Curitiba Back to topAttractionsMust-see attractionsMuseu Oscar Niemeyer Designed by and named for the architect responsible for much of Brasília this striking museum features an iconic eye-shaped tower painted with whimsical… Catedral Basílica de Curitiba Inaugurated in 1893 and completely restored in 2012 Curitiba's neo-Gothic main cathedral – inspired by Barcelona's metropolitan cathedral – isn't one of… MusA-UFPR Inside the neoclassical headquarters of the Federal University of Paraná the university's museum features rotating exhibitions along with a small and… Palácio Belvedere this modest art nouveau construction was originally designed to be a city lookout It was left to die and gutted by fie in 2017 Largo da Ordem the pedestrian-only cobblestone streets are lined with beautifully restored buildings Paço da Liberdade Curitiba's historic Old City Hall was inaugurated in 1926 and designed by then-mayor Cândido Ferreira de Abreu who was responsible for many of the city's… Jardim Botânico studded with sculpture and crisscrossed by walking paths Teatro Guaíra This premier state-run theater in Curitiba is home to the Paraná Symphony Orchestra (Orquestra Sinfônica do Paraná) This southern-Brazilian city has cutting-edge transit lush gardens and memorable buildings that make an impression on any visitor Get to the heart of Curitiba with one of our in-depth Visit in ShopBrazil $28.99 Visit in ShopRio de Janeiro $21.99 Go to checkout (0 items)in partnership with getyourguide No part of this site may be reproduced without our written permission Heitor Borges is 2 years old and his best friend is a rooster named Tatá in the state of Santa Catarina in southern Brazil This little boy is delighting the internet with his adventures through an Instagram account called “country baby” (“bebê campeiro”) View on Instagram One of the posts shared by the little boy’s mother, Karoline, shows Heitor on his knees in front of a little shrine where a small statue of the Sacred Heart of Jesus and another of Our Lady of Aparecida (the patroness of Brazil) are on display Heitor’s mother wishes us all a "great and blessed week,” while the page's followers respond with phrases like these: God protect him from all the evil in this world." a little angel praying to the Mother of Heaven." View on Instagram Another video shared on the profile shows us the family visiting the monumental statue of Jesus in Cândido de Abreu (with Heitor carrying his rooster View on Instagram we couldn't leave out stopping by the Hill of Christ," the caption reads seeing the "country baby" playing innocently with the animals on the farm (and even kissing them) seems to inspire many of the page's followers to think of spiritual realities One of the many religious comments recalls the prayer attributed to St be enchanted by the innocence of little Heitor: View on Instagram Get Aleteia delivered to your inbox. It’s free! Articles like these are sponsored free for every Catholic through the support of generous readers just like you. Please make a tax-deductible donation today! Help us continue to bring the Gospel to people everywhere through uplifting Catholic news, stories, spirituality, and more. Volume 11 - 2021 | https://doi.org/10.3389/fonc.2021.686445 In approximately 15% of patients with acute myeloid leukemia (AML) total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34+ samples it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL) therapy-associated resistance is observed in 5-10% of the cases pointing to a clear need for new therapeutic strategies for those patients the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients the expression of the EGF was lower in APL plasma samples than in healthy controls The EGFR ligand AREG was detected in 29% of APL patients at diagnosis treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model while the gain in survival did not reach statistical difference compared to treatment with ATRA alone Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients the use of EGFR inhibitors in combination with standard therapy was not previously explored in APL cells resistant to ATRA and ATO Non-small cell lung cancer (NSCLC) demonstrated constitutive activation of the epidermal growth factor (EGF)/EGFR pathway, due to mutations on the EGFR (8). Although EGFR mutations are rare in AML (9–11), the level of EGF—the main EGFR ligand—was elevated in the urine of patients diagnosed with APL and decreased after ATRA-induced complete remission (12) it is conceivable that the activation of the EGF/EGFR signaling pathway could also confer APL leukemic cells with a survival advantage the prevalence and clinical significance of EGFR and its interactors in APL patients remains unknown we evaluated the effects of EGFR pharmacological inhibition in distinct APL models Gefitinib monotherapy induced apoptosis and inhibited the proliferation of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) APL cells the combination between gefitinib with ATRA and ATO rewired NB4-R2 and NB4 ATOr (ATO-resistant) cells into sensitivity to standard therapy for APL APL mice treated with ATRA alone or in combination with gefitinib exhibited increased overall survival in comparison with the vehicle-treated group Gefitinib (#S1025) and erlotinib (#S7786) were purchased from Selleck Chemicals (Houston ATRA and ATO were purchased from Sigma-Aldrich (St and ATRA were dissolved in dimethyl sulfoxide (DMSO) All compounds were stored at −20°C The human APL cell lines NB4 (ATRA-sensitive) and NB4 clone 21 (parental line of NB4 ATOr) were cultured in Roswell Park Memorial Institute 1640 medium (Gibco USA) and 10% of fetal bovine serum (FBS; Vitrocell Brazil) at 37°C in a humidified atmosphere of 5% CO2 Cell lines were tested and authenticated by STR DNA fingerprinting analysis (Laboratory of Biochemical Genetics Medical School of Ribeirao Preto – University of Sao Paulo) and plasma samples were collected from BM aspirates Mononuclear cells were isolated by Ficoll density gradient centrifugation (Histopaque-1077; Sigma-Aldrich) CD34+ cells were isolated from the BM of healthy volunteers using the CD34 Microbead Kit (#130-046-703; Miltenyi Biotec USA) according to the manufacturer’s instructions Plasma was obtained by centrifugation (500 g for 10 minutes) of heparinized BM aspirate and stored in aliquots at −80°C until use BM CD34+ cells or plasma samples from healthy donors were used as controls The study was approved by the local Research Ethics Committee of the Medical School of Ribeirao Preto Brazil (Reference: CAAE 05060818.9.0000.5440) All human samples were collected after obtaining written informed consent from patients according to the recommendations of the Declaration of Helsinki cells were washed with phosphate-buffered saline (PBS); 4 mL cold 70% ethanol was then added dropwise to the cell pellet while vortexing followed by storage at −20°C for up to 15 days before staining The cells were resuspended and washed with staining buffer (PBS with 1% FBS and 0.09% NaN3) and 100 μL of cell suspension (1 × 107/ml) was transferred to a tube containing 5 μL of Ki-67-PE antibody (#12-5698-82 USA) or PE-conjugated IgG1as an isotype control and analyzed by flow cytometry on a FACSCalibur instrument (Becton Dickinson USA) and analyzed with FlowJo software (Treestar A minimum of 10 000 events was acquired for each sample Positivity is expressed as a percentage of positive cells and mean fluorescence intensity (MFI) and NB4 clone 21 cells were collected 72 h after drug treatment and resuspended in 100 μL PBS and incubated with CD11b-PE (#347557 or the spleen of leukemia model mice were labeled with antibodies against CD11b-PE (#553311 then collected and washed and resuspended in PBS The percentage of positive cells and MFI were determined by flow cytometry Whole-cell lysates were prepared with extraction buffer (10 mM EDTA and 1% Triton X-100) followed by centrifugation at 10 000 × g for 20 min at 4°C Protein concentration was determined with the Bradford assay and 50 μg of lysate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel The proteins were transferred to a polyvinylidene difluoride membrane (Amersham Hybond-P; GE Healthcare USA) that was probed with antibodies against total EGFR (#2232 1:1000) and phosphorylated (p-)EGFR (Tyr992) (#2235 1:1000) (both from Cell Signaling Technology Protein bands were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific Plasma EGF and AREG concentrations were measured with the Human EGF Quantikine ELISA Kit (#DEGFR0) and Human Amphiregulin Quantikine ELISA Kit (#DAR00; both from R&D Systems according to the manufacturer’s instructions DNA was isolated using the QIAamp DNA Mini Kit (Qiagen The 25 μL reaction contained 2.5 μL of 5× reaction buffer and 0.2 μL GoTaq DNA polymerase (Promega A 3 μL volume of diluted DNA sample (300 ng) was used for conventional PCR and amplified products (20 μL) were visualized by electrophoresis with Tris–acetic acid–EDTA buffer on a 1.2% (w/v) agarose gel stained with ethidium bromide under ultraviolet light PCR amplification was performed on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems USA) under the following conditions: 94°C for 5 min; 35 cycles of 94°C for 30 s annealing at the melting temperature for 45 s and 60°C for 30 s; and 60°C for 7 min The following forward and reverse primers were used: PML 5’-TCAAGATGGAGTCTGAGGAGG-3’ and 5’-CTGCTGCTCTGGGTCTCAAT-3’; and β-actin 5’-TCTTGATAGTTCGCCATGGAT-3’ and 5’-GGTCATCTTTTCACGGTTGG-3’ To investigate the in vivo effects of the EGFR inhibitors gefitinib or erlotinib as monotherapy or combined with ATRA, we used a syngeneic transplantation mouse model of APL with leukemia cells from human chorionic gonadotropin (hCG)–promyelocytic locus–retinoic acid receptor A (PML–RARA) transgenic mice (B6129 mixed background), as previously described (22, 23) The hCG-PML-RARA mice were kindly donated by Dr Pier Paolo Pandolfi (Beth Israel Deaconess Medical Center Harvard University) and maintained at the Laboratory of Experimental Animal Studies (Fundação Hemocentro de Ribeirão Preto–Ribeirão Preto 8 to 12-week-old male wildtype (WT) littermates were used as transplant recipients after lethal irradiation (7 Gy split into two doses from an X-ray source i.e. 4 h apart - RS200 from Rad Source Technologies the animals were exposed to a dose of 2% isoflurane for 5 min to induce anesthesia and immediately afterward 4 × 106 viable leukemic blasts from hCG-PML-RARA mice (200 µL in PBS) were injected intravenously using a syringe with a 30-gauge disposable needle (BD Biosciences) through the retro-orbital sinus the mice were monitored and assessed for engraftment analysis The engraftment was confirmed by conventional PCR analysis of the DNA isolated from 100 μL of heparinized peripheral blood samples collected via submandibular vein by using a 5-mm lancet (Goldenrod Animal Lancet Overall survival of mice was defined as the length of time from the start of treatment until the date of spontaneous death or euthanasia All animal experiments were performed at the Laboratory of Experimental Animal Studies (Fundação Hemocentro de Ribeirão Preto – Ribeirão Preto Significant differences between groups were evaluated with the unpaired t-test or Kruskal–Wallis test Bivariate correlation analysis with Spearman’s test was performed to determine the correlation between BM plasma concentration of EGF or AREG and WBC count at the time of diagnosis The log-rank test (with Kaplan–Meier curves) was used for overall survival analysis Statistical analyses were performed using Prism v.7.03 software (GraphPad The significance level was set as P ≤ 0.05 and AREG protein expression in APL patient samples and β-actin protein levels in CD34+ cells isolated from normal BM of one healthy adult volunteer and 10 representative primary APL samples collected at diagnosis HeLa cells served as a positive control to assess EGFR and p-EGFR (Tyr992) expression and MV4-11 cell extracts were used as a positive control for SYK expression EGFR and p-EGFR (Tyr992) were detectable when exposed for 180 seconds (sec) (B) EGF levels in BM plasma samples from healthy donors (n=15) and APL patients at diagnosis (n=16) (C) Plasma AREG levels in BM from healthy donors (n=20) and APL patients at diagnosis (n=17) Table 1 The 50% effective dose (ED50) values of gefitinib and arsenic trioxide (ATO) in NB4 and NB4-R2 cells and combination index (CI) values of gefitinib plus ATO at different effective levels Figure 2 Effects of gefitinib or ATO monotherapy on APL cell apoptosis and proliferation D) treatment for 24 h decreased the fraction of apoptotic NB4 and NB4-R2 cells in a concentration-dependent manner as determined by Annexin V/PI staining and flow cytometry analysis Proliferation of NB4 (E) and NB4-R2 (F) cells was reduced at gefitinib concentrations higher than twice the ED50 after 24 h of treatment Bar graphs show mean ± SD of at least three independent experiments (****) p < 0.0001 (Kruskal–Wallis test Data represent the mean ± SD of at least three independent experiments (***) p < 0.001 (Kruskal– Wallis test Figure 4 Gefitinib therapy sensitizes APL-resistant cells to ATRA and ATO and the respective parental NB4 clone 21 (I–L) were treated with ATRA alone (0.1 µM) ATRA plus gefitinib or ATRA and ATO plus gefitinib for 72 h and the expression of the myeloid differentiation markers CD11b Gefitinib potentiated the myeloid ATRA-induced differentiation in APL cells resistant to ATRA (NB4-R2) and ATO (NB4 ATOr) (***) p < 0.001 (Kruskal–Wallis test Treatment with gefitinib at the highest dose (200 mg/kg/day) exhibited a trend to prolong the survival of APL mice (median survival=56 days; 95% CI=47–65 days) (P>0.05) Figure 5 In vivo effects of EGFR inhibitors alone or in combination with ATRA in an APL mouse model (A) Expression of the PML–RARA fusion gene in peripheral blood of WT mice 3 weeks after transplantation of leukemic blasts from hCG-PML–RARA transgenic mice detected by conventional PCR (B) Kaplan–Meier survival curves of mice treated with vehicle (DMSO; n=4) or erlotinib at 200 mg/kg/day (n=4) Survival (C) and spleen weight-to-body weight ratio (D) of mice treated with gefitinib [200 mg/kg/day; n=7 - (C) and n=5 - (D)] ATRA [2.5 mg/kg/day; n=7 - (C) and n=5 - (D)] gefitinib plus ATRA [n=9 - (C) and n=8 - (D)] Surviving mice at 200 days post transplantation were sacrificed (****) p < 0.0001 (log-rank or Kruskal-Wallis test followed by Dunn’s post hoc test) These results suggest that at least in vivo the combination gefitinib plus ATRA did not enhance the differentiation effect of ATRA monotherapy In the present study, consistent with previous findings (3, 4) we validated that the combination between gefitinib or erlotinib with ATRA and ATO enhanced the drug-induced myeloid differentiation in APL cells we demonstrate for the first time that this combination was effective for ATRA- and ATO-resistant APL cells our results provide new insights into the ongoing challenge of developing therapies to overcome ATRA and ATO resistance in APL patients SYK protein expression was not detected in our primary APL samples raising the possibility of a broader than SYK spectrum of off-target effects upon EGFR inhibitor therapy in APL Our findings highlight the relevance of repurposing the FDA-approved tyrosine kinase-targeted therapies to overcome the resistance of a specific subgroup of patients with APL who are unresponsive to standard treatment partially explaining the mild cytotoxicity antagonism interaction between ATO and gefitinib the combination of gefitinib and ATRA extended survival in mice and reduced spleen weight-to-body weight ratio compared to gefitinib or vehicle but was not superior to ATRA monotherapy One limitation of our study is the high sensitivity of hCG-PML/RARA leukemic cells to ATRA monotherapy which in the event of relapse frequently show ATRA resistance Further functional in vivo studies are necessary to verify the efficacy of EGFR or other tyrosine kinases inhibitors in APL models resistant to ATRA or ATO treatment Although the use of EGFR inhibitors did not prolong survival or increase myeloid differentiation in an APL mouse model sensitive to ATRA and re-sensitized ATRA- and ATO-resistant APL cells to ATRA and ATO induced differentiation These findings provide a basis for future studies to explore the potential role of tyrosine kinase-targeted selective therapies in combination with standard therapy which could be exploited to reverse ATRA and ATO resistance in a subset of patients with APL although some of the EGFR signaling components are expressed in APL patient blasts further investigations are necessary to understand their biological implications on leukemia progression since the effects of EGFR inhibitors seem to be a result of off-target activities The original contributions presented in the study are included in the article/Supplementary Material Further inquiries can be directed to the corresponding author The studies involving human participants were reviewed and approved by Research Ethics Committee of the Medical School of Ribeirao Preto The patients/participants provided their written informed consent to participate in this study The animal study was reviewed and approved by Animal Care and Use Committee of the Medical School of Ribeirao Preto of the University of Sao Paulo (Protocol no All authors contributed to the article and approved the submitted version This research was funded by the Fundação de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP; grant no 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 We thank Mara de Souza Junqueira and Bárbara Amélia Aparecida Santana Lemos for assistance with mouse irradiation and Patrícia Vianna Bonini Palma for advice regarding flow cytometry experiments and Ana Lúcia Pimentel (Laboratory of Biochemical Genetics Medical School of Ribeirao Preto – University of Sao Paulo) for performing cell line authentication The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021.686445/full#supplementary-material Retinoic Acid and Arsenic Trioxide for Acute Promyelocytic Leukemia Acute Promyelocytic Leukemia: Update on the Mechanisms of Leukemogenesis Resistance and on Innovative Treatment Strategies Gefitinib Potentiates Myeloid Cell Differentiation by ATRA Gefitinib Enhances Arsenic Trioxide (AS2O3)-Induced Differentiation of Acute Promyelocytic Leukemia Cell Line Erlotinib and Gefitinib for the Treatment of Myelodysplastic Syndrome and Acute Myeloid Leukemia: A Preclinical Comparison Biochem Pharmacol (2008) 76(11):1417–25 Gefitinib Induces Myeloid Differentiation of Acute Myeloid Leukemia 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FLT3 Inhibition Against FLT3-ITD-Driven Leukemia Harboring Activated SYK Kinase SYK Is a Critical Regulator of FLT3 in Acute Myeloid Leukemia Erlotinib Is Effective Against FLT3-ITD Mutant AML and Helps to Overcome Intratumoral Heterogeneity via Targeting FLT3 and Lyn PubMed Abstract | Google Scholar JNK Activation Is a Mediator of Arsenic Trioxide-Induced Apoptosis in Acute Promyelocytic Leukemia Cells Cross-Tissue Analysis of Gene and Protein Expression in Normal and Cancer Tissues Reduced Changes in Protein Compared to mRNA Levels Across Non-Proliferating Tissues Keywords: epidermal growth factor receptor (EGFR) Araujo CL and Rego EM (2021) The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells Received: 26 March 2021; Accepted: 06 September 2021;Published: 28 September 2021 Copyright © 2021 Almeida, Pereira-Martins, Weinhäuser, Ortiz, Cândido, Lange, De Abreu, Mendonza, de Deus Wagatsuma, Do Nascimento, Paiva, Alves-Paiva, Bonaldo, Nascimento, Alves-Filho, Scheucher, Lima, Schuringa, Ammantuna, Ottone, Noguera, Araujo and Rego. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Eduardo M. Rego, ZWR1YXJkby5yZWdvQGZtLnVzcC5icg== †These authors have contributed equally to this work Disclaimer: 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 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish 43,000+ global companies doing business in the region 102,000+ key contacts related to companies and projects news and interviews about your industry in English