and NAC for indicated periods was determined by MTT assays C Apoptosis of HCT116 cells treated with curcumin and/or NAC for 48 h was determined by Western blot analysis of cleaved PARP protein levels D Immunofluorescence detection of NRF2 protein after treatment with DMSO or 15 μM curcumin and 5 mM NAC for 48 hours E Western blot analysis of NRF2 protein levels in cytoplasmic and nuclear cellular fractions after curcumin treatment F qPCR analysis of NQO1 expression in HCT116 cells after treatment with DMSO or 15 μM curcumin with 5 mM NAC for 48 hours G Western blot analysis of NQO1 protein levels after treatment with DMSO A–F qPCR analysis of pri-miR-34a and pri-miR-34b/c expression after treatment of the indicated cells with 15 μM curcumin for the indicated periods G qPCR analysis of mature miR-34a expression in HCT116 cells after treatment with 15 μM curcumin for 48 hours I qPCR analyses of pri-miR-34a and pri-miR-34b/c expression in HCT116 cells after treatment with curcumin and/or NAC for 48 hours In (A)–(I) mean values ± SD are shown (n = 3) pri-miR-34b/c expression in HCT116 cells treated as indicated for 48 hours C qPCR analysis of indicated mRNAs in SW480 cells after transfection with empty pcDNA3.1 or NRF2 pcDNA3.1 vectors for 72 hours D Western blot analysis of NRF2 and NQO1 proteins in SW480 cells after transfection with pcDNA3.1 and NRF2 pcDNA3.1 for 72 hours E ARE consensus sequence defined as 5ʹ-A/G TGA C/G NNNGC A/G-3ʹ where “N” represents any nucleotide according to jaspar.genereg.net (MA0150.1) F Map of human miR-34a and miR-34b/c genomic regions with indicated NRF2 binding sites G Sequences of NRF2 binding sites within the miR-34a and miR-34b/c genomic regions H qChIP analysis of NRF2 occupancy at the human miR-34a and miR-34b/c genomic regions in HCT116 p53 −/− cells after treatment with curcumin or DMSO for 48 hours NQO1 and 16q22 served as positive and negative controls and (H) mean values ± SD are shown (n = 3) A The indicated cell lines were treated with increasing concentrations of curcumin for 48 hours B Analysis of apoptosis after treatment with 15 μM curcumin for 48 hours determined by Annexin V-FITC and PI staining and cleaved caspase 3 after treatment with 15 μM curcumin for 48 hours was determined by Western blot analysis D Detection of senescent cells after curcumin treatment for 48 hours by pH 6 β-gal staining E Evaluation of migration by wound healing assay 24 hours after treatment with curcumin F Analysis of invasion using Boyden chamber assays 48 hours after treatment with curcumin or DMSO Analysis of cell viability in HCT116 p53-deficient cells by MTT assay after transfection NRF2 siRNA pool or control siRNA pool 24 hours with curcumin for 48 hours H Wound healing assay in HCT116 p53-deficient cells treated with curcumin for 24 h after transfection with NRF2-specific siRNA pool or control siRNA pool I Invasion assay of HCT116 p53-deficient cells exposed to curcumin for 48 hours after transfection with NRF2-specific siRNA pool or control siRNA pool J Evaluation of migration by wound healing assay 24 hours after transfection with pcDNA3.1 or NRF2 pcDNA3.1 K Analysis of invasion using Boyden chamber assays after transfection with pcDNA3.1 or NRF2 pcDNA3.1 and D–H mean values ± SD (n = 3) are shown in miR-34a/b/c-deficient HCT116 cells ectopic NRF2 had no effect on migration and invasion demonstrating that miR-34a and miR-34b/c are required mediators of NRF2 function our results demonstrate that the NRF2-mediated activation of miR-34a and miR-34b/c genes is a required mediator for the effects on curcumin on apoptosis and pri-miR-34b/c (C) after treatment with H202 and transfection with control or NRF2 siRNA for 24 hours E Expression of pri-miR-34a (D) and pri-miR-34b/c (E) in p53-deficient HCT116 cells after the indicated transfections/treatments G Expression of pri-miR-34a (F) and pri-miR-34b/c (G) in indicated cells after transfection with control or NRF2 siRNA and treatment with curcumin and/or IL-6 (200 ng/ml) for 48 hours In (A)–(G) mean values ± SD are shown (n = 3)*P < 0.05 indicating that this effect of curcumin was mediated by NRF2 B Expression of pri-miR-34a (A) and mature miR-34a (B) in SW620-luc2 cells treated with curcumin or DMSO for 48 hours C Analysis of SW620-luc2 cell migration after transfection with miR-34 antagomirs and/or curcumin for 72 hours D Analysis of SW620-luc2 cell invasion after transfection with miR-34 antagomirs and/or curcumin for 72 h E qPCR analyses of EMT-related genes in SW620-luc2 cells 72 hours after the indicated treatments/transfections F–I Formation of lung metastases by SW620-Luc2 cells which were treated as indicated for 48 hours and then injected into tail-vein of NOD/SCID mice Representative images of luciferase signals at the indicated time points after xenografting (F) and the quantification of total photon flux (G) H right: representative lungs 5 weeks after tail vein injection Left: representative images of H&E-stained resected lungs Quantification of metastatic nodules in the lungs of indicated mice In (A)–(E) mean values ± SD (n = 3) are shown A–D HCT116 cells with the indicated genotypes were exposed to curcumin (15 μM) After 48 hours cell viability was determined by MTT assays F The viability of HCEC-1CT and CCD-18Co cells was determined by MTT assay after the indicated treatments for 48 hours Schematic model of the findings obtained in this study Here we identified a new regulatory connection that explains how curcumin up-regulates the expression of miR-34a and miR-34b/c in a p53-independent manner (for a summary see Fig. 8G) we show that miR-34a and miR-34b/c mediate senescence and apoptosis invasion and metastasis of CRC cells after exposure to curcumin the induction of miR-34a and miR-34b/c by curcumin was dominant over their repression by exposure to IL-6 or hypoxia curcumin may re-activate miR-34 expression which is repressed due to signals generated by the tumor microenvironment inhibition of miR-34a function in CRC cells prevented the curcumin-mediated suppression of lung metastases formation of CRC cells transplanted into immune-incompetent mice these results establish a central role of the ROS/KEAP1/NRF2/miR-34a/b/c axis in mediating the tumor suppressive effects of curcumin be mediated activation of miR-34a and miR-34b/c Since curcumin was able to antagonize the repression of miR-34a and miR-34b/c by hypoxia or IL6 exposure a therapeutic treatment with curcumin may therefore reactivate miR-34 expression in primary CRC and metastases the findings presented here may be exploited for the development of therapeutic approaches that aim at restoring the tumor suppressive function of the p53/miR-34 pathway and SW620-Luc2 cell lines were cultured in McCoy’s 5 A medium (Invitrogen USA) with 10% fetal bovine serum (FBS) (Invitrogen) containing 100 units/ml penicillin and 0.1 mg/ml streptomycin HCEC-1CT immortalized human colonic epithelial cells (Evercyte GmbH) were maintained in DMEM supplemented with 2% FBS and sodium-selenite; Thermo Fisher Scientific) 20 ng/mL epidermal growth factor (EGF; AF-100–15 For the 5-FU experiment dialyzed FBS (A3382001; Thermo Fisher Scientific) #C1386) was dissolved in DMSO (stock concentration 50 mM) and the working concentration was 15 μM #A0737) was dissolved in water (stock concentration 100 mM) and the working concentration was 5 mM IL-6 (Immunotools) was dissolved in water and used at a final concentration of 200 ng/ml # 343922) was dissolved in DMSO (stock concentration 5 mg/ml) and the working concentration was 2 µg/ml siGENOME Human NRF2 siRNA SMARTPool (Horizon Discovery USA) and siGENOME Non-Targeting siRNA Control Pools (Horizon Discovery and respective negative controls were transfected at a concentration of 10 nM using HiPerFect transfection reagent (Qiagen Hypoxia (0.5% O2) was achieved using a CD210 incubator (Binder and analyzed by measuring the absorbance at 570 nm by a Varioscan system (Thermo Fisher) Cells were cultured and treated with curcumin on glass cover slides fixed in 4% paraformaldehyde/PBS for 15 min permeabilized in 0.2% Triton X-100 for 5 min and then blocked in 1% BSA/PBS for 1 h at room temperature cells were incubated with NRF2 primary antibody (D1Z9C Cell Signaling Technology) for 1 h at room temperature washed 3 time with PBS-Tween and then incubated with cy3-labelled secondary antibody (ab6939 The images were acquired by confocal laser scanning microscopy (CLSM) using the LSM700 microscope with a Plan Apochromat 20×/0.8 M27 objective and ZEN 2009 software (Zeiss) HCT116 p53-/- were treated with curcumin for 48 hours to activate NRF2 before cross-linking. ChIP experiments were performed using the iDeal ChIP-qPCR kit (Diagenode, Belgium) according to manufacturer’s instructions. The sequences of qChIP primers are provided in Table S2 Reactive oxygen species (ROS) were detected by DCFDA/H2DCFDA staining HCT116 cells were seeded at a density of 104 cells per well in 96 well plates The positive control tBHP was added to HCT116 cells for 4 hours before staining After removal of medium 1X Buffer (100 µl/well) was added cells were stained with DCFDA solution (100 µl/well) for 45 min at 37 °C in the dark plates were analyzed immediately on a fluorescence plate reader at Ex/Em = 485/535 nm Image J software was used to analyze the images and compared the control group SW620 cells stably expressing Luc2 were described previously [30] Cells were injected into the lateral tail vein of NOD/SCID mice using 25-gauge needles (4 × 106 cells /0.2 ml) in HBSS with D-luciferin (150 mg/kg) and imaged for 5 minutes using the IVIS Illumina System (Caliper Life Sciences) in weekly intervals mice were sacrificed and examined for lung metastases using H&E staining Animal experimentations and analyses were approved by the Government of Upper Bavaria The statistical differences between two groups were calculated using a Student’s t test (two-tailed; unpaired) the one-way analysis of variance (ANOVA) with the Tukey multiple comparison post-test was used Asterisks generally indicate: *p < 0.05 Additional method descriptions can be found as supplementary information and study materials will be made available to other researchers upon reasonable request GLOBOCAN Global Cancer Observatory (GCO). https://gco.iarc.fr (accessed on 18 April 2023) Metastatic patterns and survival outcomes in patients with stage IV colon cancer: A population-based analysis Curcumin and colorectal cancer: An update and current perspective on this natural medicine Curcumin against gastrointestinal cancer: A review of the pharmacological mechanisms underlying its antitumor activity Anticancer properties of curcumin against colorectal cancer: A review Curcumin combined with FOLFOX chemotherapy is safe and tolerable in patients with metastatic colorectal cancer in a randomized phase IIa trial Effects of curcuminoids on systemic inflammation and quality of life in patients with colorectal cancer undergoing chemotherapy: a randomized controlled trial Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer Curcumin in combination with mesalamine induces remission in patients with mild-to-moderate ulcerative colitis in a randomized controlled trial Diet as a trigger or therapy for inflammatory bowel diseases Combination treatment with curcumin and quercetin of adenomas in familial adenomatous polyposis Curcumin targeting non-coding RNAs in colorectal cancer: therapeutic and biomarker implications MicroRNAs in the p53 network: micromanagement of tumour suppression Frequent concomitant inactivation of miR-34a and miR-34b/c by CpG methylation in colorectal and renal cell carcinomas and soft tissue sarcomas miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial-mesenchymal transitions Antagonistic effects of p53 and HIF1A on microRNA-34a regulation of PPP1R11 and STAT3 and Hypoxia-induced epithelial to mesenchymal transition in colorectal cancer cells The p53/miR-34 axis in development and disease IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis Combined inactivation of TP53 and MIR34A promotes colorectal cancer development and progression in mice via increasing levels of IL6R and PAI1 miR-34a and miR-34b/c suppress intestinal tumorigenesis Curcumin stabilizes p53 by interaction with NAD(P)H:quinone oxidoreductase 1 in tumor-derived cell lines Curcumin reverses NNMT-induced 5-fluorouracil resistance via increasing ROS and cell cycle arrest in colorectal cancer cells The molecular mechanisms regulating the KEAP1-NRF2 pathway Small Maf proteins serve as transcriptional cofactors for keratinocyte differentiation in the Keap1-Nrf2 regulatory pathway Value of monitoring Nrf2 activity for the detection of chemical and oxidative stress miR-34a targets BCL-2 to suppress the migration and invasion of sinonasal squamous cell carcinoma Oxidative stress dependent microRNA-34a activation via PI3Kalpha reduces the expression of sirtuin-1 and sirtuin-6 in epithelial cells SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial-mesenchymal transition Therapeutic drug monitoring of 5-fluorouracil Relationship between 5-fluorouracil (5-FU) dose intensity and therapeutic response in patients with advanced colorectal cancer receiving infusional therapy containing 5-FU Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1 mediates an intricate crosstalk between mitochondrial turnover Curcumin promotes cancer-associated fibroblasts apoptosis via ROS-mediated endoplasmic reticulum stress Curcumin exerts cytotoxicity dependent on reactive oxygen species accumulation in non-small-cell lung cancer cells The effects of the phenylalanine 256 to valine mutation on the sensitivity of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) Ca2+ pump isoforms 1 and 3 to thapsigargin and other inhibitors Highlighting curcumin-induced crosstalk between autophagy and apoptosis as supported by its specific subcellular localization Effect of curcumin and curcumin copper complex (1:1) on radiation-induced changes of anti-oxidant enzymes levels in the livers of Swiss albino mice Tumor suppression in the absence of p53-mediated cell-cycle arrest a p53-inducible regulator of glycolysis and apoptosis The critical role of catalase in prooxidant and antioxidant function of p53 a p53-inducible regulator of glutamine metabolism and reactive oxygen species a novel p53 target gene regulating energy metabolism and antioxidant function Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death Hormetic effects of curcumin: What is the evidence Expression of miR-34 is lost in colon cancer which can be re-expressed by a novel agent CDF Novel evidence for curcumin and boswellic acid-induced chemoprevention through regulation of miR-34a and miR-27a in colorectal cancer Curcumin modulates DNA methylation in colorectal cancer cells Curcumin inhibits cancer stem cell phenotypes in ex vivo models of colorectal liver metastases and is clinically safe and tolerable in combination with FOLFOX chemotherapy Consumption of the putative chemopreventive agent curcumin by cancer patients: assessment of curcumin levels in the colorectum and their pharmacodynamic consequences A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Download references are recipients of Chinese Scholarship Council fellowships received grants from the Rudolf Bartling-Stiftung Open Access funding enabled and organized by Projekt DEAL and MR designed experiments; CL performed experiments and analyzed results; MR performed and analyzed mouse experiments All authors read and approved the final manuscript Download citation DOI: https://doi.org/10.1038/s41418-023-01178-1 Photochemical & Photobiological Sciences (2025) Metrics details the development of diabetes mellitus (DM) may also be accompanied by liver damage and inflammatory disorders Sitagliptin is an inhibitor of dipeptidyl peptidase-4 (DPP4 the mechanism of sitagliptin’s efficiency in liver diseases is largely unknown mice suffering from streptozotocin (STZ) exhibit elevated liver DPP4 expression and activity as well as inflammatory and chronic liver injury phenotype whereas specifically inhibiting the activity of DPP4 in mouse liver tissues and hepatocytes by sitagliptin contributes to decreased cytokines and inflammation in STZ-induced diabetic mice sitagliptin reduced TNFα or LPS-induced cellular reactive oxygen species (ROS) level and protein expression in the NFκB signaling pathway in HepG2 cells or primary mouse hepatocytes our study confirms that sitagliptin may protect liver tissue by alleviating ROS production and NFκB signaling activation providing a putative mechanism for preventing the development of diabetic liver disease there are fewer studies on the effect of DPP4 inhibitors on hepatic oxidative stress and liver disease we explored the possible mechanisms between sitagliptin and diabetic liver disease We hypothesized a functional role of sitagliptin in protecting cell apoptosis and inflammation in hepatocytes as well as delaying the progression of liver disease Our study provided strong theoretical evidence for the progression management of diabetes patients A After 24 h TNFα (20 ng/ml) or TNFα + SITA (100 μM) treatment DPP4 activity in HepG2 cells were detected B HepG2 cells were co-stimulated recombinant human DPP4 (hDPP4 0.1 and 1 μg/ml) protein with sitagliptin for 24 h after that the activity of DPP4 was determined C Western blots and quantitative analysis of the effects of sitagliptin on the protein levels of DPP4 and p-IKBαS36 in TNFα or TNFα + SITA-treated HepG2 cells and primary mouse hepatocytes D Western blottings and quantitative analysis of the effects of different concentrations of hDPP4 on the protein levels of DPP4 The results are presented as mean ± SEM of three independent experiments; *P < 0.05 A Cellular ROS levels of HepG2 cells after TNFα and TNFα + NAC treatment were detected by flow cytometry B Cellular ROS levels of HepG2 cells after hDPP4 and hDPP4 + SITA treatment were detected by flow cytometry and TNFα + NAC stimulation was detected by flow cytometry D The apoptosis of HepG2 cells after hDPP4 and hDPP4 + SITA stimulation was detected by flow cytometry A DPP4 activity in liver tissue from STZ-induced diabetic mice with or without 12 weeks of sitagliptin-containing diet treatment and sLDH in STZ-induced diabetic mice with or without sitagliptin treatment for 12 weeks E H&E staining of liver tissue (scale bar 100 and 200 μm) in STZ-induced diabetic mice with or without sitagliptin treatment G Sirius red and α-SMA staining of liver tissue (scale bar 100 and 200 μm) in STZ-induced diabetic mice with or without sitagliptin treatment H After 12 weeks sitagliptin-supplemented diet mice were fasted overnight and injected with glucose; then the blood glucose were measured at the specific time points (0 Treatment with sitagliptin significantly improved STZ-induced hepatic oxidative stress A RNA-seq heatmap of the expression of mentioned genes in liver tissues of STZ-induced diabetic mice and Col3 levels in diabetic mice with or without sitagliptin treatment C Gene signatures for NFκB activation (HALLMARK_TNFA_SIGNALING_VIA_NFKB) were enriched within STZ group comparing with STZ + SITA group D F4/80 staining of liver tissues (scale bar 100 and 200 μm) in STZ-induced diabetic mice with or without sitagliptin treatment E Western blottings and quantitative analysis of the effects of sitagliptin on the protein levels of DPP4 and p-IKBαS36 in mice liver tissues (n = 5 mice per group) B TUNEL and ROS staining of liver tissue (scale bar 100 and 50 μm) Specifically inhibiting the activity of DPP4 in hepatocytes by sitagliptin contributes to decreased activation of NFκB pathway and oxidative stress as well as cell apoptosis under diabetic conditions The ROS cleaning function of sitagliptin promotes the deactivation of NFκB pathway These contradictory results may be due to the differences in duration of treatment sitagliptin also plays a role of ROS scavenger which can inhibit the activation of NFκB pathway and effectively alleviate chronic liver injury and hepatotoxicity induced by STZ as evidenced by decreased serum ALT sitagliptin can prevent hepatocytes apoptosis and inflammatory response of hepatocytes stimulated by TNFα/LPS our in vitro data suggested that sitagliptin is performed as an antioxidant on hepatocytes by reducing the cellular ROS level and avoiding the activation of a validated signaling pathway these functions are systemic anti-inflammatory and glucose management independent the present study showed that in STZ-induced diabetes model increased liver DPP4 activity significantly enhanced the tissue oxidative stress and activated NFκB signaling pathway and further induced chronic liver inflammation Sitagliptin played as an antioxidant and especially inhibited the activity of DPP4 in hepatocytes we concluded that sitagliptin therapy may be effective in improving the progression of liver disorders in patients with diabetes wild-type C57BL/6J mice (8 weeks old) were purchased from GemPharmatech (Nanjing Mice were adapted to the new environment for 1 week C57BL/6J mice were randomly assigned to three groups of six mice each two experimental groups were injected with 60 mg/kg STZ (Sigma-Aldrich dissolved in 0.1% mol/L citrate buffer [pH 4.5]) for three times every other day to destroy islet function and generate a diabetic mouse model mice were injected with 0.1% mol/L citrate buffer and fed with standard chow diet (Control) blood glucose ≥ 20 mmol/L was considered as a successful induction of diabetes mice were fed with standard chow (STZ) or sitagliptin-supplemented diet (40 mg/kg All animals were treated humanely and maintained in specific pathogen-free conditions The experimental protocols were approved by the guideline of the Ethics Committee of the Capital Medical University mice were killed by injecting pentobarbital (150 mg/kg body weight) at the termination of the experiments Liver tissues were fixed in 4% paraformaldehyde and embedded in paraffin for hematoxylin and eosin staining and Sirius red staining USA) primary antibodies at 1 : 300 dissolved in 3% bovine serum albumin (BSA)–phosphate-buffered saline at 4 °C overnight Quantitative results were calculated by Image-Pro Plus software to average the percentage of positive area for five images in each section Serum samples were collected for the assay of ALT and LDH levels with Hitachi 7600-020 clinical analyzer (Tokyo After 4 weeks of sitagliptin-supplemented diet with only water available for glucose tolerance tests Glucose (2.5 g/kg body weight) was injected intraperitoneally Blood samples were collected from the mice’s tails and blood glucose was measured at the time points before the injection and after 15 Blood glucose was measured by using ACCU-CHEK® Performa (ROCHE To determine the cell apoptosis in liver tissues the TUNEL assay was performed according to the manufacturer’s instructions with a commercial kit (Roche Quantitative results were calculated by ImageJ by averaging the numbers of TUNEL-positive cells for five fields in each section HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) Primary mouse hepatocytes were isolated from livers of 6–8 weeks male C57BL/6J mice and which were cultured with RPMI 1640 (Gibco All cells were sustained at 37 °C with 5% CO2 in an incubator The inflammatory response was stimulated by two common inducers of cell inflammation—PS (5 μg/ml UK) in the presence or absence of different concentrations (0 and 200 μM) of sitagliptin (dissolved in DMEM UK) was used to upregulate the expression level of DPP4 in HepG2 cells The level of liver lipid peroxidation product MDA was measurement by using a commercial kit (Applygen Technologies The level of liver GSH content and activities of antioxidant defense enzymes including CAT and GPx were measured by Elisa kit (Enzyme-linked Biotechnology According to the manufacturer’s instructions, total RNA from mouse liver and HepG2 cells were extracted with TRIzol (Invitrogen, USA). A HiScript® II 1st Strand cDNA Synthesis Kit was used to get the cDNA sequence (Vazyme Biotech, Nanjing, China). To quantify the expression of mRNA, the SYBR Green Supermix (Bio-Rad, CA, USA) was used. All PCR primers are in Table 2 Proteintech) in liver tissues and cell lines were examined by western blotting diluted 1 : 5000) was purchased from Huaxingbio (Beijing The polyvinylidene difluoride membrane was blocked for 1 h at room temperature by 5% fat-free milk or 5% BSA (dissolved in Tris-buffered saline containing 0.05% Tween-20) After the overnight incubation of the primary antibody at 4 °C the appropriate horseradish peroxidase-conjugated secondary antibody (Huaxingbio The immunoreactive bands were visualized by ECL chemiluminescent system and exposed with ChemiDoc™ XRS + Imager (Bio-Rad a flow-based Annexin V/fluorescein isothiocyanate (FITC) assay (BD) was used to measure the apoptosis of HepG2 cells by following the manufacturer’s instructions The cells in FITC-positive fraction were considered apoptotic 2 × 105 HepG2 cells were seeded in 12-well plates dichloro-dihydro-fluorescein diacetate (Applygen Technologies China) were used to incubated cells at 37 °C for 30 min in the dark Analyses were performed with a FACScan-420 flow cytometry instrument (Becton-Dickinson False discovery rate (FDR) provides the estimated probability that a gene set with a given normalized enrichment score (NES) represents a false-positive finding; FDR < 0.25 is an accepted cutoff for the identification of biologically significant gene sets UK) was used for the detection of DPP4 activity by following the manufacturer’s instructions The results were measured by an Enspire™ Multimode Microplate Reader (PerkinElmer Student’s t-test or analysis of variance was used to determine the statistical significance between two or more than two groups Data were analyzed using GraphPad Prism 8 (GraphPad Software Data are excluded if it deviates from mean with >3 SDs All P-values are two-sided and P < 0.05 was considered statistically significant All data and material in our study are valid and veritable A different perspective for management of diabetes mellitus: controlling viral liver diseases and mortality among people with type 2 diabetes and alcoholic or nonalcoholic fatty liver disease hospital admission Liver disease in patients with diabetes mellitus Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties Increased hepatic expression of dipeptidyl peptidase-4 in non-alcoholic fatty liver disease and its association with insulin resistance and glucose metabolism Dipeptidyl peptidase IV (DDP IV) in NASH patients Dipeptidyl peptidase-4: a key player in chronic liver disease Dipeptidyl peptidase IV inhibition with MK0431 improves islet graft survival in diabetic NOD mice partially via T-cell modulation Reversal of new-onset diabetes through modulating inflammation and stimulating beta-cell replication in nonobese diabetic mice by a dipeptidyl peptidase IV inhibitor Four-year clinical remission of type 1 diabetes mellitus in two patients treated with sitagliptin and vitamin D3 Dorothy Hodgkin Lecture 2012: non-alcoholic fatty liver disease insulin resistance and ectopic fat: a new problem in diabetes management Impact of liver diseases on the development of type 2 diabetes mellitus Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes fibrosis and preserves diastolic function in a rat model of heart failure with preserved ejection fraction DPP-4 (CD26) inhibitor sitagliptin exerts anti-inflammatory effects on rat insulinoma (RINm) cells via suppressing NF-kappaB activation improves endothelial function and reduces atherosclerotic lesion formation in apolipoprotein E-deficient mice Sitagliptin reduces plaque macrophage content and stabilises arteriosclerotic lesions in Apoe (-/-) mice Caveolin-1 triggers T-cell activation via CD26 in association with CARMA1 The origin of myofibroblasts in liver fibrosis Reactive oxygen and mechanisms of inflammatory liver injury: present concepts Uncoupling protein-2 mediates DPP-4 inhibitor-induced restoration of endothelial function in hypertension through reducing oxidative stress Sitagliptin ameliorates oxidative stress in experimental diabetic nephropathy by diminishing the miR-200a/Keap-1/Nrf2 antioxidant pathway Neuroprotective effects of vildagliptin in rat rotenone Parkinson’s disease model: role of RAGE-NFkappaB and Nrf2-antioxidant signaling pathways Increased mitochondrial fission promotes autophagy and hepatocellular carcinoma cell survival through the ROS-modulated coordinated regulation of the NFKB and TP53 pathways Comprehensive analysis of liver and blood miRNA in precancerous conditions The effects of aerobic exercise frequencies on liver fibrosis alpha-fetoprotein and cytokeratin 19 in experimental type 2 diabetes-induced rats: an immunohistochemistry study Roles of adipokines in digestive diseases: markers of inflammation metabolic alteration and disease progression Gene expression of tumor necrosis factor alpha and TNF-receptors Management of diabetes mellitus in patients undergoing liver transplantation The treatment of diabetes mellitus of patients with chronic liver disease Pharmacokinetics of linagliptin in subjects with hepatic impairment Comparative clinical pharmacokinetics of dipeptidyl peptidase-4 inhibitors and efficacy of DPP-4 inhibitors and GLP-1 receptor agonists in patients with type 2 diabetes mellitus and renal or hepatic impairment Twelve week liraglutide or sitagliptin does not affect hepatic fat in type 2 diabetes: a randomised placebo-controlled trial Effect of sitagliptin on intrahepatic lipid content and body fat in patients with type 2 diabetes Elevated hepatic DPP4 activity promotes insulin resistance and non-alcoholic fatty liver disease Dipeptidyl peptidase-4 regulation of SDF-1/CXCR4 axis: implications for cardiovascular disease Sitagliptin ameliorates thioacetamide-induced acute liver injury via modulating TLR4/NF-KB signaling pathway in mice DPP4 Inhibitor Attenuates Severe Acute Pancreatitis-Associated Intestinal Inflammation via Nrf2 Signaling Nuclear factor-kappa B activation in skeletal muscle of patients with chronic heart failure: correlation with the expression of inducible nitric oxide synthase Hepatocyte-secreted DPP4 in obesity promotes adipose inflammation and insulin resistance The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes Download references This study was supported by the National Natural Science Foundation of China (81800768 and 81800723) and Beijing Natural Science Foundation (7212055) These authors contributed equally: Xin Wang Center for Endocrine Metabolism and Immune Diseases Beijing Key Laboratory of Diabetes Research and Care L-YY and DZ supervised the experiments and revised the manuscript This paper has been approved by the Medical Ethics Committee of the Beijing Luhe Hospital Download citation DOI: https://doi.org/10.1038/s41420-021-00625-7 and provides many of the important features we needed This article explains the existing ROS build tooling and why colcon was chosen as the build tool for ROS2 Colcon does a ton of the heavy lifting for us allowing us to focus on our specific functionality colcon build replaces catkin_make and ament_make with no modifications to the packages being built AWS RoboMaker users can install a single tool and execute the full workflow to generate a bundle for use with AWS RoboMaker I will go over the normal workflow for creating a bundle to use with AWS RoboMaker and some solutions for frequently-encountered issues I will dive deeper into the newest version of our ‘bundle’ file format and provide an overview of what happens when you execute colcon bundle to create a bundle ROS simulation workflows require an environment with ROS and other runtime dependencies of the application installed This is usually fulfilled by executing various apt-get install commands and cannot be reproduced reliably because upstream package updates can occur at any time we were concerned about the reliability of using apt to install a new environment for every simulation run so we looked into various complete packaging formats The most compelling and reliable distribution format for an entire ROS application was a Debian package It can contain everything required to run the robot: ROS We decided not to go with a preexisting format because we wanted flexibility to add optimizations for downloading So we created the ‘bundle’ format and the corresponding tooling to support it We are excited to announce the second version of the bundle format with optimizations to reduce or remove these limitations The format now supports partial downloads and partial extraction AWS RoboMaker supports this new version transparently. If you would like to try out these new improvements run sudo pip3 install -U colcon-bundle in your development environment. I will dive deeper into the implementation of this new format in the second part of this blog. If you have any questions, ideas, or issues, we’d love to read them in our issues on GitHub Bundling your workspace with colcon takes only two steps: Every ROS package that can be built with catkin_make can also be built with colcon build. Over months working with ROS packages, we’ve encountered only minor issues building packages with colcon build. If you run into issues building packages with colcon build that build successfully with catkin_make, please report them in colcon-ros issues NOTE: The most noticeable change from catkin_make to colcon build is that there is no longer adevel directory If you want to add your workspace to your local environment After your workspace has been built with colcon build you should have a build and an install directory and compresses them along with your built workspace into a bundle to be used on AWS RoboMaker The built bundle is located at bundle/output.tar The standard build instructions for ROS1 development use catkin_make many ROS1 developers might not be familiar with it but it is fully compatible with catkin builds While building existing ROS packages (previously built with catkin_make) with colcon build we ran into a couple of minor issues and want surface them here to save other developers some time and effort A common build issue we encountered is colcon failing to properly enumerate all the packages in the workspace leading to failed colcon bundle invocations and odd installation directory structures This is usually caused by nested folder structures withCMakeLists.txt in the intermediate directories This is required for building nested packages within directories withcatkin_make Colcon supports arbitrarily deep nested folder structures These are the most frequent issues we’ve run into while working with bundles of applications on AWS RoboMaker The most common problem we saw while bundling various open source ROS libraries was missing dependencies the build environment already has a dependency installed because of another package but after the package is bundled that dependency is missing The common error messages when a dependency is missing are Could not load libxzy.so or Could not load module 'python_dependency' add the dependency to the package.xml of the package that requires it If you use dependencies in your applications from your own apt or pip repository you need to include those repositories in your colcon bundle invocation Now you can build and bundle your application to simulate on AWS RoboMaker! To upgrade to the latest version of our packages run: sudo pip3 install -U colcon-bundle colcon-ros-bundle in your development environment. If you have any questions or run into issues, please post on the relevant GitHub repo If you’re interested in what is actually happening when colcon bundle is executed This section is a technical deep dive into the internals of the bundle format and the implementation of colcon bundle At the end of this section you will come out with an understanding of what’s happening when colcon bundle executes we hope you’ll want to build your tooling on top of the colcon ecosystem and contribute it back to the community installs those dependencies into a local staging area and then packages them along with the local built workspace into a bundle for consumption by AWS RoboMaker That workflow produces what we call a ‘bundle’ (we know, it’s a boring name – if you have a naming idea for this format, please open an issue in our repository!). The specification of the bundle format is located in the colcon-bundle repository This diagram shows V2 of our bundle format. You can view the history here. A ‘bundle’ file follows the standard GNU tar format I outlined the benefits of the latest version of our format up above These are the major changes between V1 and V2: Overall, we are very happy with these improvements. If you have any ideas, we’d love to read them in our issues on GitHub In this section I will dive into what is actually happening when you execute colcon bundle (It has been a great experience building our tooling on top of colcon If you are looking to create specialized build tooling I highly recommend seeing if colcon could provde value as a base framework.) To extend the command line interface, colcon supports adding commands via VerbExtensionPoints. To build our functionality, we created the bundle verb and registered it in our setup.cfg That’s all we had to do to get an integrated command into this awesome tool (All the work from there on was specific to our domain.) If you’re looking to build tooling around your ROS workflows some provided to us by thecolcon infrastructure but I encourage you to dive into the code for a deeper understanding of how everything works The bundle format can be used locally just like we use it on AWS RoboMaker Doing this could be useful in your application We will soon publish the GoLang library we have built to consume colcon bundle archives in our services I’ll summarize the steps of how we currently read and consume a colcon bundle archive: Matthew is a software engineer on the RoboMaker team he likes to eat great food and play computer and board games Metrics details Prices may be subject to local taxes which are calculated during checkout Reactive oxygen species as intracellular messengers during cell growth and differentiation Role of peroxisomes as a source of reactive oxygen species (ROS) signaling molecules Oxidation of fatty acids is the source of increased mitochondrial reactive oxygen species production in kidney cortical tubules in early diabetes Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution Role of glutaredoxin in metabolic oxidative stress Glutaredoxin as a sensor of oxidative stress mediated by H2O2 Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress The Nrf2 cell defence pathway: Keap1-dependent and -independent mechanisms of regulation Nrf2:INrf2 (Keap1) signaling in oxidative stress Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells Mitochondrial dysfunctions in neurodegenerative diseases: relevance to Alzheimer’s disease Oxidative damage to neurons caused by the induction of microglial NADPH oxidase in encephalomyocarditis virus infection Hepatitis C virus core protein increases mitochondrial ROS production by stimulation of Ca2+ uniporter activity Superoxide dismutase 1 protects hepatocytes from type I interferon-driven oxidative damage Cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells Reactive oxygen species are induced by Kaposi’s sarcoma-associated herpesvirus early during primary infection of endothelial cells to promote virus entry The Marburg virus VP24 protein interacts with Keap1 to activate the cytoprotective antioxidant response pathway Dimerization controls Marburg virus VP24-dependent modulation of host antioxidative stress responses Neuroglobin protects astroglial cells from hydrogen peroxide-induced oxidative stress and apoptotic cell death ROS-dependent caspase-9 activation in hypoxic cell death Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via mitochondrial mediated caspase-dependent mechanism Aldehyde dehydrogenase 2 deficiency negates chronic low-to-moderate alcohol consumption-induced cardioprotecion possibly via ROS-dependent apoptosis and RIP1/RIP3/MLKL-mediated necroptosis Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease A model of chronic inflammation and pulmonary emphysema after multiple ozone exposures in mice Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2 is a novel substrate for the redox-regulated Keap1-dependent ubiquitin ligase complex PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to mitochondria Folding and biogenesis of mitochondrial small Tim proteins Molecular characterization of mitochondrial apoptosis-inducing factor AIF: not just an apoptosis-inducing factor A conserved motif mediates both multimer formation and allosteric activation of phosphoglycerate mutase 5 Viral immune modulators perturb the human molecular network by common and unique strategies Marburgvirus hijacks nrf2-dependent pathway by targeting nrf2-negative regulatorkeap1 HCV and oxidative stress: implications for HCV life cycle and HCV-associated pathogenesis Protective Role of Sirtuin3 (SIRT3) in oxidative stress mediated by hepatitis B virus X protein expression Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins Critical contribution of oxidative stress to TNFα-induced necroptosis downstream of RIPK1 activation Ferroptosis: an iron-dependent form of nonapoptotic cell death The mitochondrial phosphatase PGAM5 is dispensable for necroptosis but promotes inflammasome activation in macrophages Production of large amounts of hydrogen peroxide by human tumor cells a phytochemical extract from the Scutellaria barbata plant disrupts proliferation of human breast and prostate cancer cells through distinct mechanisms dependent on the cancer cell phenotype Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues RIPK3 activates parallel pathways of MLKL-driven necroptosis and fadd-mediated apoptosis to protect against influenza A virus Sequestration by IFIT1 impairs translation of 2’O-unmethylated capped RNA The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host Estimation of globular protein secondary structure from circular dichroism MaxQuant enables high peptide identification rates individualized p.p.b.-range mass accuracies and proteome-wide protein quantification Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction Dual Role of IL-22 in allergic airway inflammation and its cross-talk with IL-17A Download references Weber (Veterinary University Giessen) for pI.18_3xFlag_NSs and pI.18_3xFlag_DMx_1xFlag; F Meissner (Max-Planck Institute of Biochemistry Klein (Max-Planck Institute of Neurobiology Bowie (Trinity College Dublin) for HEK293 cells; A Musacchio (Max-Planck Institute of Cell Biology Kochs (University of Freiburg) for antibody to FluAV NS1; A Knapp (University of Oxford) for amino-terminal His-tagged PGAM5 (pNIC28-Bsa4-PGAM5(Δ2–28); the innate immunity laboratory for critical discussions and suggestions; A Mann for technical assistance; the MPI-B core facility for technical assistance with protein purification Wilson and ECM Biosciences for raising the antibody to AIFM1 phosphorylated at Ser116; K Sowa for maintaining mass spectrometers; and S Kaphengst and the MPI-B animal facility for breeding mice Supported by the Max-Planck Free Floater program (A.P.) the German research foundation (PI 1084/2 and TRR179 to A.P the European Research Council (starting grant ERC-StG iVIP Infect-Era and the German Federal Ministry of Education and Research (ERASE) (A.P.) the US National Institutes of Health (R01 AI107056 and 1R41AI127009 (Moir) to D.W.L and G.K.A.) the Centre National de la Recherche Scientifique (B.R.) and the European Regional Development Fund (BIO-TARGET 2016-00110366 to Region Centre Val de Loire and la Région Centre (Respir_Ozone Spemann Graduate School of Biology and Medicine Jennifer Wettmarshausen & Fabiana Perocchi Institute of Infectious Diseases and Molecular Medicine German Center for Infection Research (DZIF) designed the experiments and wrote the paper Supplementary Figures 1-6 and Supplementary Tables 1-4 Download citation DOI: https://doi.org/10.1038/s41590-017-0013-y Metrics details Redox imbalance generates multiple cellular damages leading to oxidative stress-mediated pathological conditions such as neurodegenerative diseases and cancer progression maintenance of reactive oxygen species (ROS) homeostasis is most important that involves well-defined antioxidant machinery we have identified for the first time a component of mammalian protein translocation machinery Magmas to perform a critical ROS regulatory function Magmas overexpression has been reported in highly metabolically active tissues and cancer cells that are prone to oxidative damage We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging Magmas promotes cellular tolerance toward oxidative stress by enhancing antioxidant enzyme activity thus preventing induction of apoptosis and damage to cellular components Magmas enhances the activity of electron transport chain (ETC) complexes Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance The function of Magmas as a ROS sensor was found to be independent of its role in protein import The unique ROS modulatory role of Magmas is highlighted by its ability to increase cell tolerance to oxidative stress even in yeast model organism The cytoprotective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress-related diseases Although overexpression of Magmas in energy-demanding tissues and cancer cells is well known the physiological advantage provided by the enhanced expression of the protein is still enigmatic we have provided compelling evidence favoring an additional function of Magmas as a ‘ROS regulator’ The overexpression of Magmas led to reduction in ROS and increased cellular tolerance to oxidative stress whereas its downregulation elevated the cellular ROS level and made the cells more susceptible to ROS-mediated apoptosis To maintain the redox equilibrium and provide cytoprotection against oxidative stress Magmas controls ROS production by enhancing the ETC complex activity it increases the activity of two major antioxidant enzymes such as MnSOD and glutathione peroxidase (GPx) to promote ROS scavenging we propose a novel essential role of Magmas as a ‘ROS regulatory protein’ in the maintenance of cellular redox homeostasis and imparting cytoprotection under oxidative stress Modulation of ROS by differential Magmas expression (a) Western blot of Magmas comparing the protein’s expression level in PC-3 and LNCaP Anti-Tim 23 antibodies were used as a loading control (b) Quantification of western blot showing expression level of Magmas in PC-3 and LNCaP (blot shown in (a)) Quantification was performed using ImageJ software (c) PC-3 and LNCaP cells were stained with DCFDA dye; relative amount of cellular ROS was measured by flow cytometry and represented as fold change in mean fluorescence intensity (MFI) of oxidized dye over LNCaP cells (d) The mitochondrial superoxide level was measured by staining the cells with MitoSOX and quantified by analyzing the fluorescent intensity through flow cytometry (e) Western blot showing the level of Magmas downregulation (Mag ↓) in PC-3 cells after siRNA transfection (f and g) Measurement of an increase in oxidative stress in PC-3 cells through DCFDA (f) and MitoSOX (g) staining after siRNA-mediated downregulation of Magmas ROS level is presented as fold MFI over untransfected PC-3 cells (UT) and rotenone-treated cells were used as a positive control for the production of superoxides in mitochondria A nontargeting siRNA (NT) was used as a negative control for siRNA-mediated knockdown (h and i) Fluorescent images of PC-3 stained with DCFDA (h) and MitoSOX (i) after Magmas downregulation (j) Western blot showing accumulation of precursor form of mtHsp70 in PC-3 cells with reduced expression of Magmas and exposed to heat stress for 2 h; p n=3; **P<0.001 (two tailed) and ***P<0.0001 (two tailed); scale bar: 10 μm these findings suggest that elevated expression of Magmas leads to decrease in cellular ROS and downregulation of the protein leads to increment in ROS level that is independent of its import function Modulation of ROS by extrinsic overexpression of Magmas: involvement of J-like domain (a and b) Quantification of decrease in ROS by increased Magmas expression (Mag ↑) in HEK293T cells presented as fold decrease in fluorescent intensity of DCFDA (a) and MitoSOX (b) over untransfected HEK293T cells Empty vector was transfected as a transfection control (c and d) HEK293T cells were transfected with Magmas mutants to generate a semidominant negative phenotype and an increase in oxidative stress was observed by flow cytometry as depicted by fold changes in MFI values of DCFDA (c) and MitoSOX (d) over untransfected cells (e and f) HeLa cells overexpressing Magmas and Magmas mutants were stained with DCFDA (e) and MitoSOX (f); the comparative ROS levels were inferred from the intensities of the oxidized fluorescent form of dyes presented as images of the cells n=3; ***P<0.0001 (two tailed); scale bar: 10 μm Magmas enhances the activity of respiratory chain complexes for the reduced production of ROS (a) Mitochondria were isolated from PC-3 and LNCaP cells and subjected to measurement of activities of respiratory chain complexes (b and c) Isolated mitochondria from HEK293T cells overexpressing Magmas (b) and Magmas mutants (c) were utilized for the determination of changes in activities of ETC complexes upon Magmas overexpression and functional defect respectively All enzyme activities are presented as the rate of the reaction in μM/s (d) Mitochondrial ATP level was quantified in isolated mitochondria through Promega TOXGlo detection kit **P<0.01 (two tailed) and ***P<0.001 (two tailed) Magmas prevents accumulation of ROS under conditions of oxidative stress (a) Magmas was downregulated in PC-3 (Mag ↓) cells followed by an exposure to increasing concentration of NaAsO2 (10–100 μM) for 90 min The increment in lethality was assessed by cellular uptake of PI cells transfected with empty vector and HEK293T cells overexpressing Magmas (Mag ↑) were treated with increasing concentrations of NaAsO2 for 90 min and the cellular viability was estimated by PI staining and represented in terms of percentage lethality with increasing oxidative stress (c and d) Activation of caspase 3 and 7 was measured by ApoGlo kit from Promega PC-3 cells with downregulation of Magmas (c) and HEK293T cells with overexpression of Magmas (d) were treated with 25 μM NaAsO2 and the activation of caspases was detected by fluorescence generated through the cleavage of substrate (e) HEK293T cells with higher expression of Magmas were exposed to oxidative stress (25 μM NaAsO2) and stained with DCFDA dye and the level of cellular ROS was depicted as fold MFI over the untransfected untreated HEK293T cell HEK293T cells with enhanced expression of Magmas were subjected for measurement of the level of mitochondrial superoxides The cells were stained with MitoSOX and superoxide level was presented as fold MFI over untransfected untreated HEK293T cells as was analyzed through flow cytometry cells were recovered with 2 mM N-acetyl cysteine (NAC) for 1 h after NaAsO2 treatment n=3; **P<0.05 (two tailed) and ***P<0.0001 (two tailed) (g and h) To validate the ROS modulating property of Magmas HeLa cells overexpressing Magmas and Magmas mutants were subjected to 25 μM NaAsO2 treatment followed by staining with DCFDA dye (g) and MitoSOX (h) The fluorescent intensity of the oxidized dye was observed through microscopy our results highlight the critical role of Magmas in prevention of apoptotic cell death and increased cellular stress tolerance by maintaining the free radical balance Conserved function of Magmas as ROS modulator in lower eukaryotes: involvement of J-like domain Magmas was expressed under ADH and TEF promoter in yeast cells deleted for wt PAM16 (P-16) (a) Yeast cells expressing wt P-16 and Magmas were pretreated with increasing concentrations of H2O2 (2–8 mM) followed by spotting over YPD medium for recovery at 30°C for different time intervals as indicated (b and c) Yeast strains expressing different levels of Magmas and P-16 were subjected to oxidative stress by peroxide treatment and then stained with DCFDA (b) and MitoSOX (c) The cellular level of ROS and mitochondrial superoxide level were presented in the form of fold MFI of respective dyes over the untreated wt P-16 as analyzed by flow cytometry (d) The ability of Magmas mutants to survive under oxidative stress was analyzed by their ability to recover after an exposure of peroxide (e) DCFDA staining of mutant expressing yeast cells grown at 25°C (permissive temperature) (f and g) Mutant strains were revived at 25°C followed by a heat shock at 37°C (nonpermissive temperature) for 2 h to generate a mutant phenotype before staining with MitoSOX (f) and DCFDA (g) that were analyzed by flow cytometry to measure ROS level Data represented as mean±S.E.M.; n=3; *P<0.01 (two tailed) **P<0.001 (two tailed) and ***P<0.0001 (two tailed) it is clear from our findings that Magmas maintains redox equilibrium by both reducing production and promoting scavenging of ROS Overexpression of Magmas is associated with elevated enzymatic activity of antioxidants and mitochondrial DNA copy number (a) For the quantitative measurement of mtDNA isolated mitochondria expressing a basal level of Magmas (UT) and higher amount of protein (Mag ↑) were stained with SYBr green and the fluorescence emitted upon its binding to mtDNA was represented as fold fluorescent at 520 nm (F520) over the control (UT) were amplified by qPCR and data are presented as fold change in the copy number upon Magmas overexpression over untransfected control (c and d) Level of cellular GSH (c) and GSSG (d) was measured in cells overexpressing Magmas in a luminescence-based assay in the presence or absence of oxidative stress (e) The enzyme activity of glutathione peroxidase was measured spectrophotometrically under untreated condition and after treatment of 25 μM NaAsO2 (f) Isolated mitochondria were utilized to measure the activity of MnSOD upon Magmas overexpression The activity is presented in terms of rate of the reaction thereby preventing the accumulation of ROS under oxidative stress Magmas increases the activity of MnSOD and GPx thereby causing a decrease in mitochondrial superoxide level and total cellular ROS level respectively Physiological relevance of maintenance of REDOX equilibrium by Magmas: protection to lipid and protein (a) ROS-mediated oxidation of lipid in cells exposed to oxidative stress was detected by TBARS assay via spectrophotometric detection of its end product malondialdehyde at 532 nm (b) The damage to Fe-S cluster leads to the loss of enzyme activity To determine the oxidative damage to Fe-S cluster in Magmas mutant-expressing cells aconitase activity was measured in isolated mitochondria (c) The oxidative damage to the protein leads to carbonylation of its residues The extent of protein carbonylation was analyzed by the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) The derivatization of protein carbonyls to 2,4-dinitrophenylhydrazone (DNP) was analyzed by using an anti-DNP antibody Anti-β-actin antibody was used as loading control Magmas by virtue of its overexpression acts as a ROS regulator to maintain cellular redox homeostasis and its ROS modulatory function is independent of its function in the import of precursor proteins higher expression of Magmas found in metabolically active tissues is beneficiary to these cells that are more susceptible to ROS production because of high rate of respiration it can be concluded that Magmas maintains the sublethal level of ROS and promotes ‘redox adaptation’ in cancer cells traditionally regarded as a co-chaperone of mtHsp70 has evolved to perform an additional function as ROS regulator in higher eukaryotes The identification of novel cytoprotective ROS modulators such as Magmas holds special importance toward its prospective future implication in understanding the different pathophysiological conditions associated with oxidative stress HEK293T cells were cultured in advanced Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen USA) containing 10% fetal bovine serum (Gibco USA) and 1% penicillin–streptomycin (Gibco) at 37°C in 5% CO2 RPMI-1640 (Invitrogen) with 10% serum and 1% Glutamax and Penstrep were used as medium Gene encoding Magmas with a C-terminus HA tag was cloned in plasmid pCDNA3.1a HEK293T cells were transfected with a construct of Magmas in pCDNA3.1a by using Lipofectamine 2000 (Invitrogen) Magmas mutants were cloned in pCI-neo vector and transfected in HEK293T cells to generate a semidomiant negative phenotype Cells expressing Magmas and Magmas mutants were harvested for different experiments after 48 h of transfection MAGMAS was cloned in pRS415 vector under TEF promoter and in pRS414 vector under ADH promoter for its differential expression Pam16 and Magmas mutants were cloned in pRS415 TEF The constructs containing cDNA encoding for Magmas and Pam16 were transformed in Δpam16 haploid yeast cells containing wild-type PAM16 in a URA3-based plasmid The transformants were selected on minimal medium and streaked on 5-FOA plates to select for cells having lost the URA3 plasmid The generated strains expressing wild-type and mutant Magmas were revived on YPD medium before assays All genetic analyses were performed in PJ53 background strain of Saccharomyces cerevisiae The total cellular ROS level was measured using DCFDA dye and for mitochondrial superoxide detection 50 000 cells were stained with 15 μM DCFDA for 15 min at 37°C The cells were washed with 1 × phosphate buffer saline (PBS) and subjected to a flow cytometric analysis at excitation wavelength of 492 nm and emission of 520 nm using BD FACSCanto II flow cytometer (San Jose 50 000 cells were harvested and stained with 5 μM dye for 10 min at 37°C The stained cells were excited at 510 nm and the emitted fluorescence was detected at 580 nm by flow cytometry ROS in yeast cells was quantified after growing them in YPD medium at 30°C to late log phase 0.3 OD of cells were harvested and stained with 50 μM DCFDA for 15 min at 30°C for flow cytometric analysis Mitochondrial superoxides were detected by adding 5 μM MitoSOX dye to 0.3 OD of cells followed by incubation at 30°C for 15 min yeast cells were treated with 4 mM H2O2 for 3 h and 30 min at 30°C Human cells were treated with 25 μM NaAsO2 for 90 min at 37°C to induce oxidative stress before the detection of ROS The formation of peroxides in cell was validated by Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen Molecular Probes USA) and fluorescence was observed at 585 nm For the downregulation of Magmas in HEK293T cells a pool of dsiRNA (IDT HSC.RNAi.NO16069.12.1 and IDT HSC.RNAi.NO16069.12.2) was transfected using Lipofectamine 2000 and cells were harvested after 48 h of transfection Magmas was downregulated in PC-3 cells through Lipofectamine-based transfection of dsiRNA and the cells were harvested after 24 h of transfection Nontargeting dsiRNA was used as a negative control for siRNA-mediated downregulations The expression level of Magmas was semiquantitatively measured through western blotting 100 μg protein corresponding to mitochondria was loaded on SDS-PAGE and transferred to PVDF membrane Protein was probed with anti-Magmas or anti-Pam16 antibody for 1 h at room temperature HRP-conjugated anti-rabbit secondary antibody was used for its detection Anti-Tim23 antibody was used as loading control of mitochondria-enriched fractions As a loading control in detection of protein expression in yeast anti-Mge1 and anti-Tim50 antibodies were used Activation of caspases was detected using Promega Apo-ONE Homogeneous Caspase-3/7 assay kit (Promega Yeast cells expressing wild-type Pam16 and Magmas under TEF promoter were grown at 30°C to the late log phase 0.5 OD of cells were harvested and pretreated with different concentrations of H2O2 for 3 h 30 min at 30°C followed by spotting on YPD medium for recovery Growth of the cells exposed to the oxidative stress was observed at different time intervals as indicated Strains expressing Magmas mutants were revived at permissive temperature and pretreated with 4 mM H2O2 The ability of mutants to withstand the oxidative insult was assessed by recovering them on YPD HeLa cells (cell count 5000) were grown in DMEM at 37°C with 5% CO2 cells were transfected with Magmas and Magmas mutants and grown in DMEM Cells were stained with 3 μM DCFDA for 10 min and mounted in ProLong Gold Antifade reagent (Invitrogen) or in phenol red-free DMEM before imaging cells overexpressing Magmas and Magmas mutants were stained with 25 μM MitoSOX for 15 min and mounted in ProLong Gold Antifade reagent with DAPI (Invitrogen) All images were analyzed under Zeiss AxioObserver Z1 Apotome 2.0 (Jena Images were acquired using Zeiss AxioCam MRm Camera (Jena Germany) at constant exposure of 300 ms and processed using Zeiss Axio vision Rel Images were zoomed digitally by Adobe Photoshop CS4 (San Jose The images were quantified by selecting a uniform area to measure the change in fluorescent intensity of respective dyes using ImageJ software (National Institute of Health The level of ATP was measured in isolated mitochondria by Promega TOX Glo detection kit (Promega) The extent of protein carbonylation was assessed by OxyBlot Protein Oxidation Detection kit (Millipore Procedure followed as directed by the instructions of the manufacturer cells were lysed for the preparation of protein extract 5 μg protein was derivatized to 2,4-dinitrophenylhydrazone (DNP) DNP was detected using an anti-DNP antibody through immunoblotting Cells were treated with increasing concentrations of NaAsO2 (10 50 and 100 μM) for 90 min at 37°C to induce generation of ROS Cells were harvested by using 0.25% Trypsin-EDTA and stained with 0.8 μg of dye just before analyzing through flow cytometry A total of 50 000 cells were stained with 10 nM NAO (Invitrogen Molecular Probes) and fluorescence was measured flow cytometrically at 530 nm mitochondria were incubated in phosphate buffer containing 1% DOC and 2 mM sodium azide for 15 min on ice Permeabilized mitochondria were then incubated with 1 mM NADH and the reaction was initiated with the addition of 60 μM ubiquinone The decrease in absorbance by the oxidation of NADH was recorded at 340 nm for 2 min Complex II activity was measured in a coupled assay Mitochondria were incubated with 20 mM succinate in a phosphate buffer for 10 min at 30°C to activate complex II 2 μg/μl antimycin and 2 mM sodium azide were added to inhibit other complexes the reduction of ubiquinone was linked to the reduction of DCPIP (50 μM) The production of colorless reduced DCPIP was measured as a decrease in absorbance at 600 nm mitochondria were permeabilized in phosphate buffer containing 1% DOC and 2 mM sodium azide 0.25% v/v Tween-20 and 75 μM oxidized cytochrome c The reaction was initiated by the addition of 0.1 μM ubiquinol and reduction of cytochrome c was detected as an increase in absorbance at 550 nm Assessment of complex IV activity was done by measuring the decrease in absorbance at 550 nm corresponding to the oxidation of reduced cytochrome c A parallel reaction was set up for every experiment with the inhibitor of respective complexes to assure the specificity of the reaction GPx activity was measured using the glutathione reductase (GR) coupled assay by spectrometrically following the decrease in the concentration of NADPH at 340 nm GR (1.7 Units) and cell lysate corresponding to 50 μg of protein in 100 mM Reaction was initiated by adding H2O2 (240 μM) and decrease in absorbance at 340 nm was observed for 45 s MnSOD activity was analyzed in isolated mitochondria using SOD Assay Kit (Sigma-Aldrich and the linearity was confirmed within an appropriate concentration range using genomic DNA from untransfected HEK293T cells The mitochondrial DNA content was quantified by utilizing a fluorometric assay where an increase in SYBr Green (Invitrogen Molecular Probes) fluorescence because of higher DNA binding was quantified as a measure of the amount of DNA content Equivalent amounts of mitochondria were incubated with saturating amounts of SYBr-Green dye and fluorescence was observed at 520 nm Damage to DNA was detected as formation of double-stranded breaks Cells adhered over a coverslip were fixed with IC Fixation buffer (Invitrogen) for 15 min at room temperature followed by treatment with 90% alcohol for 7 min Cells were washed with PBS and incubated with blocking solution containing 0.05% IgG free BSA and 0.05% Triton-X 100 for 30 min anti-γH2AX antibody was added and incubated for 1 h and 30 min Cells were washed three times with PBS and incubated with secondary antibody conjugated with Alexa-488 cells were mounted on ProLong Gold Antifade reagent with DAPI (Invitrogen) Cells were treated with 5 μM cisplatin as positive control for the generation of double-stranded breaks by intercalating DNA strands or with 500 μM CuSO4 as control for ROS-mediated DNA damage The formation of γH2AX foci was analyzed under Zeiss AxioObserver Z1 Apotome 2.0 63 × oil NA 1.45 Images were acquired using Zeiss AxioCam MRm Camera and processed using Zeiss Axio vision Rel Images were zoomed digitally by Adobe Photoshop CS4 Cells were pretreated with 25 μM NaAsO2 (HEK293T cells) or 4 mM H2O2 (yeast cells) cells were harvested and washed with 1 × PBS to completely remove the medium Cells were resuspended in hypotonic buffer and sonicated at 30% amplitude for 3 pulses of 20 s 1% thiobarbituric acid was added to the cell lysate followed by an incubation at 90°C for 1 h followed by addition of 250 μl of n-butanol The solution was centrifuged at 16 000 × g for 2 min for the separation of two phases The supernatant was used to measure absorbance at 530 nm Calculation of statistical significance was done using two-tailed Student’s t-test through Graph-Pad Prism 5 software (San Diego USA) unless otherwise mentioned; P-values are mentioned in figure legends granulocyte-macrophage colony-stimulating factor Oxidative stress: oxidants and antioxidants Angew Chem Int Ed Engl 1986; 25: 1058–1071 Generation of reactive oxygen species by the mitochondrial electron transport chain NOX enzymes and the biology of reactive oxygen Signal transduction by reactive oxygen species Role of oxidative stress in development of complications in diabetes Reactive oxygen species and the mitochondrial signaling pathway of cell death The redox state of the lung cancer microenvironment depends on the levels of thioredoxin expressed by tumor cells and affects tumor progression and response to prooxidants Role of superoxide dismutase in cancer: a review Physiological roles of mitochondrial reactive oxygen species Cytochrome c release from mitochondria proceeds by a two-step process Proc Natl Acad Sci USA 2002; 99: 1259–1263 Caspase-1 and -3 are sequentially activated in motor neuron death in Cu,Zn superoxide dismutase-mediated familial amyotrophic lateral sclerosis Proc Natl Acad Sci USA 2000; 97: 13901–13906 Hydrogen peroxide-induced apoptosis: oxidative or reductive stress Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach The cytoprotective role of the Keap1-Nrf2 pathway Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs Magmas expression in neoplastic human prostate Identification and characterization of Magmas a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction Role of Magmas in protein transport and human mitochondria biogenesis Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry Simple quantitative detection of mitochondrial superoxide production in live cells Biochem Biophys Res Commun 2007; 358: 203–208 Essential role of Drosophila black-pearl is mediated by its effects on mitochondrial respiration The impairment of MAGMAS function in human is responsible for a severe skeletal dysplasia Mitochondrial formation of reactive oxygen species Developmental expression of Magmas in murine tissues and its co-expression with the GM-CSF receptor NF-kappaB activation and cell proliferation in human breast cancer MCF-7 cells free radical production and antioxidant status in breast cancer iron homeostasis and oxidative stress in Friedreich ataxia Gamma-H2AX - a novel biomarker for DNA double-strand breaks Mitochondrial protein translocases for survival and wellbeing Methylation-controlled J-protein MCJ acts in the import of proteins into human mitochondria Unraveling the intricate organization of mammalian mitochondrial presequence translocases: existence of multiple translocases for maintenance of mitochondrial function Mitochondrial AtPAM16 is required for plant survival and the negative regulation of plant immunity Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction Parkinson’s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled an endogenous mitochondrial repressor of the respiratory chain that controls metabolic alterations Parkinson’s disease: mechanisms and models Protein aggregation in the brain: the molecular basis for Alzheimer’s and Parkinson’s diseases Protein aggregation and neurodegenerative disease Advanced lipid peroxidation end products in oxidative damage to proteins Potential role in diseases and therapeutic prospects for the inhibitors Magmas overexpression inhibits staurosporine induced apoptosis in rat pituitary adenoma cell lines ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis Regulated cycling of mitochondrial Hsp70 at the protein import channel Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Analysis of iron-sulfur protein maturation in eukaryotes Download references Elizabeth A Craig for yeast strains and yeast-specific antibodies We are obliged to Professor Rajan R Dighe and Ankur Sharma for their generous help in conducting ETC complex activity experiments We acknowledge IISc FACS facility for performing flow cytometry experiments Umakant Sahu and Keshav for their help in the study This work was supported by Department of Science and Technology DST/SJF/LS-01/2011-2012 (to PD’S) and DST-INSPIRE fellowship (to SS) Council of Scientific and Industrial Research Fellowship (to DS and PPS) Indian Institute of Science Research Associate Fellowship (to DS) and DBT Research Associate Fellowship (to HM) The authors declare no conflict of interest Supplementary Information accompanies this paper on Cell Death and Disease website Download citation DOI: https://doi.org/10.1038/cddis.2014.355 Metrics details Five-year survival rate of esophageal squamous cell carcinoma (ESCC) patients treated with radiotherapy is <20% Our study aimed to investigate whether cancer-associated fibroblasts (CAFs) one major component of tumor microenvironment were involved in tumor radioresistance in ESCC human chemokine CXCL1 was found to be highly expressed in CAFs compared with that in matched normal fibroblasts Inhibition of CXCL1 expression in CAFs significantly reversed CAF-conferred radioresistance in vitro and in vivo CAF-secreted CXCL1 inhibited the expression of reactive oxygen species (ROS)-scavenging enzyme superoxide dismutase 1 leading to increased ROS accumulation following radiation by which DNA damage repair was enhanced and the radioresistance was mediated CAF-secreted CXCL1 mediated the radioresistance also by activation of Mek/Erk pathway The cross talk of CAFs and ESCC cells induced CXCL1 expression in an autocrine/paracrine signaling loop which further enhanced tumor radioresistance our study highlighted CAF-secreted CXCL1 as an attractive target to reverse tumor radioresistance and can be used as an independent prognostic factor of ESCC patients treated with chemoradiotherapy lymph node involvement and distant metastases of ESCC patients we investigated whether CAF-secreted chemokines were involved in tumor radioresponse and how chemokines were involved in the interaction of CAFs and tumor cells human chemokine CXCL1 was discovered as an attractive target to reverse CAF-conferred radioresistance Further studies revealed that CAF-secreted CXCL1 conferred tumor radioresistance by activation of DNA damage repair and Mek/Erk signaling pathway CXCL1 expressed in CAFs was found to be an independent prognostic factor of ESCC patients treated with chemoradiotherapy Human chemokine CXCL1 was expressed in an autocrine/paracrine signaling loop (a) CXCL1 was highly expressed in CAF medium compared with in matched NF medium as determined with human cytokine/chemokine array (b) The fold change of CXCL1 mRNA level in different cells by qRT-PCR analysis (c) The concentration of CXCL1 in culture medium of KYSE-30 (d and e) The mRNA level changes of CXCL1 and its receptor CXCR2 after 8 Gy of radiation in KYSE-30 and in KYSE-150 by qRT-PCR analysis compared with CXCL1 mRNA level in KYSE-30 or KYSE-150 before radiation compared with CXCR2 mRNA level in KYSE-30 or KYSE-150 before radiation (f) The fold change of CXCL1 mRNA level in KYSE-30 and KYSE-150 that were cultured in CAF medium for 24 h compared with cultured in normal medium and in CAF-1 and in CAF-2 that were cultured in the CM of KYSE-30 or KYSE-150 for 24 h compared with cultured in normal medium by qRT-PCR analysis Negative control (NC): the cells that were transfected with scrambled siRNA compared with CXCL1 mRNA level in NC that was transfected with scrambled siRNA before cultured in normal medium for 24 h These results suggested the cross talk of CAFs and tumor cells may result in CXCL1 expression in an autocrine/paracrine signaling loop CXCL1 conferred radioresistance by enhancement of DNA damage repair (a and b) The determination of the radiosensitivity of KYSE-150 and KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h by clonogenic survival assay compared with cultured in CAF medium with 500 ng/ml CXCL1 antibody (c) The expressions of cellular DNA damage repair proteins including γ-H2AX p-Chk2 and DNA-PKcs 30 min after radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium for 24 h DNA-PKcs and p-DNA-PKcs 30 min after radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h (e) The expressions of p-ATM and γ-H2AX 30 min after radiation in KYSE-150 and KYSE-30 that were cultured in normal medium or in CAF medium with or without 10 μM ATM kinase inhibitor Ku55933 for 24 h These results suggested CAF-secreted CXCL1 enhanced DNA damage repair after radiation Inhibition of ATM kinase reversed CAF-conferred radioresistance The radiosensitivity of KYSE-150 and KYSE-30 that were cultured in normal medium or in CAF medium with or without 10 μM ATM kinase inhibitor Ku55933 for 24 h as determined by clonogenic survival assay compared with cultured in CAF medium with 10 μM ATM kinase inhibitor Ku55933 compared with cultured in normal medium without 10 μM ATM kinase inhibitor Ku55933 CXCL1 enhanced DNA damage repair in a SOD1–ROS-axis-dependent manner (a and b) The ROS level following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or CAF medium with or without 500 ng/ml CXCL1 antibody or 400 nM CXCR2 inhibitor SB225002 for 24 h (c and d) The fold change of SOD1 mRNA level before or after radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody or 400 nM CXCR2 inhibitor SB225002 for 24 h (e) The expression of SOD1 protein following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h p-DNA-PKcs and SOD1 following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 100 ng/ml human SOD1 protein for 24 h CXCL1 conferred radioresistance by activation of Mek/Erk signaling pathway p-Erk1/2 and p-Mek1/2 following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium for 24 h p-Erk1/2 and p-Mek1/2 following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h p-Mek1/2 and γ-H2AX following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 10 μM Mek1/2 kinase inhibitor U0126 for 24 h (d) The radiosensitivity of KYSE-150 and KYSE-30 that were cultured in normal medium or in CAF medium with or without 10 μM Mek1/2 kinase inhibitor U0126 for 24 h as determined by clonogenic survival assay compared with cultured in CAF medium with 10 μM Mek1/2 kinase inhibitor U0126 compared with cultured in normal medium without 10 μM Mek1/2 kinase inhibitor U0126 Blockage of CAF-secreted CXCL1 reversed radioresistance of xenograft tumor models (a and b) The growth curve of xenograft tumors implanted with KYSE-150 or KYSE-30 alone or combined with CAFs after treatment with tumor injection of 1 μg/ml CXCL1 antibody fractionated radiation at a total dose of 12 Gy alone or their combinations Tumors treated with only PBS was chosen as a control (c and d) The weight of excised xenograft tumors in (a) and (b) at the end of experiment at day 28 (e and f) The photos of excised xenograft tumors in (a) and (b) at the end of experiment at day 28 CXCL1 expressed in CAFs is an independent prognostic factor of ESCC patients treated with chemoradiotherapy (a) The concentration of CXCL1 in plasma of ESCC patients (n=35) and in healthy controls (n=29) by ELISA (b) Kaplan–Meier analysis showed overall survival of ESCC patients with positive CXCL1 expression in CAFs (n=75) was significantly poorer than those CXCL1-negative patients in CAFs (n=66) after chemoradiotherapy The grading of CXCL1 expression was described in ‘Materials and Methods’ (c) One representative result of CXCL1 expression in CAFs and in tumor tissues in 141 ESCC patients by IHC analysis CD31 and vimentin were used for staining blood vessels and fibroblasts the correlation of CAFs and tumor radioresistance has never been reported in ESCC CAF-secreted CXCL1 enhanced DNA damage repair in a SOD1–ROS-dependent manner Treatment with SOD1 protein reversed CXCL1-conferred radioresistance These results suggested CAFs had important effects on redox system by which tumor radioresistance was conferred our study found inhibition of Erk activation by CAF-secreted CXCL1 could also reverse tumor radioresistance which further enhanced the pathological roles of CXCL1 in ESCC our study highlighted CAF-secreted CXCL1 as an attractive target to radiosensitize ESCC in vitro and in vivo CAF-secreted CXCL1 can be used as an independent prognostic factor of ESCC patients treated with chemoradiotherapy The concentration of CXCL1 in plasma of ESCC patients was significantly higher than that in plasma of healthy controls suggesting CXCL1 may be a potential tumor biomarker in ESCC Our study for the first time discovered molecular mechanisms of radioresistance from the viewpoint of TME and these findings may provide important implications for exploiting novel strategies against tumor radioresistance in ESCC tumor tissues and matched noncancerous esophageal tissues were obtained from two pathologically diagnosed ESCC patients who had not been treated with preoperative chemoradiotherapy before esophagectomy tissues specimens were cut into as small pieces as possible rinsed with PBS solution and then digested with 1 mg/ml collagenase type II for 2 h at 37 °C in 5% CO2/95% air cell precipitation was collected and seeded into 25 cm2 culture flask the medium was replaced with fresh medium to remove non-adherent cells (mainly tumor cells) to obtain pure fibroblasts because adherent time of fibroblasts (<30 min) is much shorter than that of tumor cells (usually more than 1 h) a unique homogeneity of stromal fibroblasts were obtained and cultured for further study The fibroblasts isolated from tumor tissues were defined as CAFs and the fibroblasts from matched noncancerous esophageal tissues as NFs Two pairs of CAFs and their matched NFs (CAF-1 CAF-2 and matched NF-1 and NF-2) were successfully isolated and used in our study The human ESCC cells KYSE-150 and KYSE-30 were obtained from American Type Culture Collection and cultured in RPMI-1640 medium (Gibco USA) supplemented with 10% fetal bovine serum (Gibco Life Technologies Inc.) at 37 °C in 5% CO2/95% air CAFs and NFs were seeded into 75 cm2 culture flask the culture medium was collected and centrifuged at 3000 r.p.m The supernatant was collected as CM and kept at −80 °C until use The CM from CAFs and NFs used in our study were defined as CAF medium and NF medium Normal medium referred to fresh RPMI-1640 medium with 10% fetal bovine serum SOD1 (#2770) and GAPDH (#2118) were purchased from Cell Signaling Technology (Beverly Human SOD1 protein (#PHG 9214) was purchased from Epigentek Group Inc α-SMA (#ab5694) and Ki-67 (#ab92742) were purchased from Abcam (Cambridge Antibody against FSP-1 (#07-2274) was purchased from Millipore (Billerica The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651) ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston the following information on patients participating in this study was provided including age KYSE-30 or CAFs were plated into six-well plates at a density of 1 × 105 cells per well and cultured to adherent growth the mixture of CXCL1 siRNA (sense 5′–3′: GAUUAACUCUACCUGCACATT; antisense 5′–3′: UGUGCAGGUAGAGUUAAUCTT) and Lipofectamine 2000 (Hanbio Biotechnology Co. the medium was replaced with normal medium or CAF medium The RNA was extracted to examine CXCL1 downregulation in cells that were transfected with CXCL1 siRNA The cells that were transected with scrambled siRNA were used as a negative control Protein expression was examined by western blotting analysis cells receiving indicated treatments were collected by trypsin–EDTA exposure and washed twice with ice-cold PBS before adding into protein extraction buffer Equal amount of proteins was fractionated on 12% SDS-PAGE gel and transferred to polyvinylidence difluoride membranes The membranes were incubated with the indicated primary and secondary antibodies Proteins were ultimately visualized by enhanced chemiluminescence and autoradiography (Thermo Scientific Intracellular ROS level was determined with ROS assay kit (Beyotime China) according to the manufacturer’s instructions cells were equipped with 10 mM DCFH-DA 30 min before radiation DCFH-DA is hydrolyzed by cellular esterase into DCFH which then transfers to fluorescent DCF in response to radiation-induced ROS Immunofluorescence images were taken using a confocal laser scanning microscope (CLSM Exponentially growing tumor cells were seeded into six-well plate under different conditions adhesive cells were exposed to radiation at 0 6 and 8 Gy with an average dose rate of 100 cGy/min the cells were cultured for another 10 days at 37 °C in a 5% CO2 environment to allow colony formation Only colonies containing ⩾50 cells were counted as clonogenic survivors Unirradiated cells were chosen as a control CAFs and NFs were seeded into 25 cm2 culture flask and cultured to adherent growth the medium was replaced with 4 ml fresh serum-free medium at 4 °C for 30 min to obtain the supernatant The detection of CXCL1 concentration in the supernatant of 106 KYSE-150 CAFs and NFs was performed with Human CXCL1 Quantikine ELISA Kit (R&D Systems USA) according to the manufacturer’s instructions Cells were seeded into six-well plate and cultured to adherent growth cells were fixed with acetone/methanol (1:1) and permeabilized with 0.1% Triton X-100 in PBS Nonspecific binding was blocked with 3% (m/v) bovine serum albumin (BSA) in PBS the cells were incubated with antibody against γ-H2AX for 2 h in PBS containing 0.1% (m/v) BSA Indirect immunofluorescence was performed by incubation with Alexa Fluor 488-conjugated secondary antibodies (Invitrogen Immunofluorescence images were taken using a CLSM (Nikon-A1 system) washed twice with ice-cold PBS and then fixed with 70% ethanol diluted in PBS cell samples was washed with 0.5% BSA in PBS and then incubated with Alexa Fluor 488-conjugated γ-H2AX antibody (CST cells were washed twice with PBS and cultured in 4 ml fresh serum-free medium culture medium was collected and centrifuged at 3000 r.p.m at 4 °C for 30 min to obtain the conditioned medium of CAFs and NFs The expression profile of human cytokines/chemokines was analyzed with human cytokine/chemokine antibody array (RayBiotech USA) according to the manufacture’s instructions the conditioned medium was incubated with human cytokine/chemokine antibody array for 1 h at room temperature and then at 4 °C overnight the human cytokine/chemokine antibody was incubated with biotinylated antibody cocktail before with HRP–Streptavidin Cytokines/chemokines were ultimately visualized by enhanced chemiluminescence and autoradiography (Thermo Scientific) KYSE-30 and CAFs were collected by exposure to trypsin–EDTA and washed with ice-cold PBS KYSE-150 or KYSE-30 alone or combined with CAFs at a ratio of 8:1 (KYSE-150 or KYSE-30/CAFs) were implanted into the right flanks of female BALB/c nude mice When xenograft tumors had reached a mean diameter of around 0.5 cm mice were randomly assigned into different groups (six mice in each group) Tumors were treated with 12 Gy of radiation in six fractions tumor injection of 1 μg/ml CXCL1 antibody for 11 consecutive days alone or in their combinations (fractionated radiation was performed at day 1 the day when tumor injection of CXCL1 antibody began was defined as day 1) Tumors treated with only PBS was used as a control Each animal was earmarked and followed individually throughout the experiments Tumor volume (mm3) was calculated using the following formula: V(mm3)=A(mm) × B(mm)2/2 where A and B were the longest and widest diameter of tumor all mice were killed according to the institutional guidelines IRs of tumor growth were calculated using the following equation: IR=100% × (mean tumor weight of control group − mean tumor weight of experimental group)/mean tumor weight of control group IHC staining of CXCL1 was performed on paraffin-embedded sections of tumor biopsy specimens of ESCC patients sections of 4 μm thick were deparaffinated and rehydrated trough a series of graded alcohols Endogenous peroxidase activity was quenched with 3% (v/v) H2O2 for 20 min Nonspecific binding was avoided by immersing sections into 3% BSA in PBS for 30 min at room temperature the sections were incubated with anti-CXCL1 primary antibody and HPR-conjugated secondary antibody The intensity of CXCL1 expression was graded as 0 strong staining in <30% of CAFs cells; and 3+ strong staining in more than 30% of CAFs cells 0 and 1+ were defined as CXCL1-negative; and 2+ and 3+ as CXCL1-positive The slides were scored by a pathologist and two experienced researchers independently All of the experiments in our study were independently performed in triplicate Statistical analyses were performed with SPSS software 16.0 (SPSS Survival curves were estimated by Kaplan–Meier method and the difference between the curves were evaluated by log-rank tests The Cox proportional hazards regression model was used for the univariate and multivariate survival analysis with 95% CI and P-value calculated The other statistical analyses were performed with Student’s t-test Differences among groups were considered statistically significant at a level of P<0.05 Epidemiology of upper gastrointestinal malignancies Global incidence of oesophageal cancer by histological subtype in 2012 INT 0123 (radiation therapy oncology group 94-05) phase III trial of combined-modality therapy for esophageal cancer: high-dose versus standard-dose radiation therapy STAT3 inhibitor stattic enhances radiosensitivity in esophageal squamous cell carcinoma Inhibition of human positive cofactor 4 radiosensitizes human esophageal squmaous cell carcinoma cells by suppressing XLF-mediated nonhomologous end joining 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DNA damage response abrogates glioblastoma cancer stem cell radioresistance Redox-modulated phenomena and radiation therapy: the central role of superoxide dismutases A growth-related oncogene/CXC chemokine receptor 2 autocrine loop contributes to cellular proliferation in esophagealcancer Role of receptor and nonreceptor protein tyrosine kinases in H2O2-induced PKB and ERK1/2 signaling Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G(2)/M checkpoint arrest in cells exposed to ionizing radiation ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53 Irradiation-induced G2/M checkpoint response requires ERK1/2 activation Mitogen-activated protein kinase (MAPK) expression and activation in non-small cell lung cancer (NSCLC): investigating their prognostic significance and role in radiation responses MEK/ERK signaling pathway regulates the expression of Bcl-2 and Mcl-1 and promotes survival of human pancreatic cancer cells Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-x(L) expression through a MEK-dependent pathway Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-Flip and implications for anoikis Epidermal growth factor and ionizing radiation up-regulate the DNA repair genes XRCC1 and ERCC1 in DU145 and LNCaP prostate carcinoma through MAPK signaling and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated The role of CXC chemokines in the transition of chronic inflammation to esophageal and gastric cancer A key role for early growth response-1 and nuclear factor-kappa b in mediating and maintaining GRO/CXCR2 proliferative signaling in esophageal cancer Association of activated transcription factor nuclear factor kappa B with chemoradiation resistance and poor outcome in esophageal carcinoma Download references This work was financially supported by the National Natural Science Foundation of China (No Zhejiang Provincial Natural Sciences Foundation of China (No Zhejiang Provincial Medical Scientific Research Foundation of China (No Hangzhou City Medical Scientific Research Foundation of Zhejiang Province 2015Z04) and Hangzhou City Scientific Technology Research Foundation of Zhejiang Province Supplementary Information accompanies this paper on Cell Death and Disease website Download citation DOI: https://doi.org/10.1038/cddis.2017.180 Journal of Hematology & Oncology (2025) Metrics details Reactive oxygen species (ROS) constitute a group of highly reactive molecules that have evolved as regulators of important signaling pathways It is now well accepted that moderate levels of ROS are required for several cellular functions The production of ROS is elevated in tumor cells as a consequence of increased metabolic rate and excess ROS are quenched by increased antioxidant enzymatic and nonenzymatic pathways in the same cells Moderate increases of ROS contribute to several pathologic conditions among which are tumor promotion and progression as they are involved in different signaling pathways and induce DNA mutation ROS are also able to trigger programmed cell death (PCD) Our review will emphasize the molecular mechanisms useful for the development of therapeutic strategies that are based on modulating ROS levels to treat cancer we will report on the growing data that highlight the role of ROS generated by different metabolic pathways as Trojan horses to eliminate cancer cells ROS are generated after exposure to physical agents (ultraviolet rays and heat) and after chemotherapy and radiotherapy in cancer Tight regulation of ROS levels is crucial for cellular life; in fact moderate ROS contribute to the control of cell proliferation and differentiation eukaryotic cells benefit from a complex scavenging system based on superoxide dismutases (SODs) mitochondria and the extracellular matrix; glutathione peroxidase (GPX); glutathione reductase (GR); peroxiredoxin; thioredoxin; and catalase which convert superoxide anions into water and recycle the antioxidants in the reduced state we focus on the molecular mechanisms that support the elaboration of anticancer therapies that modulate the production and scavenging of ROS and on the opportunities raised by their ability to induce cell death upon exceeding a threshold level excessive intracellular levels of ROS may damage lipids and this ability has been exploited in a series of anticancer strategies small increases in ROS would be expected to activate the PI3-K/Akt pathway preferentially while further increases would be expected to trigger p38MAPK-dependent apoptosis Most ROS-sensitive pathways transduce cytoplasmic signals to the nucleus where they influence the activity of transcription factors that control the expression of a wide array of genes cancer cells respond to oxidative stress by inducing the transcription of antioxidant enzymes highlighting the relevance of an in-depth knowledge of these pathways for use in elaborating therapies that alter ROS levels ROS also induce mutations in mitochondrial DNA with the potential generation of a feedback loop in which mutations in genes encoding complexes of the ETC may directly affect the efficiency of electron transport The major sensitivity of mitochondrial DNA to ROS-induced mutagenesis is intuitive and mitochondria lack the nucleotide excision repair (NER) enzymatic system The main consequences of redox signaling and oxidative stress in normal and cancer cells are presented in Fig. 1. The major signaling cascades induced by growth factor-stimulated ROS are highlighted on the left The same pathways influence the cell cycle and affect the activity of transcription factors and genes that play roles in the cellular response to the hypoxic microenvironment ROS also induce lipid peroxidation with commensurate electron leakage in mitochondria and the release of Ca2+ from intracellular stores The main consequences of oxidative stress in cancer cells are illustrated on the right Moderately elevated ROS induce oncogenes and inhibit tumor suppressor genes that while the expression of genes involved in the formation of new blood vessels and in the establishment of a boosted antioxidant system is enhanced ROS also activate HDACs and have a dual effect on DNMTs with important outcomes for the expression of oncogenes and tumor suppressor genes Oxidized bases trigger mutations and engage DNA repair enzymes in response to death-inducing ligands (TNFα and Fas) enhance the assembly of DISCs and the activation of effector caspases and reduce Bcl-2 activity or as a consequence of increased permeability of mitochondrial PTPs stimulate the intracytoplasmic release of cytochrome c which interacts with Apaf-1 and procaspases and forms the apoptosome (apoptosis) ROS can also inhibit the negative regulators of autophagy (TORC1) and increase the formation of LC3-dependent autophagosomes (autophagy) induced by several receptor-interacting protein kinases (RIPs) which increases ROS levels via a mechanism that depends on intracellular iron (ferroptosis) the inhibition of DNA damage repair by PARP may sensitize cancer cells to the oxidative stress induced by platinum-containing drugs ROS generated during nuclear receptor-induced transcription of target genes by the activity of lysine demethylases on lysine 9 in histone H3 must be controlled to prevent their accumulation while phosphorylation of H3S10 inhibits the rapid remethylation of the same lysine If inhibitors of the H3S10 kinases are introduced as a Trojan horse together with nuclear receptor ligands The first approach is based on lowering ROS levels to counteract their role in cellular transformation; it is aimed at reducing the number of transformed cells by depriving them of fuel (represented in the upper right side of the figure as a lower proportion of transformed cells with respect to that of normal cells) The second approach is based on the consideration that cancer cells with an antioxidant system already triggered are more sensitive than their normal counterparts to further increases in ROS and are unable to achieve redox balance by inducing ROS under these metabolic conditions a high percentage of the cells undergo death (represented in the lower right side of the figure where transformed cells are depicted as apoptotic) in contrast to the mechanism of sister pathways redox signaling is based on migrating electrons the signaling in this pathway is much more diffuse although more studies are required to increase the selectivity of these anticancer ROS-related drugs the common mechanisms elicited by oncogenes to promote the adaptation to a large set of stress conditions are being revealed in more depth every day we expect that targeting ROS will represent fruitful ground for future molecular anticancer strategies An overview of mechanisms of redox signaling The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin Decreased complex III activity in mitochondria isolated from rat heart subjected to ischemia and reperfusion: role of reactive oxygen species and cardiolipin metals and antioxidants in oxidative stress-induced cancer Exploring oxidative modifications of tyrosine: an update on mechanisms of formation advances in analysis and biological consequences Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1 Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo Reactive oxygen species promote TNF alpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases Protein kinase B activation by reactive oxygen species is independent on tyrosine kinase receptor phosphorylation and required Src activity Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors Latimer, H. 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cancer cells by targeting GAPDH NRF2 activation by antioxidant antidiabetic agents accelerates tumor metastasis Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiation resistance Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis BACH1 stabilization by antioxidants stimulates lung cancer metastasis Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer ROS homeostasis and metabolism: a dangerous liaison in cancer cells ROS homeostasis and metabolism: a critical liaison for cancer therapy Molecular effectors of multiple cell death pathways initiated by photodynamic therapy Download references This study was supported by Italian Ministry of University and Scientific Research [P.R.I.N 2017EKMFTN_002 to G.C.]; P.O.R-Regione Calabria [Progetto “Razionale” to A.M.] Giovanni Galasso is supported by a fellowship of P.R.I.N Pia Giovannelli is supported by VALERE (Vanvitelli per la Ricerca) Program Marzia Di Donato is supported by a fellowship of ‘Fondazione Umberto Veronesi’ [FUV Postdoctoral fellowship-2019] Istituto per l’Endocrinologia e l’Oncologia Sperimentale Gabriella Castoria & Antimo Migliaccio Dipartimento di Medicina Molecolare e Biotecnologie Mediche Reprints and permissions Download citation DOI: https://doi.org/10.1038/s12276-020-0384-2 Highly reactive molecules called reactive oxygen species (ROS) which at low levels are natural regulators of important signaling pathways in cells might be recruited to act as “Trojan horses” to kill cancer cells Researchers in Italy led by Bruno Perillo of the Institute of Food Sciences in Avelllino review the growing evidence suggesting that stimulating production of natural ROS species could become useful in treating cancer Although ROS production is elevated in cancer cells it can also promote a natural process called programmed cell death but could be selectively activated to target diseased cells The authors discuss molecular mechanisms underlying the potential anti-cancer activity of various ROS-producing strategies including drugs and light-stimulated therapies They expect modifying the production of ROS to have potential for developing new treatments Metrics details Epithelial–mesenchymal transition (EMT) is a crucial step in tumor progression and the TGFβ–SMAD signaling pathway as an inductor of EMT in many tumor types is well recognized the role of non-canonical TGFβ–TAK1 signaling in EMT remains unclear we show that TAK1 deficiency drives metastatic skin squamous cell carcinoma earlier into EMT that is conditional on the elevated cellular ROS level The expression of TAK1 is consistently reduced in invasive squamous cell carcinoma biopsies Tumors derived from TAK1-deficient cells also exhibited pronounced invasive morphology TAK1-deficient cancer cells adopt a more mesenchymal morphology characterized by higher number of focal adhesions increase surface expression of integrin α5β1 and active Rac1 these mutant cells exert an increased cell traction force an early cellular response during TGFβ1-induced EMT transcription factors associated with mesenchymal phenotype is also upregulated in TAK1-deficient cancer cells compared with control cancer cells We further show that TAK1 modulates Rac1 and RhoA GTPases activities via a redox-dependent downregulation of RhoA by Rac1 which involves the oxidative modification of low-molecular weight protein tyrosine phosphatase the treatment of TAK1-deficient cancer cells with Y27632 a selective inhibitor of Rho-associated protein kinase and antioxidant N-acetylcysteine augment and hinders EMT Our findings suggest that a dysregulated balance in the activation of TGFβ–TAK1 and TGFβ–SMAD pathways is pivotal for TGFβ1-induced EMT TAK1 deficiency in metastatic cancer cells increases integrin:Rac-induced ROS which negatively regulated Rho by LMW-PTP to accelerate EMT the various stages consist of regulatory networks with many opportunities for cancer therapy The potential for new therapeutics avenues and their clinical impact to reduce cancer mortality rate underscores the immense importance to identify novel target modulators of these regulatory networks during metastasis whereby actin stress fibers are formed in conjunction with an increased establishment of integrin-containing focal contacts effectively enhancing cell attachment to the extracellular matrix Cancer cells undergoing EMT are also enriched with mesenchymal type intermediate filaments (i.e The expression of mesenchymal features and suppression of epithelial traits are induced by early EMT-associated transcriptional factors such as SNAIL and ZEB1 Studies suggested that this mesenchymal transformation enhances the ability of cancer cells to intravasate and disseminate the identification of mechanistic drivers of EMT would provide crucial insights and therapeutic targets to halt cancer spreading the effect of ROS on EMT is not well understood we investigated the role of TAK1 in TGFβ-induced EMT of tumor cells We found that TAK1 deficiency in tumor cells accelerated EMT in ROS-dependent manner thus providing a previously unexplored mechanistic regulation of EMT by TAK1 TAK1 deficiency promotes TGFβ1 induced EMT in A5RT3 cells (a and b) Relative mRNA (a) and protein (b) expression of TAK1 in human squamous cell carcinomas (SCC) biopsies and their cognate perilesional normal skin (PNS) Biopsies #1–9 and #10–15 are benign and invasive grades SCC samples Biopsies #5–7 and #10–14 were used for western blot analysis data points from the same individual are linked by colored lines (c) Immunofluorescence staining of laminin-332 (green) in cryosections of A5RT3CTRL- and A5RT3TAK1-derived tumors Nuclei were counterstained with DAPI (blue) (d) Expression of invasive markers laminin-332 and MMP-9 in A5RT3CTRL- and A5RT3TAK1-derived tumors as determined by immunoblot and gelatin zymography Values below each band represent the mean fold differences (n=3) in expression level when compared with A5RT3CTRL-derived tumor (e) Phase-contrast microscopy images of A5RT3CTRL and A5RT3TAK1 cell colonies 48 h after TGFβ1 treatment (f) Immunostaining of E-cadherin and N-cadherin (green) in A5RT3CTRL and A5RT3TAK1 cells under the indicated treatment (g) qPCR analysis of EMT markers in A5RT3CTRL and A5RT3TAK1 24 h after TGFβ1 treatment Samples were normalized with reference gene (h) Representative western blots of EMT markers in A5RT3CTRL and A5RT3TAK1 48 h after TGFβ1 treatment The densitometry values as indicated below respective lanes were normalized with respect to the control all samples were normalized with housekeeping gene L27 gene The TAK1 mRNA sequence transcribed from pCDNA–TAK1 contains silent mutations that are not targeted by the siRNA produced in A5RT3TAK1 TGFβ1-induced EMT is effected through SMAD signaling (a) Phase-contrast images of SMAD3 siRNA-transfected A5RT3TAK1 and respective control cells with 48 h of TGFβ1 (10 ng/ml) treatment (b) qPCR analysis of EMT markers in SMAD3 siRNA-transfected A5RT3TAK1 cells with and without TGFβ1 treatment (c) Immunoblot analysis of SMAD3 siRNA-transfected A5RT3TAK1 cells for their knockdown and EMT markers upon TGFβ1 treatment Representative blots were shown with respective densitometry values indicated below lanes Expression of surface integrin in A5RT3CTRL and A5RT3TAK1 (a and b) FACS analysis of A5RT3CTRL and A5RT3TAK1 cells immunostained with antibodies against indicated surface integrin β subunits (a) and with integrin α5β1 (b) The distribution of tumor cells with regard to their extent of integrin staining was presented in histogram plots with the identity of the stained integrin subunit indicated (top left) Cells stained with only control IgG served as the negative controls Each image is representative of at least three different experiments Values shown indicate mean fluorescence intensity (c) PLA assay of α5β1 integrin expression (upper panel) and active integrin β1:active Rac1 (lower panel) n A5RT3CTRL and A5RT3TAK1 cells Representative images were shown with mean number of PLA spots per nucleus±S.D TAK1 deficiency increases cell traction force in A-5RT3 facilitating EMT (a) Vinculin immunostaining (green) and phalloidin F-actin (red) staining were conducted of A5RT3CTRL and A5RT3TAK1 cell colonies with and without TGFβ1 treatment for 48 h with representative images shown (b and c) Cell traction force profiling (b) and mean measured cell traction stress (c) of A5RT3CTRL and A5RT3TAK1 with and without TGFβ1 (10 ng/ml) treatment for 24 h Color scale bar denotes traction stress (kPa) Values (mean+S.D.) of three independent measurements (d) A5RT3CTRL and A5RT3TAK1 cells were seeded on a transwell membrane of pore size and evaluated for their migration through the pore with and without TGFβ1 treatment for 48 h Migrated cells were stained with crystal violet and destained with a fixed volume of 0.5% Triton-X solution Absorbance at 595 nm of the solutions was measured to quantify transwell migration Graph displays mean absorbance values±S.D.; n=3 Role of elevated ROS during TGFβ1-induced EMT in A5RT3TAK1 (a) PLA assay of active Rac1 and Nox1 in A5RT3CTRL and A5RT3TAK1 cell (b) A5RT3CTRL and A5RT3TAK1 with or without 48 h of TGFβ1 (10 ng/ml) treatment were stained with DCF and analyzed with flow cytometry Antioxidant NAC-treated cells (100 μM) served as a negative control Image shown is representative of three different experiments (c) Phase-contrast images showing DCF staining of A5RT3CTRL and A5RT3TAK1 after 48 h of TGFβ1 (10 ng/ml) treatment (d) Phase-contrast images A5RT3TAK1 cells subjected to indicated treatements (e) qPCR analysis of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment (f) Representative blots of EMT markers in TGFβ1 induced A5RT3TAK1 with NAC treatment were shown with densitometry values indicated below respective lanes Samples were normalized with tubulin as a loading control Redox regulation of RhoA by Rac1 facilitates TGFβ1 induced EMT (a) RhoA and Rac1 activities and LMW-PTP oxidation were compared with immunoblotting as respectively indicated 10 ng/ml TGFβ1 and 100 μM of antioxidant NAC were used respectively for treatment duration of 48 h (b) FACS analysis of tumor cells transfected with or without constitutive-active (G12V) and dominant-negative Rac1 (T17N) (c) Representative images of E-cadherin (green/488 nm) immunostaining of tumor cells with respectively indicated treatments and transfection (d) Phase-contrast images of A5RT3TAK1 cells with and without TGFβ1 induction were inhibited with Y27632 (10 μM) for 24 h or transfected with siRNA for RhoA Representative phase-contrast images of treated cells (e) Representative blots of EMT markers in TGFβ1-induced A5RT3CTRL and A5RT3TAK1 treated with ROCK Y27632 or transfected with pooled siRNA for RhoA Values shown with densitometry values indicated below respective lanes our observations showed that TAK1 deficiency increased integrin:Rac1-induced ROS which negatively regulated RhoA by LMW-PTP to accelerate EMT Proposed mechanism by which TAK1 inhibits TGFβ1-induced EMT TAK1 likely counters the well-established TGFβ1-SMAD3 induction of EMT with the suppression of integrin expression and hence Rac1 activation Rac activity otherwise promotes ROS production via the recruitment of Nox1 which oxidizes LMW-PTP and correspondingly inhibit RhoA activity a better understanding the role of TGFβ–TAK and TGFβ–SMAD signalings in CAFs and their influence on epithelial cell behaviors requires our future attention we showed that TAK1 deficiency increases CTF by a ROS-dependent manner to drive EMT in metastatic A5RT3 cells Antibodies were purchased against integrin α5β1 (Millipore Nox1 and secondary HRP-conjugated antibodies (Santa Cruz Biotechnology Transfection reagent ExGen 500 was purchased from Fermentas Double-promoter lentivirus-based siRNA vector and pFIV packaging kit were acquired from System Biosciences (Mountain View Pooled siRNA and transfection reagent were purchased from Dharmacon (Pittsburgh All restriction enzymes and DNA/RNA-modifying enzymes were obtained from Fermentas TGFβ1 and IL-1 were purchased from Peprotech (Rehovot chemicals were purchased from Sigma-Aldrich (St Cells or tissue sections were fixed with 4% paraformaldehyde for 15 min blocked and permeabilized in 3% BSA with 0.05% Triton-X for 1 h Cells or tissue sections were incubated with either anti-vinculin (1 : 100) or anti-N-cadherin (1 : 25) or in anti-laminin-332 (1 : 50) in 0.3% BSA overnight at 4 °C Secondary goat anti-rabbit Alexa Fluor 488 antibodies (1 : 200) in 0.2% BSA were added with incubation for 1 h at room temperature in the dark Cells were then incubated with 1 : 50 dilution of Alexa Fluor 594 phalloidin (Molecular Probes USA) in 2% BSA for 2 h and counterstained with DAPI (Vectashield Immunofluorescent and phase-contrast images were taken using Nikon 80I Eclipse microscope equipped with a Nikon DS-SI1 camera using a (20x/0.5) DIC objective and the NIS element D3.0 software with constant exposure and gain Respective PLA assays were carried out as per manufacturer protocol (Olink Bioscience Images were taken using Carl Zeiss (Thornwood USA) confocal microscope LSM 710meta using a Plan-Apochromat × 63/1.4 oil DIC objective and ZEN 2009 LE software with constant exposure and gain The resulting fluorescent spots were quantified using Duolink ImageTool (Olink Bioscience) Cells were seeded and grown on 75 cm2 culture flask (Corning Cells were then washed with PBS and incubated with 25 mM EDTA/PBS for 20 min EDTA suspended cells were then spun down at 250 × g and fixed with resuspension in 4% paraformaldehyde for 20 min at room temperature and washed with PBS twice through resuspension Washed cells were then treated with anti-β1 respectively at concentration (1 : 25) in 10% BSA and incubated at room temperature for 1 h Cells were washed twice with PBS resuspension before incubation in secondary antibody conjugated with Alexa Fluor 488 at concentration (1 : 100) in 10% BSA at room temperature in the dark for 1 h Stained cells were analyzed with Becton Dickinson (BD Analysis of flow cytometry results were performed with Flowjo software (Ashland cells were incubated with 10 μM CM-H2DCFDA (Invitrogen USA) for 30 min at 37 °C prior to trypsinization and analysis by flow cytometry (LSRII from BD Biosciences) Results were analyzed by the Flowjo software ROS was quenched with the addition of N-acetyl-L-cysteine (NAC) to a final concentration of 100 μM CM-H2DCFDA-labeled cells were also imaged with the Nikon 80I Eclipse microscope equipped with a Nikon DS-SI1 camera using a ( × 20/0.5) DIC objective and the NIS element D3.0 software with constant exposure and gain The first band in each blot was given an arbitrary density of 1 and relative densities of the remaining bands were determined Densitometry results of all individual bands are divided by those from tubulin and the ratio is presented as values below individual lanes Human embryonic kidney (HEK293-T) and human skin squamous cell carcinoma (A5RT3) cells were routinely maintained in DMEM and FBS (Hyclone Transfection of A5RT3CTRL and A5RT3TAK1 cells was carried out as per manufacturer’s protocol (Fugene USA) with either plasmids expressing constitutive-active Rac1 G12V or dominant-negative Rac T17N cells were cultured on PDMS substrate embedded with fluorescent beads glass coverslips were cleaned by use of oxygen plasma before it was placed in a vacuum desiccator containing a small drop of 1H,1H,2H,2H-perfluorooctyltrichlorosilane for 30 min the surface-treated coverslips were removed and 100 μl of diluted yellow–green FluoSpheres microspheres (0.2 μm Molecular Probes) was added to the coverslips with the treated surface facing upwards This step forms a monolayer of fluorescent beads on the coverslips Thin film of PDMS were generated by placing 15 μl of PDMS mixture (before curing 1 : 60 w/w of cross-linker to base ratio) on the fluorescent microspheres coated glass coverslips One layer of Scotch tapes was applied as a spacer before putting the other bare glass coverslip on the top of fluorescent microspheres coated glass coverslips The PDMS droplet was flattened under the weight of the upper coverslip The entire assembly was left undisturbed for 5 min and then placed in 37 °C incubator overnight before carefully peeling off the upper bonded PDMS thin film with fluorescent microspheres on its top Only samples with well-dispersed beads were used in the study As PDMS surface does not allow cells to adhere the substrate was coated by incubation with human plasma derived fibronectin solution (50 μg/ml in 0.02 M acetic acid for thin coating) overnight to promote cell attachment and stored in the dark at 4 °C prior to culture 1 × 103 cells/inserts were seeded for 48 h under cell culture conditions Cells were subsequently treated with 10 ng/ml TGFβ1 for 24 h before traction force measurement The cell traction mapping was based on the DIC image of cells on PA substrate and the corresponding traction stress mapping in pseudocolor which indicated regions of traction stresses (dark blue to light pink according to stresses from low to high) yi) was calculated based on the following formulation: where denoted the Greens’ tensor and denoted the experimental displacements of fluorescent beads at position (xi The overall force of the cell (F) is an integral of the traction field magnitude over the area where T(x,y)=[Tx(x,y)+Ty(x,y)] is the continuous field of traction vectors defined at any spatial position (x,y) within the cell 5 × 104 cells were dispensed into the top chamber of transwell migration chambers (8 μm Millicell USA; Hanging Cell Culture Inserts) loaded on a 6-well plate and allowed to attach overnight in cell culturing conditions One thousand microliters of complete medium (supplemented with 10 ng/ml TGF-β1) was added to each of the underlying wells This construct was then incubated for 48 h the inserts were washed with PBS and fixed with 1% glutaraldehyde for 10 min The inserts were rinsed with PBS and subsequently stained in 0.1% crystal violet for 25 min Cells were thoroughly rinsed with PBS after crystal violet staining Cotton buds were used to swipe the upper surface of the transwell membrane ensuring thorough removal of unmigrated cells The inserts were then immersed in a 500 μl of 0.5% Triton-X solution dispensed in a clean 6-well plate and incubated overnight in the dark The 6-well plate was sealed with parafilm tape to prevent evaporation Absorbance of the 500 μl volume of 0.5% Triton-X at 595 nm was then measured using Nanodrop The absorbance difference for the test and control wells were used as an index for the comparison of cellular migration The basics of epithelial-mesenchymal transition TGFbeta signalling: a complex web in cancer progression TGF-beta and epithelial-to-mesenchymal transitions 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Fibroblasts and Their Matrix Know thy neighbor: stromal cells can contribute oncogenic signals TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia Loss of TGF-beta type II receptor in fibroblasts promotes mammary carcinoma growth and invasion through upregulation of TGF-alpha- Angiopoietin-like 4 protein elevates the prosurvival intracellular O2(-):H2O2 ratio and confers anoikis resistance to tumors A simple method to systematically study oxidatively modified proteins in biological samples and its applications Stresses at the cell-to-substrate interface during locomotion of fibroblasts Download references This work is supported by research grant from Ministry of Education (MOE 2010-T2-2-009) to NST A5RT3 cell line was kindly provided by Dr Petra Boukamp of German Cancer Research Center DKFZ School of Materials Science and Engineering Supplementary Information accompanies this paper on Cell Death and Disease website This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Download citation DOI: https://doi.org/10.1038/cddis.2013.339 Metrics details 2′,2′-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma The purpose of our work was to improve GEM activity by addition of cannabinoids we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments The antiproliferative synergism is prevented by the radical scavenger N-acetyl-L-cysteine and by the specific NF-κB inhibitor BAY 11-7085 demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells we have investigated the effect of the combination between GEM and three different CB ligands arachidonoyl cyclopropamide (ACPA) and SR141716 (SR1) for CB1 and GW405833 (GW) for CB2 on pancreatic adenocarcinoma cell growth Our results show that GEM induces both CB1 and CB2 receptors by an NF-κB-dependent mechanism we demonstrate that all the three cannabinoids determine ROS production and that these effects are potentiated by GEM GEM/cannabinoid treatment produces a strong synergistic inhibition of pancreatic cancer cell growth in vitro and significantly enhances the antitumoral effect of GEM in vivo The same treatment is ineffective on normal fibroblasts and does not determine apparent toxicity in nude mice these data strongly support the development of GEM/cannabinoid treatment in the management of pancreatic cancer or SR1 on growth of pancreatic adenocarcinoma cell lines and normal fibroblasts (a) Cells were seeded in 96-well plates and incubated overnight The compounds were added at the concentrations of 200 nM GEM and 90 μM ACPA; cells were incubated for additional 48 h Values are reported as percentage of treated versus untreated cells and are the means of triplicate samples from three independent experiments (±S.D.) each cannabinoid versus its combination with GEM in Panc1 cells no significance between the various treatments in fibroblasts (b) Panc1 cells were seeded in 96-well plates and incubated overnight The compounds were added at the following concentrations for 24 h: 25 nM GEM The growth ratio on the y axis was obtained by dividing the absorbance of untreated or treated cell lines by the mean absorbance of each cell line measured at time 0 Values are the means of triplicate samples from three independent experiments (±S.D.) The statistical analysis was performed for each combined treatment versus control (c) Antiproliferative synergism by GEM/cannabinoids The percentage values were obtained by analyzing CI/effect curves % CI<1 in all cancer cell lines versus normal fibroblasts GEM/cannabinoid combinations did not determine any synergism in normal fibroblasts Effect of GEM and/or cannabinoids on intracellular ROS production Panc1 cells were treated with increasing concentrations of the compounds for 4 h at constant dose ratios corresponding to the level of ROS production Values are the means of triplicate samples from three independent experiments The statistical analysis was performed for each combined treatment versus single treatments Role of NF-κB in CB1 and CB2 activation by GEM and in the antiproliferative synergism by GEM/cannabinoids (a) Panc1 cells were seeded in 60-mm plates and incubated overnight Cells were pretreated with 5 μg/ml ActD for 1 h Total RNA extraction and real-time PCR were performed Values are the means of triplicate samples from four independent experiments (±S.D.) (b) qPCR analysis of CB1 and CB2 mRNAs from cells treated with 2 μM GEM or 10 μg/ml IL-1 control versus GEM or GEM+NAC and P<0.001 IL-1 versus IL-1+MG132 (for both CB1 and CB2) (c) Analysis of the antiproliferative synergism by 2 μM GEM and 40 μM GW or 40 μM SR1 in the absence or presence of 100 nM MG132 or 1 μM BAY Values are the means of three independent experiments (±S.D.) Statistical analysis for total synergism (0.3<CI<1): P<0.001 GEM+SR1 versus GEM+SR1+MG or GEM+SR1+BAY; P<0.05 Statistical analysis for high synergism (CI<0.3): P<0.001 GEM+GW versus GEM+GW+MG or GEM+GW+BAY; P<0.001 Effect of GEM and/or cannabinoids on ER stress-related gene expression Panc1 cells were treated with 500 nM GEM and/or 40 μM GW RT-PCR for XBP-1(S) and qPCR for Grp78 and CHOP were performed Densitometric analysis of XBP-1(S) was normalized to β-actin and performed GEM versus each cannabinoid and each cannabinoid versus its combination with GEM (for all the three genes) (a) Panc1 cells were treated with 200 nM GEM and/or 16 μM GW or 16 μM SR1 for 48 h and analyzed by flow cytometry to determine the percentages of apoptotic cells control versus each combination; P<0.01 no significance between control and each cannabinoid (b) Cells were treated with 200 nM GEM and/or 16 μM GW Cell cycle distribution was analyzed by a flow cytometer after DNA staining with PI Statistical analysis: no significance of GEM versus each combination (c) Western blot analysis of LC3 was performed using total protein extracts from Panc1 cells treated with 500nM GEM and/or 40 μM GW or 40 μM SR1 for 24 h in the presence of acid lysosomal protease inhibitors E64d (10 μM) and pepstatin A (10 μg/ml) Densitometric quantification of LC3-II bands was normalized to α-tubulin and performed or SR1 also were able to induce autophagy and whether GEM could additionally enhance this effect Involvement of ROS in GEM/cannabinoid-induced autophagy (a) Fluorescence microscopy analysis of autophagosome formation in Panc1 cells after acridine orange staining treated with 500 nM GEM and/or 40 μM GW or 40 μM SR1 in the absence or presence of 20 mM NAC or 10 μM CQ or 1 mM 3-MA for 24 h at FACS after trypsinization of the acridine orange-labeled cells each cannabinoid or GEM versus their combination; and P<0.001 GEM/cannabinoids versus GEM/cannabinoids+NAC (c) Flow cytometric analyses of autophagosomes formation (MDC incorporation) in Panc1 cells treated with 500 nM GEM and/or 40 μM GW and ACPA induced a similar increase in AVO formation which was more prominent in GW-treated cells This effect was strongly potentiated by the GEM/cannabinoid combinations and antagonized by the autophagy inhibitors CQ or 3-MA or the scavenger NAC Involvement of ROS and autophagy in the antiproliferative synergism by GEM/cannabinoids and kinetic analysis of ROS (a) Analysis of the antiproliferative synergism by 500 nM GEM and 40 μM GW or 40 μM SR1 in the absence or presence of 20 mM NAC or 10 μM CQ or 2.5 mM 3-MA (b) Panc1 cells were treated with 500 nM GEM and 40 μM GW or 40 μM SR1 for the indicated time points Values are the means of three independent experiments (c) Schematic representation of the kinetic analysis of oxidative stress (ROS) and autophagy (LC3-II) marker induction by GEM/cannabinoids Effect of GEM+SR1 treatment on xenografts of PaCa44 cells in nude mice Cells were subcutaneously injected into female nude mice or/and SR1 were administered twice a week for 4 weeks (a) Values are the means of mice tumor volume measured at 3 days after each injection (b) Values are the means of mice body mass measured at 3 days after each injection (c) Values are the means of mice tumor mass (±S.D.) measured after 8 injections we report that GEM-resistant cell lines showed a significantly higher synergism (CI<0.3) of cell growth inhibition by GEM/cannabinoids compared with GEM-sensitive cell lines and that the synergism was dependent on the increase in intracellular ROS induced by the combinations This mechanism is supported by the observation that the radical scavenger NAC totally inhibited the synergistic antiproliferative effect induced by GEM/cannabinoids These findings strongly support the idea that the increase in ROS production may be a good strategy to overcome GEM resistance in the therapeutic management of pancreatic cancer Both CB1 and CB2 gene induction by GEM was totally prevented by these inhibitors Similar results were also obtained using TNF-α (data not shown) As NF-κB induction is described to be mediated by oxidative stress we analyzed CB gene induction by GEM in the presence of NAC Our data show that NAC failed to prevent CB induction by GEM indicating that GEM induces CB gene expression by a ROS-independent mechanism The molecular mechanism that is the basis of NF-κB induction by GEM is still unknown and its clarification needs additional investigations we show that all the three ER stress-related genes induced by cannabinoids supporting their involvement in GEM/cannabinoid-mediated antiproliferative synergism and autophagic cell death were sequential events in our experimental conditions or other apparent toxicity-related features the results presented here provide the first evidence that the GEM/cannabinoid combinations exerted a strong synergistic antiproliferative effect on pancreatic adenocarcinoma GEM-resistant cell lines by ROS-dependent mechanisms whereas it is scarcely toxic toward normal cells in vivo studies strongly boost the addition of cannabinoids to GEM in designing new therapeutic strategies for pancreatic cancer treatment GEM (Gemzar) was provided by Eli Lilly (Florence SR141716 (N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-pyrazole-3-carboxamide); SR1 (rimonabant; Acomplia Italy); ACPA was obtained from Cayman Chemicals (Inalco Italy); and GW405833 hydrochloride (1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-(1-morpholine)ethyl)-5-methoxyindole; GW) from Sigma (Milan chloroquine diphosphate (CQ; N4-(7-chloro-4-quinolinyl)-N1,N1-dimethyl-1,4-pentanediamine) MG132 and BAY 11-7085 (2E)-3-94-(1,1-dimethylethyl)-phenyl-sulfonyl-2-propenenitrile) (BAY) were obtained from Enzo Life Sciences (VinciBiochem and the recombinant human IL-l from PeproTech (Inalco and 50 μg/ml gentamicin sulfate (BioWhittaker Italy) were grown in DMEM supplemented with 2 mM glutamine Cells were plated in 96-well cell culture plates (5 × 103 cells per well) and treated with various compounds cell proliferation was evaluated by Crystal Violet (Sigma) staining The dye was solubilized in 1% SDS in PBS and measured photometrically (A595 nm) to determine cell viability Three or four independent experiments were performed for each assay condition with IC50 mean values corresponding to 18 μM for SR1 and GW and 100 μM for ACPA By inverting the molar ratios of the compounds between the two groups of cell lines no significant alteration of the results was observed (data not shown) indicating that the data obtained were not influenced by the specific experimental condition tested The non-fluorescent diacetylated 2′,7′-dichlorofluorescein (DCF-DA) probe (Sigma) which becomes highly fluorescent upon oxidation was used to evaluate intracellular ROS production cells were plated in 96-well plates (5 × 103 cells per well) and cells were incubated with 10 μM DCF-DA for 15 min at 37°C and the DCF fluorescence was measured by using a multimode plate reader (Ex 485 nm and Em 535 nm) Three independent experiments were performed for each assay condition and reported as fold induction relative to controls 2.5 × 105 Panc1 cells were seeded in 60-mm plates and incubated overnight The percentage of apoptotic cells was evaluated by staining 3 × 105 cells with annexin V-FITC (Bender Med System Italy) and 5 μg/ml PI in binding buffer (10 mM HEPES/NaOH (pH 7.4) and 2.5 mM CaCl2) for 10 min at room temperature in the dark The samples were analyzed by flow cytometry (FACScalibur USA) to determine the percentage of cells displaying annexin V+/PI− (early apoptosis) or annexin V+/PI+ staining (late apoptosis) we report the percentage values corresponding to the addition of early and late apoptosis Cell cycle distribution was analyzed by staining 3 × 105 cells with PI incubated with 0.1% sodium citrate dihydrate and analyzed using a flow cytometer (FACScalibur The percentage of cells in the various stages of the cell cycle was determined using the ModFitLT software (Verity Software House cells were incubated with 50 μM MDC in PBS at 37°C for 15 min and immediately analyzed by flow cytometry All fluorescences were analyzed with a FACScalibur flow cytometer (Becton Dickinson) The fluorescent emissions were collected through a 530 nm band pass filter (FL1 channel) At least 10 000 events were acquired in log mode CellQuest software (Becton Dickinson) was used to calculate mean fluorescence intensities (MFIs) The MFIs were calculated by the formula (MFItreated/MFIcontrol) where MFItreated is the fluorescence intensity of cells treated with the various compounds and MFIcontrol is the fluorescence intensity of untreated and unstained cells Values reported in the figures are the means±S.D.s from three independent experiments the cytoplasm and nucleus are bright green and dim red whereas acidic compartments are bright red The intensity of the red fluorescence is proportional to the degree of acidity cells were incubated with acridine orange solution (1 μg/ml) at 37°C; 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Joint Project of University of Verona SR141716 (Rimonabant) was provided by Sanofi-Aventis (grant to M Bifulco Federica Cioffi (University of Verona) for FACS analysis support Giorgio Malpeli (University of Verona) for qPCR technical support I Dando and T Zaniboni: These authors equally contributed to this work Department of Life and Reproduction Sciences Interdepartmental Laboratory for Medical Research Department of Pharmaceutical and Biomedical Sciences Supplementary Information accompanies the paper on Cell Death and Disease website This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License Download citation DOI: https://doi.org/10.1038/cddis.2011.36 Naunyn-Schmiedeberg's Archives of Pharmacology (2024) Journal of Analytical Science and Technology (2023) Drug Delivery and Translational Research (2022)