Exposure to MLo1302 reduced DNMT protein expression, similar to decitabine, and showed a partial effect in cell differentiation, reducing protein expression of pluripotency markers. RT2 profiler expression array indicated several dysregulated targets, related to activation of apoptosis, differentiation, and cell cycle arrest. We validated these data by showing increased apoptosis, increased protein expression of cleaved caspase 8 and activated caspase 2, and reduced proliferation (BrdU assay), with increase in CDKN1A and decrease in MIB-1 expression. Therefore, synthetic drugs designed to target DNA methylation in cells may uncover effective treatments for TGCT patients.Less toxic treatment strategies for testicular germ cell tumor (TGCT) patients are needed, as overtreatment is a concern due to the long-term side effects of platin-based chemotherapy. Although clinical benefit from classical hypomethylating agents has to date been limited, TGCTs show an abnormal DNA methylome indicating the potential of treating TGCTs with hypomethylating drugs. We tested, for the first time in TGCT cell lines, a new synthetic flavonoid compound (MLo1302) from the 3-nitroflavanone family of DNA methyltransferase (DNMT) inhibitors. We show that MLo1302 reduces cell viability (including of cisplatin resistant cell line NCCIT-R), with IC Testicular germ cell tumors (TGCTs) are among the most common solid cancers in young-adult men of Caucasian descent, and incidence is increasing worldwide due to lifestyle-related factors . SurvivaThe molecular background and the morphology of TGCTs are very rich. The most common TGCTs (type II) derive from the common precursor lesion germ cell neoplasia in situ, and are categorized as seminomas or non-seminomas, the latter including embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma and mixed tumors, containing at least two or more of these elements. This tumor model resembles embryonic and germ cell development, where epigenetic changes are critical events . Since tDNA methylation is one of the most well-known epigenetic mechanisms regulating gene expression and is catalyzed by DNA methyltransferases (DNMTs), leading to gene repression (namely of tumor suppressor genes within cancer cells) when occurring on gene promoters . DNMT inIn this study, we aimed to test the in vitro activity of a newly synthesized chemical compound (MLo1302) belonging to the 3-nitroflavanone family of compounds, engineered to inhibit DNMTs in cells. The compound corresponds to “compound 1” in reference , where cTGCT cell lines  were provided by Prof. Leendert Looijenga, and cultured as described . AdditioNCCIT (p53 mutated) is representative of a primary mediastinal mixed non-seminoma with embryonal carcinoma; 2102Ep is representative of a primary testicular mixed non-seminoma with embryonal carcinoma, p53 wild-type; and NTERA-2 is representative of a primary testicular embryonal carcinoma, p53 wild-type. Cells were maintained in low passages and were confirmed negative for Mycoplasma spp. .https://www.lgcstandards-atcc.org/STR%20Database.aspx?geo_country=pt and to the ones available in previous publications [The cell lines used in this study were authenticated via STR profiling, with profiles compared to the database ications ,17. All TM Technologies, Vancouver, BC, Canada) for 10 days ; DAC  for 72 h  and MLo1302 , for 7250 values were computed as described [Cell viability was assessed at 24 h, 48 h, and 72 h of treatment with DAC and MLo1302, as described in . Cells wescribed .2O2. The OD obtained for apoptosis assay was normalized for the OD obtained by viability assay at the same time point.Apoptosis assay was performed at 72 h of treatment with MLo1302 according to the protocol detailed in , using tFor assessing cell proliferation, BrdU assay (Cell Proliferation ELISA 5-bromo-2′-deoxyuridine) was performed 72 h after treatment with MLo1302, following the protocol fully described in . Density50, IC50, and 2 × IC50 values every 24 h until 72 h. The supernatants were collected every 24 h before MLo1302 treatment renewal and stored at −20 °C. For LDH quantification, samples were incubated with 0.21 mM β-NADH in 0.05 M phosphate buffer at 30 °C for 5 min, followed 23 mM sodium pyruvate. The kinetics of NADH disappearance was followed at 340 nm during 3 min at 30 °C. The levels of LDH released were normalized to positive control and to the number of live cells. LDH levels in MLo1302 conditions were normalized for control condition.The cytotoxic effect of MLo1302 compound was evaluated through LDH activity in supernatant cell culture medium by NADH kinetics function. Briefly, TGCT cells were plated at desired density in 96 well plate and allowed to adhere overnight at 37 °C, 5% CO2. Then, TGCT cells were treated with 1/2IC®, Foster City, CA, USA) as reported in [RASSF1A gene promoter  and 500 ng were bisulfite-treated using the EZ-96 DNA Methylation-Gold Kit , according to manufacturers’ protocol. qMSP reactions were run in a 7500 Real-Time PCR System . Resultof TGCTs , and comTotal RNA was extracted using TRIzol method, quantified and reverse transcribed as reported in . RT-qPCRWestern blot was performed according to the protocol detailed in . ProteinImmunofluorescence for 5-methylcytosine (5 mC) was performed after 72 h of MLo1302 treatment. Cells were seeded in dark 96-well plates at 4000, 3000, and 2000 cells/well density . Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X solution in 0.5% Tween-20 for 20 min. Before blocking with 5% bovine serum albumin 1 h at room temperature, cells were treated with 4M HCl during 20 min following trypsin at 37 °C for 2 min. Then, cells were incubated with primary antibody for 5 mC  , overnigAdditionally, NCCIT cells were incubated with anti-5hmC antibody and the same protocol was followed, using secondary antibody anti-rabbit Alexa Fluor 488 .DNA was extracted and quantified as mentioned before. One thousand nanograms of DNA were diluted in TE buffer. Subsequently, DNA was denatured in 0.1 M of NaOH at 95 °C for 10 min and single chains were stabilized in 1 M of ammonium acetate. DNA was pipetted into nitrocellulose membranes, which were allowed to dry for 30 min at 37 °C. Membranes were exposed to UV light for 1 min to produce crosslinks between DNA and membranes. Then, membranes were blocked with 5% dry milk in TBS with 0.1% Tween and incubated with primary antibody against 5mC  at 4 °C, overnight. On the next day, membranes were incubated with secondary horseradish peroxidase conjugated antibody  anti-mouse (1:5000), for 1 h at room temperature. As for western blot, the blots were detected using enhanced chemiluminescence and quantified using ImageJ software. Sybergreen I nucleic acid stain  was used as loading control.2 Profiler PCR Array System Kit  included 96 genes corresponding to cancer research molecular pathways and adequate controls, in quadruplicates. The expression levels were determined by real-time quantitative PCR in a LightCycler 48 platform  and ACTINβ, β2M, GAPDH, HPRT1, and RPLP0 were used as endogenous controls. The RT2 profiler PCR array analysis was performed in Qiagen specific platform. The data analysis in web portal calculates fold change using ΔΔCt method. Genes with a logarithm fold change above 1.5 or below −1.5 were considered. Additionally, DNA genomic contamination, as well as first strand synthesis and real-time PCR efficiency were monitored in Qiagen platform for RT2 profiler PCR array analysis. The lower limit detection was set at Ct ≥ 35.Total RNA was extracted from NCCIT cell line  using TRIzol  and quantified in NanoDropTM Lite Spectophotometer . Four hundred nanograms of cDNA were synthetized using Transcriptor High Fidelity cDNA Synthesis Kit , according to manufacturer’s instructions. The RTp-values were adjusted for multiple comparisons (Dunn’s test). Statistical significance was set at p < 0.05 and is reported in graphs/figures as such: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.Data was tabulated using Microsoft Excel 2016 and analyzed using GraphPad Prism 6. Nonparametric tests  were used for comparing levels among samples. All 50 of 400 nM and 600 nM, respectively), followed by 2102EP (IC50 of 2.2 µM)  B. Additiof 1.2µM C.50 values of 35 nM for NCCIT, 9 nM for NTERA-2, and 100 nM for 2102EP).Regarding DAC treatment D, cell v50 for NCCIT/NTERA-2 at 72 h), 250 nM (1/2IC50) and 1 µM (2IC50) of MLo1302 were selected to perform the phenotypical and molecular assays (see below).The concentrations of 10 nM (previously showed to have antitumor effect in TGCT cell lines ) and 1 µWe found a significant increase in apoptosis levels in treated NCCIT and NTERA-2 cell lines A at 72 hMoreover, we found that exposure to MLo1302 for 72 h induced a significant decrease in cell proliferation in all cell lines B. MLo130This was accompanied by a decrease in MIB-1 transcript levels  and by an increase in transcript levels of CDKN1A, indicating cell cycle arrest  D.ATRA classically induces differentiation of human germ cell tumor lines, which is phenotypically appreciated based on change of morphology  . DNMT inAs described, ATRA induced differentiation in NCCIT and NTERA-2 cell lines. Morphometric analysis of the cells with most prominent and sustained phenotypical changes (NTERA-2) showed a statistically significant increase in cell perimeter, and decrease in area and sphericity, approximating a neuronal shape . These pWe then evaluated effect on differentiation at the molecular level, by assessing protein levels of pluripotency factors. Regarding DAC treatment, the concentration of 10 nM decreased pluripotency factors , and the higher concentration of 1 µM produced a more pronounced effect across the cell lines for SOX2, NANOG, and OCT3/4 B. HoweveRASSF1A promoter methylation in differentiated NCCIT and NTERA-2 cells (RASSF1A promoter methylation (Upon ATRA-induced differentiation a significant decrease in DNMT3B expression was verified (as reported elsewhere ), with n-2 cells . DAC con-2 cells , and a dhylation , as prevhylation , a decrehylation .RASSF1A promoter was detected for 2102EP, no significant effect was achieved in the remaining two cell lines , SNAI3 l tumors ), PGF (rntiation ), and PPpoptosis ). Additiegulated .Novel treatment options for TGCT patients, less toxic than the classical  cisplatin-based chemotherapy are needed. These new therapies may allow dose reduction, sparing very young patients from the undesirable side effects of chemotherapeutic drugs, which may severely affect their quality of life ,35. Thes50 values in the nanomolar range, for NCCIT and NTERA-2 cell lines. The optimal dose of DAC for treating patients in the clinic is still under debate, as is the schedule of administration, another very relevant variable [2 was established. Considering an average 70 kg male, this corresponds to an approximate 100 uM concentration of the drug, so we hypothesize that MLo1302 concentrations used in our work may be effective and nontoxic in the clinic. 2102EP cell line was the least sensitive to treatment, also observed upon exposure to DAC. For both drugs, the effect was dependent on time of exposure and concentration; overall, at 72 h, IC50 values were lower for DAC compared to MLo1302. With DAC treatment, however, we found no additional effect on viability beyond the dose of 250 nM, even upon exposure to very high doses . The surviving cells may endure the exposure to the drug by activating cell differentiation, as demonstrated by downregulation of pluripotency factors upon exposure to high doses of DAC. On the contrary, exposure to high doses of MLo1302 reduced viability to zero in all cell lines. Interestingly, Albany et al. [50 values in the same range  upon exposure to DAC. These values reflect a higher sensitivity of TGCTs to this family of compounds, rendering lower IC50 values than those observed for many other solid tumors [50 values in higher ranges than those observed with TGCT cell lines. Importantly, MLo1302 also reduced cell viability in a cisplatin-resistant cell line (NCCIT-R), with an IC50 of 1.2 µM. This cell line shows a much higher IC50 to cisplatin (31.59 µM), which is the mainstay available drug in the clinic. Further studies should be pursued in the context of cisplatin resistance to support that this compound can also be clinically useful in this context.Remarkably, MLo1302 reduced cell viability in three non-seminoma cell lines, with ICvariable ,43. In tvariable , guadeciy et al.  obtainedd tumors . This is+ consumption and LDH release to the cell media) and increased apoptosis in NCCIT and NTERA-2 cell lines, but not in 2102EP, the cell line showing the least pronounced effect in cell viability. However, a significant decrease in cell proliferation was observed for all cell lines upon treatment with MLo1302, and this was supported by increased expression of CDKN1A and decreased MIB-1, indicating cell cycle arrest.In consonance with viability, functional cell assays demonstrated both cytotoxicity  and an increase of PAX6— differentiation-related marker in NCCIT and NTERA-2 cells. This is less apparent in 2102EP cells, which is less prone to differentiation upon ATRA treatment [RASSF1A promoter DNA methylation levels in NCCIT and NTERA-2 cell lines, in line with the reported gradient of increasing global DNA methylation found in these tumors and related to more differentiated histological subtypes [Since hypomethylating agents are known to induce cell differentiation , we assereatment . Furtherreatment . Additiosubtypes . As for DNMT1, DNMT3A, and DNMT3B protein levels (completely abrogating expression for DNMT1 and DNMT3A). Additionally, we found a decrease in RASSF1A promoter methylation after DAC treatment, in line with previous studies [DNMT1 expression in all cell lines, and a decrease of DNMT3A and DNMT3B expression in some cell lines, when treated with 500 nM. However, the compound had no effect on DNMT3B in NCCIT and NTERA-2 cells, which actually showed increased expression (but not reaching significance). This is in line with results obtained for DAC treatment precisely for NCCIT and NTERA-2 cells  and induced by hypoxia (by impeding cytochrome c release from mitochondria). Finally, it also interferes with TNF-induced necrosis, blocking this pathway by interacting with TNFRSF1A [CASP8 in particular also has a role in other forms of cell death, like necroptosis and pyroptosis, which can be further explored [BIRC3 was also downregulated, and this protein was shown to impair apoptosis by inhibiting ripoptosome formation, by ubiquitinating RIPK1 and, again, CASP8. This player is also a relevant regulator of the NF-kappa-B signalling pathway and also of the innate immune response [PPP1R15A is activated in response to stress and involved in activation of p53 by phosphorylation of Ser-15 and in activation of p21/WAF1, promoting apoptosis and leading to cell cycle arrest [CASP3 [KRT14 has been demonstrated to promote squamous cell differentiation in a NOTCH1-dependent manner [SNAI3, a member of the SNAIL family, is involved in mesodermal differentiation during embryogenesis and regulates epithelial-to-mesenchymal transition [PGF is a key factor in trophoblast differentiation, which binds to the receptor FLT1/VEGFR-1 and activates stress activated protein kinase (SAPK) pathways, leading to endothelial cell growth and neoangiogenesis typical of this tissue [CPT2 is transported from the nucleus to the inner membrane of the mitochondria. This carnitine palmitoyltransferase (together with acyl-CoA synthetase and carnitine/acylcarnitine translocase) leads to beta-oxidation of fatty acids. A summary of these signalling pathways is represented in We further explored the downstream mechanisms elicited upon MLo1302 treatment. We performed an expression array, which indicated that the apoptotic pathway was the most significantly involved in the drug’s mechanism of action. Seven genes were differentially expressed with fold change above the defined criteria. Specifically, a statistically significant downregulation of the anti-apoptotic gene aspase 2 ,29. Inteaspase 2 . In factchondria . Moreovechondria . More spTNFRSF1A . CASP8 iexplored . Other tresponse . Moreovet [CASP3 . On anott manner ; SNAI3, ansition ; while Ps tissue . Of releWe explored, for the first time, the effects of a newly synthesized non-nucleoside flavanone compound, in four TGCT cell lines, including a cisplatin resistant clone. DNMT downregulation was observed along with relevant effects on cell differentiation. Importantly, we also demonstrated that MLo1302 cytotoxic effect, is due mostly to stimulation of apoptosis, namely by targeting the anti-apoptotic factor NOL3, and by inducing cell cycle arrest. The effects of MLo1302 may be in part a consequence of DNA hypomethylation, and this may stem indirectly from DNA methylation inhibition or may derive from a non-methylation-dependent effect . An indiFuture studies are needed to definitively answer these questions. One limitation of our work is related to assessment of putative toxicity of the compound in the normal germ cell counterpart, as a measurement of the compound safety. However, to date, no normal germ cell line is available, and cell lines derived from stromal cells do not represent truly the germ cell lineage and react differently to exposure to drugs. In order to answer this important question we intend to assess MLo1302 effect in vivo, using a control group, providing histological and biochemical evidence of the effect in normal subjects. Moreover, MLo1302 may be explored in combinatory strategies, either with cisplatin or other targeted therapies, to improve treatment of TGCT patients. Specifically, following one line of investigation of our group and our previous results ,58, we i


ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents.The genetic variants of the ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment.We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p = 0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide  and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p = 0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml).No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of  ARID5B gene  family and acts as a transcription coactivator that binds to the 5′-AATA[CT]-3′ core sequence  were determined using the alamarBlue cell viability assay  as previously reported . Cells . Mann–Whitney U test was always applied for comparisons between two groups of cell lines using R (version 3.5.1) statistical software.We applied Fisher’s exact test for comparison of allele frequencies between cell lines and Japanese population in HapMap project database -rearranged, 4 ETV6/RUNX1-positive, 3 TCF3/HLF-positive, and 2 BCR/ABL1-like cell lines, but no hyperdiploid cell lines .We first analyzed patients . Our celARID5B are located in intronic regions, we next performed an expression quantitative trait locus (eQTL) analysis. We quantified the ARID5B gene expression level in each cell line by real-time RT-PCR using ACTB gene expression as an internal control. However, in the eQTL analysis of 72 BCP-ALL cell lines, neither genotypes of susceptibility-linked rs7923074 and rs10821936 nor those of relapse-linked rs4948488, rs2893881, and rs6479778 were significantly associated with ARID5B expression level  of nine representative chemotherapeutic agents  used for children with ALL. Of note, IC50 values of VCR (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous risk allele (C) genotypes in the relapse-linked rs4948488 were significantly higher (p = 0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T) , and Ara-C in BCR/ABL1-negative and BCR/ABL1-like-negative ALL cell lines.Next we verified whether the genotypes of relapse-linked SNPs of We further analyzed any association of the susceptibility-linked rs7923074 and rs10821936 with drug sensitivities. In contrast to the genotypes of the relapse-linked SNPs, no significant associations were observed in genotypes of rs7923074 and rs10821936 with sensitivities to VCR, CY and AraC  was significantly higher (p = 0.023 in Mann–Whitney U test) than those in the other 36 cell lines with lower expression (16.9 ng/ml)  Fig. a. A simiups Fig. b–d. FurtARID5B with cell cycle progression in BCP-ALL cell lines. First, we compared cell cycle progression between cell lines with homozygous genotype of risk allele in each relapsed-linked SNP and those with the non-risk allele -rearranged, and 3 cell lines were TCF3/HLF-positive. Furthermore, we later discovered that 15 cell lines were positive for MEF2D-fusions [ETV6/RUNX1-positive, and no cell lines were hyperdiploid. Thus, the majority of our cell lines were established from BCP-ALL with a poor prognosis.In the present study, using a series of BCP-ALL cell lines, we tried to verify the significance of genotype in the susceptibility-linked and relapsed-linked SNPs of -fusions , which a-fusions , 20. In ARID5B and the sensitivities to representative drugs for ALL treatment. We analyzed the sensitivities to nine agents, and found that sensitivities to VCR, CY, and AraC were associated with relapsed-linked SNPs; cell lines with homozygous genotypes of risk alleles in the relapsed-linked rs4948488, rs2893881, and rs6479778 were significantly more resistant to VCR, CY, and AraC than cell lines with non-risk alleles. These relapsed-linked SNPs of ARID5B were located in intron 2 of the ARID5B gene. However, no significant association was observed between genotypes of the relapse-linked SNPs of ARID5B and ARID5B gene expression level in BCP-ALL cell lines. Moreover, ARID5B gene expression level was not associated with sensitivities to VCR, CY, and AraC. Thus, genotypes of the relapse-linked SNPs of ARID5B are associated with VCR, CY, and AraC sensitivities of BCP-ALL cell lines independent of their ARID5B expression levels. Additionally, although cell cycle progression is highly associated with the drug sensitivity of leukemia cells, no clear association was observed between cell cycle progression and genotypes of the relapse-linked SNPs of ARID5B in BCP-ALL cell lines. Thus, further analyses are required to clarify the underlying mechanism behind the association between genotypes of the relapse-linked SNPs of ARID5B with the sensitivities to VCR, CY, and AraC in BCP-ALL cell lines.Using these biased samples, we analyzed associations between genotypes of the relapsed-linked SNPs of ARID5B gene expression level was associated with resistance to MTX. This finding seems to be partly consistent with a recent report by Xu et al. [ARID5B knockdown in ALL cell lines led to specific resistance to MTX and 6MP. The authors showed that knockdown of ARID5B by using shRNA and CRISPR/Cas9 in ALL cell lines induced partial cell-cycle arrest at G0/G1 phase through upregulation of p21 [ARID5B expression were similar to those in cell lines with higher ARID5B expression, suggesting that cell cycle progression was not a direct mediator in the association between ARID5B gene expression level and sensitivity to MTX in BCP-ALL cell lines. Further mechanism(s) other than cell cycle progression may be involved in the association of ARID5B expression with MTX sensitivity.Regarding the association of ARID5B with drug sensitivities in BCP-ALL cell lines, we also found that lower u et al.  who founn of p21 , suggestARID5B may be associated with higher relapse rates because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. Moreover, lower ARID5B expression may be associated with MTX resistance. Limitations of the present study include that the study sample was restricted to limited numbers of leukemic cell lines with biased karyotypes and that underlying biological mechanisms for the associations remain unclarified. Since our findings were obtained from leukemia cell lines, further studies are needed before making a firm conclusion.In summary, our observations in 72 BCP-ALL cell lines suggest that the risk allele of the relapse-linked SNPs of Additional file 1: Table S1. Karyotype, age, patient status at the cell establishment, and SNP genotypes and gene expression level of ARID5B in cell lines.Additional file 2: Table S2. IC50 of each drug in cell lines.Additional file 3: Fig. S1. Association of relapse- and susceptibility-linked SNP genotypes with sensitivities to Pred (a), Dex (b), DNR (c), L-Asp (d), MTX (e), and 6MP (f). Vertical axis indicates log-scaled IC50 values of Pred (a), Dex (b), DNR (c), L-Asp (d), MTX (e), and 6MP (f). The IC50 values of cell lines with homozygous genotype of risk allele and those with heterozygous or homozygous genotypes of non-risk allele in each SNP were compared. P-value in Mann–Whitney U test is indicated at the top of each SNP.Additional file 4: Fig. S2. Association of relapse- and susceptibility-linked SNP genotypes with sensitivities to VCR (a), CY (Maf) (b), and AraC (c) in 56 BCP-ALL cell lines excluding 14 BCR/ABL1-positive and 2 BCR/ABL1-like ALL cell lines. Vertical axis indicates log-scaled IC50 values of VCR (a), CY (Maf) (b), and AraC (c). The IC50 values of cell lines with homozygous genotype of risk allele and those with heterozygous or homozygous genotypes of non-risk allele in each SNP were compared. P-value in Mann–Whitney U test is indicated at the top of each SNPAdditional file 5: Fig. S3. Association of ARID5B gene expression with sensitivities to MTX in 56 BCP-ALL cell lines excluding 14 BCR/ABL1-positive and 2 BCR/ABL1-like ALL cell lines. Vertical axis indicates log-scaled IC50 value of MTX. The IC50 values of 28 cell lines with higher than median value ARID5B expression veles and the other 28 cell lines with lower than median value expression levels were compared. P-value in Mann–Whitney U test is indicated at the top of each SNPAdditional file 6: Fig. S4. Association of ARID5B gene expression with sensitivities to Dex (a), Pred (b), DNR (c), LAsp (d), and 6MP (e). Vertical axis indicates log-scaled IC50 values of Dex (a), Pred (b), DNR (c), LAsp (d), and 6MP (e). The IC50 values of 36 cell lines with higher ARID5B expression and the other 36 cell lines with lower expression were compared. P-value in Mann–Whitney U test is indicated at the topAdditional file 7: Fig. S5. Association of relapse- and susceptibility-linked SNP genotypes with cell cycle progression. Percentage of G0/G1 phase was compared between cell lines with homozygous genotype of risk allele and those with heterozygous or homozygous genotypes of non-risk allele in each SNP. P-value in Mann–Whitney U test is indicated at the top of each SNPAdditional file 8: Fig. S6. Association of ARID5B gene expression with cell cycle progression. Percentages of G0/G1 phase in 36 cell lines with higher than median value ARID5B expression levels were compared with those in the other 36 cell lines with lower median value ARID5B expression levels. P-value in Mann–Whitney U test is indicated at the top


During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm–sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury. Alcohol is a major lifestyle-related risk factor for many human diseases including oral and esophageal cancers. The oral cavity and the esophagus are unique sites that are directly exposed to the high concentrations of ethanol (EtOH) present in alcoholic beverages. Alcohol consumption results in cellular DNA damage in humans , 2, whicin vivo Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: No**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The esophagus is exposed to high concentrations of ethanol during alcohol consumption and this exposure is a risk factor for ESCC. Therefore, there is a critical need to understand the effects of alcohol on the esophageal epithelium. This manuscript investigates the induction of mitochondrial dysfunction subsequent to alcohol exposure in esophageal keratinocytes. The authors use cell culture-based systems to determine the lethal dose and duration of ethanol in their cells, identify that the mitochondria (and therefore the TCA) is dysregulated and that autophagy is a potential cellular mechanism these cells are using to mitigate the damage induced by the alcohol. They, furthermore, implicate AMPK and mTOR signaling in this mechanism of autophagy. The manuscript is fairly well written with a few grammatical errors noted below. The data support the conclusions of the paper, with the exception of mTOR pathway – again discussed below. The statistics are applied in an appropriate manner and aid inn a clear understanding of the data presented.There are two significant points to address for this reviewer for this manuscript:1) In describing the dilutions used for experimental conditions and controls, it is unclear if the media nutrients are more dilute in the alcohol-containing samples versus the control samples. In the methods it is mentioned that the alcohol is diluted in media while the control cells receive only media. It would be better if the experiments also had diluted control media to a similar extent as the alcohol-containing media. This could be clarified in the methods to more fully explain how the treatments occur if this interpretation is incorrect. Alternatively, this limitation could be explored in the discussion section.2) It is unclear to this reviewer which experiments have been performed with multiple independent cell lines. The results section should clearly indicate when the data is collected in EPC1, EPC2, or EPC3 cell lines and the figures appropriately labelled for clarity.Minor points to address:1) Editing/grammar errors to fix in lines: 62, 69, 101, 283, 331, 492.2) Should delete reference to in vivo data since none is presented.3) How many reads were collected for the RNA-Seq data?4) Line 248, how was apoptosis measured?5) when quantifying data from organoids, how many were counted/slide and how many replicate slides were quantified?6) The two bar graphs in figure 5A should receive their own lettering (ie 5b and 5c) and the other lettering adjusted appropriately.7) the conclusion that mTORC1 signaling is altered from analyzing S6 phosphorylation is a bit of an overstated conclusion as stated in the subheading on line 397. The RNA-Seq analysis suggests the pathway as suppressed but one experimental piece of data was presented to support this. S6 can be phosphorylated by other pathways than mTORC1 signaling. Would suggest softening the conclusion in this section.Reviewer #2: Authors propose that autophagy mitigates ethanol-mediated mitochondrial damage and oxidative stress via the AMPK-mTORC1 axis in esophageal keratinocytes.This is a potentially interesting paper, however, several problems must be corrected.1) Some statements are simply not correct such as"RNA sequencing disclosed robust mitochondrial dysfunction".RNA sequencing may suggest (!) mitochondrial dysfunction but never shows directly mitochondrial dysfunction.You can change this to"RNA sequencing shows mitochondrial alterations."Please emphasize both in abstract and text that you have provided provide direct evidence for the mitochondrial dysfunction by following methods: Mitochondrial metabolism; Respiratory, Mitochondrial oxidative stress (MitoSOX) and cellular ATP level.2) Please explain how ethanol induces mitochondrial stress . It is most likely not a direct effect. Please explain in introduction and discussion sections. RNA sequencing data suggest that ethanol may initially affect the nuclei protein expression which later translate into mitochondrial dysfunction. Please comment and explain.Reviewer #3: This manuscript “Autophagy mitigates ethanol-mediated mitochondrial damage and oxidative stress via the AMPK-mTORC1 axis in esophageal keratinocytes” primarily aimed to investigate the effect of acute ethanol exposure on esophageal epithelial cells and its associated downstream signaling cascade. Authors had characterized the esophageal epithelial cells growth and viability at different time points in response to various concentrations of ethanol. Using RNA sequencing, LC-MS and TEM imaging, authors had showed mitochondrial dysfunction and structural changes in esophageal epithelial cells upon acute ethanol exposure. Authors had also found activation of the AMPK-autophagy axis in esophageal epithelial cells as a compensatory cytoprotective mechanism against alcohol induced cell injury.Major comments:1. The major concern of the manuscript is an inconsistency of cell lines used in each experiment and thus the statistical analysis. Most of the statistical analysis of the data was drawn from either one or 2 cell lines (except for RNA seq). It would be nice to show and draw the conclusions using 3 cell lines (n=3) in triplicates .2. In Fig.3B, authors had claimed that increase in intracellular vacuolar structures as reminiscent of autophagy vesicles (AV). However, the vacuoles in the TEM images look like having single membrane, whereas AV are double membrane structures.3. Also, in Fig. 3B, authors had showed * Mitochondria lacking cristae . Is acute ethanol exposure lead to complete collapse of cristae without any effect on mitochondria volume? Authors should comment on this.4. Authors had claimed that involvement of mitophagy to regulate mitochondrial homeostasis to promote cell survival upon ethanol exposure. Authors had drawn this conclusion based on conversion of LC3BI to II form and p62 degradation . It would be appropriate to show co-localization of PINK Parkin or mitochondrial marker with lysosomal marker as an indicator of mitophagy and its flux.5. There is an inconsistence data regarding effect of 2% ethanol on cell viability within 8hr. Fig 1. explains no effect on cell viability, whereas Fig 5D showed effect on cell viability. Authors should comment on this variability.6. No statistical analysis for some of the western blot data .7. This study is an in vitro study. So, authors should modified the sentence in the introduction (line 8081).**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  1 Sep 2020Dr. Jadeja (Academic Editor)’s comment: “As per Reviewer 3, the issues regarding replicates must be addressed during the revision”.We agree that conclusions would be strengthened with three cell lines as biological replicates and data expressed as an average of three. We have utilized EPC1, EPC2 and EPC3 to evaluate ethanol (EtOH) exposure in 3D organoids and monolayer culture . We have analyzed all three cell lines by RNA-seq to identify genes that are commonly altered upon EtOH exposure . To validate RNA-seq data, however, we focused upon EPC1 and EPC2 as biological replicates in the remainder of the manuscript. We chose EPC1 and EPC2 because they tolerated EtOH better than EPC3, as discussed . Gene expression patterns in EPC1 and EPC2 were deemed closer than EPC3 by principal component analysis . EPC1 and EPC2 displayed consistent results regarding mitochondria morphology , mitochondrial membrane potential , and autophagy . We included additional data with EPC2 to complement the above findings. We have determined cellular ATP level and metabolic profiling , EtOH oxidation by ADH1B and CYP2E1 , and pharmacological modulations of AMPK .We observed differences between EPC1 and EPC2 cells about the effect of EtOH upon AMPK-mTORC1 axis, however. In EPC1, EtOH did not induce as robust changes in mTORC1 substrates  and AMPK  as compared to EPC2 cells. We clarified in the revised text that our model  was supported by data with EPC2 cells only. Additionally, further in vivo data, beyond demonstration of mitochondria depolarization in mice receiving EtOH , are currently not available. These limitations require future investigation to reinforce the model, utilizing primary cell culture, genetically engineered mice, and human subjects. We stressed these points in revised page 26, line 629-647, and modified our conclusions  as well as the title of this manuscript to reflect cell-line specific roles of the AMPK-mTORC1 axis.In general, we had 3-4 technical replicates in all assays, and performed statistical analysis as described in the methods section and figure legends. However, we could not perform statistical analysis on densitometry of most western blots because we could not have multiple technical replicates (i.e. multiple blots from the same set of cell lysates) due to low protein yield from EPCs. We utilized EPC1 and EPC2 as two biological replicates and performed at least two independent experiments for each cell line with cell lysates prepared on different days (i.e. different sets of cell culture plates with or without treatments). Although we performed densitometry on all western blots, we did not show the data as an average of two from two independent experiments. We instead presented representative blots  for each cell line with the densitometry data .Reviewer 1: “…The manuscript is fairly well written with a few grammatical errors noted below. The data support the conclusions of the paper, with the exception of mTOR pathway…discussed below.”Major points:1) “In describing the dilutions used for experimental conditions and controls, it is unclear if the media nutrients are more dilute in the alcohol-containing samples versus the control samples. In the methods it is mentioned that the alcohol is diluted in media while the control cells receive only media. It would be better if the experiments also had diluted control media to a similar extent as the alcohol-containing media. This could be clarified in the methods to more fully explain how the treatments occur if this interpretation is incorrect. Alternatively, this limitation could be explored in the discussion section”In most experiments, cells were exposed to 2% ethanol (EtOH) for up to 8 h. To assess the impact of medium dilution, we compared media supplemented with or without phosphate buffered saline (PBS) in place of EtOH. PBS did not affect cell viability of all three esophageal cell lines tested . We concluded that the potential effect of medium dilution is negligible, if any, as noted in the revised page 13, lines 302-304 and page 14, line 325.In another experiment, now shown in S3 Fig, cell culture medium was first removed and cells were then exposed for 15 seconds to 5-80% EtOH in PBS or PBS only (control). Following removal of PBS with or without EtOH, cells were refed with regular cell culture medium and allowed to grow until cell viability was assessed at 8 h and 24 h time points. In this experiment, medium was not diluted by EtOH or PBS. We clarified the experimental condition in the revised legend for S3 Fig.2) “It is unclear to this reviewer which experiments have been performed with multiple independent cell lines. The results section should clearly indicate when the data is collected in EPC1, EPC2, or EPC3 cell lines and the figures appropriately labelled for clarity.”Please see our response to Academic Editor (Dr. Jadeja)’s comment above. In the revised results section, we have now clarified in text and figures which cell lines were used for each experiment.Minor points:1) Editing/grammar errors to fix in lines: 62, 69, 101, 283, 331, 492.We have corrected these errors throughout the manuscript.2) “Should delete reference to in vivo data since none is presented”.We have now included in vivo data. We demonstrated that a subset of esophageal epithelial cells in mice display mitochondria depolarization following a single bolus oral gavage of EtOH  as described in revised page 5, lines 94-101 (methods) and page 18, lines 426-428 (results).3) “How many reads were collected for the RNA-Seq data?”We have obtained ~32 million input reads per sample on average of which ~24 million were uniquely mapped as described . We have now included these data in S1 Table.4) “Line 248 , how was apoptosis measured?”We have performed flow cytometry for Annexin V and propidium iodide-stained cells to determine apoptosis as shown in new S2 Fig.5) “when quantifying data from organoids, how many were counted/slide and how many replicate slides were quantified?”As elaborated in the revised legend for Fig 1 , we generated organoids in triplicate per condition or time point, and performed live-cell quantitation by automated bright-field microscopy that imaged at least 100 organoids per well and determined the size of individual structures at each time point shown.6) “The two bar graphs in figure 5A  should receive their own lettering (ie 5b and 5c)…”We agree and labeled these bar graphs accordingly after moving them to S6 Fig.7) “the conclusion that mTORC1 signaling is altered from analyzing S6 phosphorylation is a bit of an overstated conclusion as stated in the subheading on line 397 (revised line 553). The RNA-Seq analysis suggests the pathway as suppressed but one experimental piece of data was presented to support this. S6 can be phosphorylated by other pathways than mTORC1 signaling. Would suggest softening the conclusion in this section”.We agree and modified the conclusion  not to overstate about mTORC1 signaling. To reinforce the possible involvement of mTORC1 signaling, we have probed 4E-BP1 as an additional mTORC1 substrate in western blots . Following EtOH exposure, we observed robust inhibition of phosphorylation of the mTORC1 target S6 in both EPC1 and EPC2 cells, as well as decreased phosphorylation of the mTORC1 target 4EBP-1 in EPC2, but little if any in EPC1 cells. This likely reflects some differences in the mTORC1 downstream pathways between the two cell lines. We extended our discussions .Reviewer 2: “This is a potentially interesting paper, however, several problems must be corrected”.Major points:1) Some statements are simply not correct such as "RNA sequencing disclosed robust mitochondrial dysfunction"….RNA sequencing may suggest (!) mitochondrial dysfunction but never shows directly mitochondrial dysfunction…You can change this to "RNA sequencing shows mitochondrial alterations."We agree that our original manuscript contained incorrect or bold statements. We appreciate the helpful advice by this Reviewer and have revised accordingly.“Please emphasize both in abstract and text that you have provided direct evidence for the mitochondrial dysfunction by following methods: Mitochondrial metabolism; Respiratory, Mitochondrial oxidative stress (MitoSOX) and cellular ATP level”.We have edited the abstract and the text to clarify the methods used to show mitochondrial dysfunction.2) “Please explain how ethanol induces mitochondrial stress . It is most likely not a direct effect. Please explain in introduction and discussion sections. RNA sequencing data suggest that ethanol may initially affect the nuclei protein expression which later translate into mitochondrial dysfunction. Please comment and explain”.We have changed mitochondrial “stress” to “dysfunction” in revised Fig 11 as recommended by this Reviewer.We agree that it is unlikely that ethanol (EtOH) induces mitochondrial dysfunction directly. It is well established that EtOH treatment results in conditions that promote mitochondrial ROS formation by mitochondria as corroborated by our data . ROS have been shown to form mitochondrial (mt) DNA adducts and mtDNA damage upon EtOH exposure . Additionally, EtOH affects the mitochondrial antioxidant mechanism, further exacerbating the damage  as discussed in revised page 28, lines 701- 704.We have explored the potential role of EtOH oxidation in mitochondrial dysfunction. To this end, we first assessed mitochondrial membrane potential (∆Ψm) by flow cytometry in EtOH-treated EPC1 and EPC2 cells  where EtOH induced a small subset of cells with decreased ∆Ψm, indicating mitochondrial depolarization. We performed RNA interference experiments directed against ADH2 and CYP2E1, two major alcohol metabolizing enzymes that catalyze EtOH oxidation . We have observed that knockdown of either molecule prevented EtOH from inducing cells with mitochondrial depolarization, suggesting that EtOH oxidation may be involved in mitochondrial dysfunction. We have described these results , and discussed .It is most likely acetaldehyde, a toxic chief metabolite of EtOH, contributes to mitochondrial dysfunction. We have previously demonstrated that acetaldehyde causes oxidative stress in murine esophageal keratinocytes and that was augmented in cells with loss of Aldh2, a mitochondrial enzyme essential in acetaldehyde clearance . Of three cell lines tested, EPC3 appeared to be more sensitive to the toxic effect of 2% EtOH than EPC1 and EPC2 . EPC1 and EPC2 cells carry wild-type ALDH2 while EPC3 carries a single nucleotide polymorphism for ALDH2. This mutant ALDH2 enhances EtOH toxicity by delaying acetaldehyde clearance. We discussed the possibility that the ALDH2 status may influence the degree of EtOH-induced mitochondrial dysfunction .Our RNA-seq data suggest that EtOH exposure may affect nuclear proteins essential in mitochondrial functions. They include TP53 and FOXO3 . Under cellular stress, TP53 is translocated to mitochondria to interact with the Bcl2 family members to trigger apoptosis . FOXO3 regulates transcription of oxidative phosphorylation (OXPHOS) genes in mitochondria. AMPK promotes FOXO3 translocation to mitochondria and mtDNA binding . Thus, FOXO3 may contribute to maintenance of mitochondrial functions in cells surviving EtOH-induced stress conditions. We have discussed these potential mechanisms in the revised manuscript .Reviewer 3:Major comments:1. “The major concern of the manuscript is an inconsistency of cell lines used in each experiment and thus the statistical analysis. Most of the statistical analysis of the data was drawn from either one or 2 cell lines (except for RNA seq). It would be nice to show and draw the conclusions using 3 cell lines (n=3) in triplicates .”Please see our response to Academic Editor (Dr. Jadeja)’s comment at the beginning of this rebuttal where we have addressed this important point raised by Reviewer 3.2. “In Fig.3B, authors had claimed that increase in intracellular vacuolar structures as reminiscent of autophagy vesicles (AV). However, the vacuoles in the TEM images look like having single membrane, whereas AV are double membrane structures.”We have rereviewed TEM images with an experienced pathologist (AJK) who has identified double-membrane vesicles as shown in revised Fig 4C. Most of the vacuoles were suspected as involutional mitochondria and some autophagy vesicles. The apparent lack of double membrane is possibly accounted for by insufficient resolution to detect distended (double) membranes in vacuolized (distended) organelles as discussed in revised page 29, lines 714-724).3. “Also, in Fig. 3B, authors had showed * Mitochondria lacking cristae . Is acute ethanol exposure lead to complete collapse of cristae without any effect on mitochondria volume? Authors should comment on this”.In the original text , we have pointed out volume increase (swelling) of mitochondria accompanying cristae changes. We have now clarified this in the revised legend for Fig 4B.4. “Authors had claimed that involvement of mitophagy to regulate mitochondrial homeostasis to promote cell survival upon ethanol exposure. Authors had drawn this conclusion based on conversion of LC3BI to II form and p62 degradation . It would be appropriate to show co-localization of PINK Parkin or mitochondrial marker with lysosomal marker as an indicator of mitophagy and its flux”.We agree visualization of mitophagy is important as we have previously demonstrated in transformed EPC2 derivative cells under oxidative stress stimulated by transforming growth factor-β . However, it was not our intention to claim involvement of mitophagy based on our western blot data in this study. We stated in the original Results section regarding transmission electron microscopy findings  that “…numerous abnormal mitochondria were positioned adjoining to these vacuolar structures, suggesting that mitochondria-targeted autophagy (i.e. mitophagy) may be occurring…” This was a bold statement without direct evidence of mitophagy provided. We have removed this statement. We now reinforced our autophagy data in both EPC1 and EPC2 cells . In the discussion section, we have pointed out the possibility that autophagy may have a role in removal of EtOH-induced dysfunctional mitochondria via mitophagy, to promote cell survival under ethanol-induced stress, a subject that warrants further study in future investigations .5. “There is an inconsistence data regarding effect of 2% ethanol on cell viability within 8hr. Fig 1. explains no effect on cell viability, whereas Fig 5D showed effect on cell viability. Authors should comment on this variability”.Original Fig 1 and Fig 5D showed differential experimental conditions in 3D organoids and monolayer culture, respectively. Additionally, we cited 3D organoid data  as a rationale to use 2% ethanol (EtOH) and 8 h exposure time for RNA-seq experiments performed in monolayer culture. We apologize for any confusion resulting from our initial presentation of these data. We have now presented 3D organoid and monolayer culture data separately . Esophageal epithelial cells appeared to be more sensitive to EtOH-induced toxicity in monolayer culture than in tissue-like 3D organoid culture conditions . We have cited monolayer culture data  as a basis of setting conditions for RNA-seq experiments.We have also recognized variability, albeit modest, in EPC1 and EPC2 cell response to 2% EtOH. In revised Fig 8D and E, 2% EtOH alone did not affect EPC1 cell viability at 8 h time point. However, EPC2 cell viability was decreased by 10% as evaluated by WST-1 assays that detect live cells. In revised Fig 2B, 2% EtOH did not increase cell death in either EPC1 or EPC2 at 8 h time point as assessed by flow cytometry of DAPI-stained cells that detects dead, but not necessarily dying, cells. This likely reflects differences in the assay mechanisms and sensitivities, as noted in revised page 22, line 538. 6. “No statistical analysis for some of the western blot data ”.Please see our discussion at the end of our response to Academic Editor (Dr. Jadeja)’s comment above about this critical point and our revised efforts to address it.7. “This study is an in vitro study. So, authors should modified the sentence in the introduction (line 8081)”.We have now included in vivo data. We demonstrated that a subset of esophageal epithelial cells in mice display mitochondria depolarization following a single bolus oral gavage of EtOH  as described in revised page 5, lines 94-101 (methods) and page 18, lines 426-428 (results).AttachmentResponse to reviewers 08-31-2020.docxSubmitted filename: Click here for additional data file. 10 Sep 2020Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytesPONE-D-20-13394R1Dear Dr. Nakagawa,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Ravirajsinh Jadeja, Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :The Authors have done a substantial revision and the revised manuscript can be accepted in its present format.Reviewers' comments: 14 Sep 2020PONE-D-20-13394R1Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytesDear Dr. Nakagawa:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org.If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access.Kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Ravirajsinh Jadeja Academic EditorPLOS ONE


MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546).Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia ( Cyclin-dependent kinases (CDKs) have been the subject of intense research for the last two decades because of their association with the cancer hallmarks of uncontrolled proliferation and increased survival. Following the successful development and approval of CDK4/6 inhibitors, the focus of pharmaceutical CDK inhibitor drug development in oncology has shifted to compounds regulating transcription of key oncogenic and pro-survival proteins .bona fide target in oncology with an acceptable therapeutic window. Two decades later, this theory is being evaluated through the development of more clinically applicable approaches to RNA pol II transcriptional inhibition, including small molecule drugs targeting CDK9, BRD4 and DOT1L methyltransferase MCL1 is o members . ImportaFLT3) internal tandem duplication (ITD) leukemia , 2.24 , 2.4 , 3.92–3.82 , 4.67–4.45 , 6.16 , 6.87 , 7.37 , 8.31 ; δC  160.11 (C), 157.68 (C), 154.57 (C), 149.42 (CH), 146.38 (C), 134.54 (CH), 129.24 (C), 124.84 (CH), 71.52 (CH), 59.65 (CH), 40.33 (CH2), 24.94 (CH2), 23.89 (CH3), 23.52 (2 x CH3), 1737 (CH3), 12.57 (CH3); m/z 398 (M + H)NMR spectrum data: δKinase assays for CDK2, CDK4, CDK7 and CDK9 were carried out as described previously using recombinant CDK/cyclins generated at Cyclacel Ltd, Dundee, UK ,34,35.50 determination. IC50 values were determined from a 10-point concentration curve using ATP concentrations approximating Km values. The value obtained for the reaction control (complete reaction mixture) was set as 0% inhibition and for the background (no enzyme) as 100% inhibition. The percentage inhibition of each test solution was calculated, which allowed the IC50 value to be determined from concentration versus percentage inhibition curves by fitting to a four parameter logistic curve.Profiling of 1 μM fadraciclib against a 256-kinase selectivity panel was performed at Carna Biosciences. Enzymes that inhibited >50% at the tested concentration were selected for IChttps://depmap.org/portal/).We analyzed data available from the Cancer Dependency Map to determine the effects of CRISPR-Cas9 knockdown of CDKs inhibited by fadraciclib on the viability of >700 cancer cell lines, including those used in the present study. Results were shown as a CERES gene effect that scores the Dependency of cell lines on a given gene Score  or partial tandem duplications (PTD): EOL-1 (MLL-PTD), ML-2 (MLL-AF6), MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), NOMO-1 (MLL-AF9), OCI-AML2 (MLL-PTD), THP-1 (MLL-AF9) and 6 contain no MLL abnormalities: HEL, HL60, Kasumi-1, KG-1, OCI-AML5 and PL-2. All AML cell lines were purchased from DSMZ with the exception of THP-1 and HL-60, which were sourced from ECACC and ATCC, respectively.Seven of the thirteen AML cell lines contain 2. Information on the cell lines including their classification, origin, supplier, doubling time, tissue culture media, MLL gene status and other molecular and genetic characteristics are summarized in All cell lines were grown in tissue culture medium recommended by the supplier in incubators at 37°C and 5% CO2. The following day, 100 μl fresh medium containing increasing concentrations of appropriate compound was added and the cells were incubated for up to 72 h at 37°C, 5% CO2. For pulse treatment of solid (adherent) tumor cell lines, medium was aspirated and replaced with fresh drug-free medium. For pulse treatment of suspension lines, plates were centrifuged at 478 xg for 5 min at the end of the incubation period with compound, medium containing compound was carefully aspirated and replaced with fresh compound-free medium. A total of 72 h after the start of treatment, 20 μl of a 10X stock of resazurin sodium salt  prepared in medium was added to the cells and incubated for up to 3 h. Absorbance was measured on the Wallac Victor plate reader  at 544–595 nm. IC50/70/90 calculations were determined in XLFit.Solid tumor cell lines were seeded in 100 μl medium into 96-well plates at an appropriate cell number determined by a cell dilution assay . AML suspension cells were seeded in 96-well plates at approximately 3000 cells per well. Cells were incubated overnight at 37°C, 5% CO5 cells/ml in a 6-well plate and incubated with the test compound or vehicle control (DMSO) for 6, 8 or 10 h. At the end of the incubation period, cells were collected into Falcon tubes, pelleted via centrifugation (5 min at 478 x g), and medium containing compound was aspirated. The cells were resuspended in 4 ml of fresh compound-free medium and returned to the 6-well plates for further incubation. Following compound addition, 100 μl (at 24 h), 75 μl (at 48 h), 50 μl (at 72 h) or 30 μl (at 96 h) of a homogeneous suspension of cells were removed from the culture, made up to 100 μl with medium and mixed with 100 μl Guava Viacount reagent  in a 96-well plate. Samples were incubated for 20 min in the dark, mixed and then analyzed by GuavaSoft 2.6 software on the Guava Easycyte 8HT (Millipore). Guava Viacount software was used to gate and calculate the proportion of viable and dead cells in the population.AML cells were seeded at 5 x 105 cells per 10 cm dish in a total of 9 ml medium and incubated overnight at 37°C, 5% CO2. The following day 1 ml of medium containing 10x test agent or vehicle was added to the cells, which were then incubated for 24 h at 37°C, 5% CO2. Cells were collected, prepared, propidium iodide stained for DNA content analysis, and analyzed as described previously  and showed excellent selectivity. The percent inhibition of each kinase by fadraciclib (CYC065) is indicated in the table. Nine kinases were inhibited by >50% and the IC(DOCX)Click here for additional data file.S7 Table50 values were determined, and compared. Nine out of thirteen cell lines were highly sensitive to fadraciclib (CYC065) and displayed 6 h pulse IC50 values similar to their 72 h continuous IC50 values. Values are the mean of 3 independent experiments.AML cell lines were incubated with fadraciclib (CYC065) for the indicated duration and IC(DOCX)Click here for additional data file.S8 TableConcomitant treatment with fadraciclib and BCL2 inhibitor venetoclax (ABT199) or BCL2/BCL2L1 inhibitors ABT263 and ABT737 was performed and analysed as described in Materials and Methods. Average combination index (CI) and SD values are listed.(DOCX)Click here for additional data file.S1 FigKasumi-1 cells were treated with 0.5 or 1.0 μM fadraciclib (CYC065) for up to 6 h, with cells harvested every hour for examination of the levels of MCL1 and cleaved PARP by Western blotting (A). Kasumi-1 cells were pulse treated with 0.5 or 1.0 μM fadraciclib (CYC065) for up to 6 h with medium replaced at the indicated times, and then samples harvested at 24 h from the start of treatment to assess viability by Viacount assay (B).(DOCX)Click here for additional data file.S2 FigMLLr/MLL-PTD appeared to be more sensitive to fadraciclib (CYC065) than non-MLLr/MLL-PTD cell lines.An ALL cell line panel was treated with fadraciclib (CYC065) for 10 h then incubated in compound-free medium until viability was assessed at 72 h. All cell lines with (DOCX)Click here for additional data file.S3 FigAML cell lines were treated with fadraciclib (CYC065) for 16 h at concentrations from 0.1–1.0 μM, then fixed and stained for cell cycle analysis by propidium iodide. The two sensitive cell lines, MOLM-13 and Kasumi-1, both show robust induction in subG1, indicative of cell death, whereas HEL cell line shows only a modest increase in sub G1 after 16 h continuous treatment with the compound.(DOCX)Click here for additional data file.S4 Fig50 values with short pulse or continuous treatment, whereas, the resistant cell lines A549, MDA-MB-231 and PC3, are only sensitive to prolonged treatment with fadraciclib (CYC065).Sensitive cell lines, A2780 and H23, show similar IC(DOCX)Click here for additional data file.S5 FigMCL1 and BCL2L1 mRNA as determined by qPCR were examined in selected sensitive and resistant solid tumour cell lines. Sensitive cell lines, H23 and A2780, had high levels of MCL1 and lower levels of BCL2L1 –confirming the results obtained by Western blotting.The levels of (DOCX)Click here for additional data file.S6 Fighttps://depmap.org/portal/; Computational correction of copy number effect improves specificity of CRISPR–Cas9 essentiality screens in cancer cells [Data were obtained from genome-wide CRISPR-Cas9 screens with the Avana sgRNA library in cancer cell lines and deposited as part of the Cancer Dependency Map project (er cells . A lower(DOCX)Click here for additional data file.


Reynoutria sachalinensis rhizomes, against two human melanoma cell lines  and two normal human cell lines—keratinocytes (HaCaT) and the primary fibroblast line. Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed. Cell viability was studied using an MTT assay. A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD). Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B. This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A. No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence. Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future. Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family. So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma. In this study, we tested these two natural products, isolated from  Melanoma is a malignant melanocytic tumor. The incidence of melanoma in the human population is ever-increasing and mortality in the group of patients with an advanced form of this disease is very high. Although melanoma accounts for only 4% of all skin cancers, it is responsible for >80% of skin cancer-related deaths . The earAmong natural products of plant origin, polyphenols have been found to target different hallmarks of cancer cells, often interfering with several of them at once, such as promoting apoptosis, regulating autophagy and inhibiting proliferation, and as well, they modulate the metabolic properties of cancer cells and the affect tumor-induced immunosuppressive behavior through the modulation of T-regulatory (Treg) cell populations and many others, including epigenetic modulation ,8. The nReynoutria sachalinensis [F.Schmidt] Nakai . This plant is native to East Asia but widely spread throughout Europe and North America becoming a noxious invasive pest. It is used traditionally as an herbal medicine in its natural distribution area for the treatment of arthralgia, jaundice, amenorrhea, cough, scalds and burns, traumatic injuries, carbuncles and sores , which precede temporal increases in fluorescence due to secondary necrosis (loss of membrane integrity). This kinetic difference in the emergence of the signals is the hallmark of an apoptotic phenotype . Our resHowever, the results of our study enable us to suppose that both vanicosides could affect the viability of malignant melanoma cells by inhibiting the BRAF(V600E) and MEK1 kinases. Molecular docking analyses show that both vanicosides were successfully docked into the active sites of the BRAF(V600E) and MEK1 kinases. Vanicosides A and B form four hydrogen bonds with BRAF kinase residues. The interaction with the Asp594 residue is present among them and this is a crucial interaction in the case of the original PLX4032 ligand (ligand of 3OG7 protein structure) . Lys483 50 values of 44 and 31 μg/ml, respectively. In this study, both vanicosides also showed cytotoxicity against the MCF cell line at submicromolar dose levels  Nakai, and identified according to the procedure described in our previous paper [Vanicoside A and vanicoside B were isolated from the rhizomes of us paper .® . Human fibroblasts were taken with the patient’s consent from the oral cavity. The cells were grown as a monolayer in Dulbecco modified Eagle medium , which was supplemented by 2 mM L-glutamine, 10% fetal bovine serum  and 50 μg/ml streptomycin  at 37 °C, 5% CO2. Before every experiment, cells were removed by 0.25% trypsin with 0.02% EDTA .Four human cell lines were tested: two melanoma cell lines - the amelanotic malignant melanoma C32(BRAFV600E) and the malignant melanoma A375(BRAFV600E) as well as two normal cell lines—keratinocytes (HaCaT) and the primary fibroblast line—. The C32, A375 and HaCaT cell lines were purchased from ATCC4 cells/well. After the incubation with selected concentrations of vanicoside A or B, the experiments were conducted according to the manufacturer’s protocol. The absorbance was determined using a multi-well scanning spectrophotometer at 570 nm . The mitochondrial metabolic function was expressed as a percentage of viable treated cells in relation to untreated control cells. Staurosporine was used as the cytotoxic positive control at 2 μM.The viability of cells was determined by an MTT assay  after experiments with different concentrations of vanicoside A or vanicoside B. Dimethyl sulfoxide  was used as a solvent for vanicosides. The following concentrations were used for the experiment: 2.5, 5, 10, 25, 50 and 100 µM. The MTT assay was used to estimate a mitochondrial metabolic function through the measurement of mitochondrial dehydrogenase after 24, 48 and 72 h incubation after experiments. For the experiment, the cells were seeded in 96-well microculture plates at 1 × 104 cells/well. A set of control wells without cells present (growth medium only) to determine background luminescence and background fluorescence was included. The appropriate dilution of vanicoside A (or B) was prepared in growth medium at 4× the desired final concentration. A set of control wells without a vanicoside present (untreated controls) was included. An amount of 50 µL of the vanicoside A (or B) dilution was added to the appropriate wells in the 96-well assay plate. Then, 100 µL of 2× concentrated RealTime-Glo™ Annexin V Apoptosis and Necrosis Detection Reagent in growth medium was added to each well. Cells were incubated in a covered 96-well assay plate at 37 °C/5% CO2 in a humidified cell culture incubator. Luminescence and fluorescence  on the GloMax® Discover instrument  using the RealTimeGlo™ Annexin V Apoptosis Assay pre-programmed protocol at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h were measured.Cells were seeded in 50 µL growth medium in white 96-well microtiter plates at the concentration of 1 × 10For the molecular docking studies, two melanoma targets were selected: B-Raf kinase V600E oncogenic mutant and MEK-1 kinase. Docking was performed with the GOLD  software  and crysThe binding site was determined based on original ligand interactions in the active site and residues within 10 Å from the original ligand. The number of poses to be generated was set to 10 and GoldScore was used as the scoring function to rank the docked compounds. The final protocol for the docking was based on successful re-docking of the reported ligands. Docking results were analyzed with the Accelrys Discovery Studio software 4.1.   and bestt-test using GraphPad Prism v. 7 . The t-test was used for the means comparison between treatments and the control (cells without vanicosides) samples. p ≤ 0.05 or p ≤ 0.0001 were considered statistically significant. The graph of time- and dose-dependent increases in luminescence and fluorescence was presented using the non-linear regression fit (least squares fit) of GraphPad Prism v. 7.All the experimental procedures were performed in triplicate. The values obtained from the MTT and RealTime-Glo™ Annexin V Apoptosis and Necrosis assay are presented as the mean of three replicates ± SD. To evaluate the distribution of the results, the Shapiro–Wilk test was used. The statistical analysis was performed by Student’s R. sachalinensis demonstrated significant cytotoxicity against amelanotic and melanotic melanoma cell lines bearing the BRAFV600E mutation. Vanicoside A decreased the viability of the amelanotic C32 cell line significantly stronger than vanicoside B, whereas both compounds showed similar effect against the less sensitive A375 cell line. The observed differences in cytotoxicity could result from the additional acetyl group in vanicoside A, and from the differences in the tested melanoma cell lines—lower melanin content in amelanotic cells, suggesting that in the melanoma cell lines, an oxidative stress-related death mechanism could be involved. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well significant phosphatidylserine (PS) exposure, but without a measurable cell death-related fluorescence. Therefore, further studies are warranted to ensure the selectivity against cancerous cells and safety. Vanicosides A and B induced an apoptotic death pathway in the melanoma cell lines. However, due to the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like PTP-mediated necrosis. Molecular docking revealed that both vanicosides may be able to bind to the active sites of the B-RafV600E and MEK-1 kinases. The resulting kinase inhibition could interfere with the RAF-MEK-ERK signaling cascade during melanocytic neoplasia, but this finding must be verified using a cell model.In conclusion, both tested phenylpropanoid esters from


Methods: The study concerned a population of 1756 subjects from 12 secondary schools (ESO). The instruments used included the Utrecht Work Engagement Scale—Student (UWES-S), the Perception of Success Questionnaire (POSQ), and the Academic Self-Concept Scale (ASCS). Results: The results revealed significant correlations between academic engagement, task-oriented goal orientation, and academic self-concept. In addition, task orientation was found to play a positive mediating role between academic engagement and academic self-concept, leading to adaptive models in secondary school students. Conclusion: These results highlight the need to promote goal orientation in order to stimulate self-determined behaviours in the school environment and improved levels of academic self-concept, which in turn will facilitate the psychological and personal development of the student and increase the chances of academic success.Some students many not possess the necessary strategies and skills to meet the demands of academic life and develop negative attitudes, physical and mental exhaustion, and other attitudes that will undermine their personal and academic development. This study analyses the relationship and possible role of goal orientation as a mediator between engagement and academic self-concept.  At school, students are exposed to numerous contextual and personal circumstances that may significantly affect their personal and academic development, especially in secondary school, during adolescence, which is a key stage in the life of a person, preceding adulthood . Some stOne of the most important psychological variables in this regard is academic engagement, a concept derived from positive psychology, which is defined as a psychological state characterised by resilience against stressful and exhausting situations . From anAs such, academic engagement responds to adaptive behaviours characterised by engagement, satisfaction, and commitment to academic tasks. Previous studies have linked academic engagement with satisfactory academic performance , high leOn the other hand, concerning academic goals, one of the most important socio-cognitive theories is goal orientation theory , a frameSpecifically, task orientation is defined as an integrated model of beliefs or pattern of attributions that directs the behavioural intentions of a person, formed by different modes of approach, commitment, affection, and response to achievement activities . In thisIn this way, numerous studies have found positive correlations between task orientation and variables such as persistence, will, and commitment ; motivatFinally, academic self-concept is an important construct that plays a key role in academic performance . It is dAcademic self-concept reflects a student’s perception of themselves in terms of academic experience and progress . AccordiPrevious studies have established the relationship between positive self-concept and several interpersonal variables such as higher levels of psychological adjustment and personal competence, a lesser likelihood of behavioural problems and depression, greater resilience against stress, and better academic behaviour ,30,31.Few studies have analysed the relationship between our target variables in adolescent populations, but they agree in relating them closely to adaptive behaviours. Extremera, Durán, and Rey  point ouIn this context, and given the dearth of works that draw specific links between our target variables, this study aims to analyse the relationship between academic engagement, goal (task)-orientation, and academic self-concept in adolescent students. The research questions are aimed, on the one hand, at the relationship between variables of engagement and academic self-concept, which play a fundamental role in the school environment. On the other hand, we wondered if the goal-task orientation, because it was more self-determined, could mediate the previous relationship between academic engagement and academic self-concept, and what weight it had in this relation on adolescent students.Two working hypotheses are set forth:(a) (Task) goal-orientation will be positively related with academic engagement and academic self-concept; and (b) (Task) goal-orientation will perform as a mediating variable in the relationship between academic engagement and academic self-concept in adolescents.This research was composed by 1756 students, both male  and female , from 12 public secondary schools. Their ages ranged from 12 to 18 years . Participants were selected through two stages: Firstly, the sample was selected through random sampling where all secondary schools had the same probability of being chosen for the research, communicated for it with sufficient advance, and gave their approval in the participation of the research. Secondly, by sample of volunteers who were the students of the educational centres initially selected. Inclusion criteria were the ability to read and communicate in the Spanish language to ensure that they could understand and answer the questionnaire. Incomplete questionnaires were discarded, including those from students who decided to drop out half way and students with cognitive disorders who could not fully understand the questionnaire were excluded from the study. The response rate was approximately 98%. The number of questionnaires discarded amounted to 35, which were not completed in their entirety or were made illegible by the students.Three widely used questionnaires were selected to channel the participant’s responses:First, the Utrecht Work Engagement Scale—Student (UWES-S)  translatGoal orientations were measured with Roberts, Treasure and Balagué’s (1998) Perception of Success Questionnaire (POSQ) , translaFinally, the Academic Self-Concept Scale (ASCS)  was usedThe questionnaires were handed out to participants with the cooperation of the schools. Both the students and parents/guardians signed informed consent forms. In coordination with the head of studies, questionnaires were handed out in a single day to all secondary school students in each school where the questionnaire was supplied in each class to all the students attending it. All subjects and parents/guardians were informed of the purpose and nature of the study, thus following the ethical guidelines conveyed by the Declaration of Helsinki . The resp ≤ 0.05 level of significance was adopted, with a 95% confidence level.Descriptive statistics were used to characterize the social and demographic characteristics of the sample and target variables. Correlations between engagement, (task) goal orientation, and academic self-concept were calculated with IBM SPSS v26.0. Finally, the SPSS v26.0’s MACRO tool was used to carry out mediation analyses by bootstrapping . For all operations, a The sample comprised 1756 students, male  and female  from 12 public secondary schools. Their ages ranged from 12 to 18 years  .As illustrated in Correlations between the target variables are presented in In order to assess whether the relationship between engagement and academic self-concept is mediated by (task) goal-orientation, calculations based on Tal-Or, Cohen, Tsarfati, and Gunther , using Hp > 0.001). Zero was not included in the bootstrap interval B = 0.14, SE = 0.03, 95%  so it can be argued that (task) goal orientation mediates in the relationship between engagement and academic self-concept.As shown in p < 0.10) but, when (task) orientation is taken into consideration there is a total effect of 0.18, p < 0.001 (direct effect + indirect effect), the proportion of variance being explained by model 2R = 0.50 ***. This suggests that goal orientation plays a mediating role between academic engagement and academic self-concept, a conclusion which has significant practical implications.It was observed that academic engagement had no direct positive effect on academic self-concept  and age  were controlled, not being significant in the mediation model.In addition, the covariates of gender  goal orientation, and academic self-concept in adolescents.The first hypothesis stated that (task) goal orientation is positively related to academic engagement and self-concept. According to this hypothesis, task-oriented students will present higher levels of academic engagement and self-concept. The results confirm this hypothesis by showing that goal orientation is positively correlated with academic self-concept, especially academic engagement.This is generally consistent with the existing literature on goal orientation. On the one hand, numerous studies have shown a clear relationship between (task) goal orientation and academic engagement. Granero, Gómez, Abraldes, and Baena  present On the other hand, other studies establish a direct link between goal orientation and academic self-concept in adolescents. Carranza and Apaza  found seThe second hypothesis stated that (task) goal orientation plays a mediating role in the relationship between academic engagement and academic self-concept in adolescents. The results also confirm this hypothesis by showing that (task) goal orientation has an effect on both academic engagement and academic self-concept, therefore playing a mediating role between them.On the one hand, the mediation analysis results show that academic engagement does not have a significant predictive value for academic self-concept. That is, the direct effects of the former on the latter were not statistically significant. On the other hand, if (task) goal orientation is taken into consideration, a significant mediating role between both constructs can be attested. These results show the important role played by (task) goal orientation in adolescents. However, it should be discussed with caution due to the cross-sectional nature of the study where temporal precedence cannot be established. Nonetheless, the mediating role of goal orientation on the other target variables has not been subject to specific studies to date with many present results that can be usefully compared with ours. Fernández, Goñi, Camino, and Zubeldia  highlighAlthough, as we have indicated previously, there are not many studies that investigate the specific variables proposed in this research, there are some studies that also approach the study of the relationship between the engagement and academic self-concept constructs in the adolescent student population. Gutiérrez, Tomás, Gómez, and Moll  showed tThese results show that target variables played a significant role in promoting adaptive behaviours characterised by greater levels of academic engagement and a task-oriented goal orientation, which in turn will lead to higher levels of self-concept. This will not only improve their academic performance, but also their personal and psychological development in such an important stage in the life cycle of an adolescence. For prospects, it would be interesting to carry out psychological and academic intervention programs to enhance academic variables as important as student engagement that would strengthen their academic self-concept, keys for proper personal and academic development. At the same time, intrinsic orientations in the assumption of school tasks would help correct the personal and academic development of students. Future studies should examine the relationship between goal orientation and other psychological and academic variables. Similarly, it would also be necessary to take academic performance into consideration, especially in order to examine its relationship with academic self-concept.The results of our research encourage the scientific community to continue investigating, incorporating new variables, and seeking new questions and research objectives that lead to the personal and academic development of adolescents.