Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN. The selective filtration of plasma is performed in the cortex of the kidney in the glomerulus of the nephron. Endothelial cells of blood arteries that enter the glomerulus, a glomerular basement membrane (GBM) between them and podocytes, and podocytes themselves form the glomerular filtration barrier (GFB). Podocytes are highly specialized cells that are dedicated to plasma filtration. They consist of a cell body and major and foot processes. Foot processes branch out from major processes and tightly cover blood arteries in the glomerulus. Between neighboring foot processes, a cell–cell junction is present, forming slit diaphragms that form a size-selective barrier for the excretion of proteins that is critical for proper kidney filtration .Diabetes mellitus (DM) affected 8.5% of adults worldwide in 2014 . It is cKlotho is an anti-aging molecule. Upon its discovery, Klotho was shown to extend its lifespan in mice by 30% and protect them from many disorders, especially disorders of renal tissue ,14. KlotKlotho has frequently been reported to decrease inflammation through suppression of the activation of numerous proinflammatory cytokines, chemoattractants, and receptors. Moreover, Klotho inhibits tissue fibrosis ,19. TherBoth membrane-bound and soluble Klotho can bind to fibroblast growth factor (FGF) receptors 1-4 (FGFR1-4), serving as a coreceptor protein for FGF23. Klotho alters the structure of these receptors and increases their binding affinity for FGF23 twenty-fold. FGFRs are widespread throughout the body ,25. HoweThe present study analyzed levels of Klotho expression in renal tissue and its levels in serum and urine under both standard and hyperglycemic conditions. Additionally, we investigated whether Klotho influences FGFR1-4 expression and glucose metabolism in podocyte cells that are cultured in media with standard glucose (SG) or high glucose (HG) concentrations. Finally, we investigated whether Klotho protein influences albumin permeability of the GFB. In brief, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.11 ± 3.47 × 1010 nm2 vs. 1.37 × 1012 ± 9.36 × 1010 nm2, respectively; During acute kidney injury, both plasma and urine levels of soluble Klotho protein decrease in affected individuals, which was proposed to be a biomarker of the disease . TherefoThe amount of soluble Klotho protein decreased in urine, and membrane-bound Klotho decreased in renal tissue and glomeruli in diabetic rats . We nextFGFR1 and FGFR2 genes significantly decreased by 20% and 21.5%, respectively , >98% decrease in FGFR2 (1.85 × 1010 ± 1.56 × 109 nm2 vs. 9.87 × 1011 ± 1.09 × 1011 nm2), ~83% decrease in FGFR3 (2.23 × 1011 ± 8.44 × 109 nm2 vs. 1.31 × 1012 ± 3.34 × 1010 nm2), and >78% decrease in FGFR4  caused a ~15% and ~18% increase in the mRNA expression of FGFR1 and FGFR2, respectively, in human podocytes that were grown under SG conditions , low levels of calcitriol are found, which is an activator of Klotho expression . We founKim et al., reported the presence of Klotho in human glomeruli . To our FGFR1 and FGFR2. We also found that the addition of recombinant Klotho to the cell medium increased the mRNA expression of FGFR1 and FGFR2 under SG conditions, with an upward tendency under hyperglycemic conditions. This tendency accelerated at the protein level, in which the addition of Klotho significantly increased the expression of both FGFR1 and FGFR2 in podocytes that were grown in HG, which was also confirmed by immunofluorescent staining.We also found that all four FGFRs were present in human podocytes at both the mRNA and protein levels. Under hyperglycemic conditions, we observed a significant decrease in the protein levels of FGFR1-4 and the mRNA expression of Studies of the level and function of FGFRs in renal tissue in diabetic individuals are scarce. Consistent with the present findings, Taylor et al., reported a decrease in the protein level of FGFR4 in mice with CKD . Cheng eIn the present study, Klotho increased glycolysis and glycolytic capacity in human podocytes under both standard and hyperglycemic conditions. To our knowledge, the only studies that investigated the influence of Klotho protein at the level of glycolysis were performed under aerobic conditions and with organs other than the kidneys. In the brain, full-length Klotho protein participates in supplying nutrients to neurons by astrocytes. Klotho stimulates a rapid increase in aerobic glycolysis and lactate release from astrocytes, which is needed for energy production by neurons . HoweverOur research group previously found that HG conditions increase albumin permeability in rat glomeruli and cultured rat podocytes, accompanied by F-actin redistribution to cortical regions of podocytes ,48,49. HTo strengthen the findings of the current study, future analyses should concern testing of human serum and/or urine samples for Klotho level assessment. These analyses could also show results of iv vivo-based studies of FGF receptors levels, glycolytic parameters and albumin permeability of glomeruli and podocytes after Klotho supplementation in diabetic rats.The experiments were performed with male Wistar rats (167–342 g) that were obtained from the Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland. The rats were maintained on a 12 h/12 h light/dark cycle with free access to a standard pellet diet and tap water. The experiments were conducted in accordance with directive 2010/63/EU, and the protocol was approved by the local Ethical Commission at the University of Science and Technology in Bydgoszcz, Poland (no. 54/2018).The experiments were conducted with male rats that were subjected to diabetes and induced by streptozotocin , and age-matched control Wistar rats. In diabetic rats, the experiments were performed 14 days after diabetes induction, only in animals with fasting blood glucose concentrations >250 mg/dL . Blood s2, 0.9 mM CaCl2, and 5.6 mM glucose. The renal capsule was removed, and a cortex was minced with a razor blade and then pressed through a series of sieves with decreasing pore diameters . The obtained cell suspension contained decapsulated glomeruli without afferent and efferent arterioles. The entire procedure was performed in an ice bath and completed in less than 1 h.Male Wistar rat kidneys were removed and placed in ice-cold phosphate-buffered saline  supplemented with 0.49 mM MgClHuman immortalized podocytes  were generated, as described previously . The expTo assess the amount of membrane Klotho protein in human podocytes, cells were incubated for 30 min with biotin solution  in PBS (pH 8.0). Excess biotin was washed out with PBS with the addition of glycine . Subsequently, the cells were lysed, as described previously . A portiHuman podocytes and rat glomerulus lysates were prepared, as described previously ,52. AmicFormalin-fixed paraffin-embedded kidney specimens that were obtained from Wistar control and Wistar STZ rats were deparaffined with Histochoice Cleaning Agent (Sigma Aldrich) and ethanol and hydrated in water. Epitopes were retrieved by incubating the specimens in sodium citrate buffer  at 97 °C for 20 min. The specimens were blocked with 5% bovine serum albumin (BSA) for 1 h. Bovine serum albumin was then removed from the slides, and primary antibodies (diluted in 5% BSA) were added to the specimens, followed by overnight incubation at 4 °C . The nexhttps://myassays.com/) (accessed on 3 August 2020).A commercially available Rat Klotho ELISA Kit  was used to determine concentrations of soluble Klotho in serum and urine samples from Wistar rats. Twenty-four hour urinary Klotho excretion was calculated based on urine Klotho concentration and the volume of 24-h urine excretion. The assay was performed in accordance with the manufacturer’s instructions. Data was analyzed using MyAssay software . Quantity and purity of the obtained RNA were determined using a NanoDrop device (Thermo Fisher Scientific). A reverse transcription reaction was performed on isolated RNA. The obtained complementary DNA was subjected to a real-time polymerase chain reaction (PCR) that was performed using a LightCycler 480 instrument  with gene-specific intron-spanning primers and 8 bp fluorescent hydrolysis probes Roche; . RelativPodocytes were grown on glass coverslips that were coated with type I collagen. After incubation under experimental conditions, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X 100, and blocked with 3% BSA in PBS (blocking solution). Subsequently, the cells were incubated overnight at 4 °C with primary antibodies  that werA glycolysis stress test was performed using a Seahorse XFp analyzer , which depends on measurements of the extracellular acidification rate (ECAR). The ECAR increases through proton release from cells to the assay medium, which occurs during the glycolysis process. Glycolytic parameters that are obtained in the analysis are mainly levels of glycolysis and glycolytic capacity . First, 2, 0.9 mM CaCl2, and 5.6 mM glucose) that contained 5% BSA. Adherent glomeruli were then incubated in 5% BSA medium that contained Klotho protein (0.5 nM) for 30 min at 37 °C. The initial incubation medium was replaced with a medium that contained 1% BSA to produce an oncotic gradient across the glomerular capillary wall (5% BSA in the lumen vs. 1% BSA in the bath medium). Control glomeruli were treated with equivalent volumes of medium that contained 5% BSA (no oncotic gradient). Glomerular volume changes were recorded by video microscopy  before and after a medium change to an oncopressive medium (1% BSA). Glomerular volume (V) was calculated from the surface area (A) of the glomerulus using the following formula using Olympus CellSens Dimension software: V = (4/3A6. Volume changes (ΔV) were calculated as ΔV = /Vinitial based on the direct relationship between the increase in ΔV and oncotic gradient that was applied across the capillary wall. This principle was used to calculate the reflection coefficient of albumin , defined as the ratio of ΔV for experimental glomeruli to ΔV of control glomeruli in response to identical oncotic gradients, where σalb = ΔVexperimental/ΔVcontrol. The reflection coefficient of albumin  was used to calculate the glomerular capillary permeability to albumin , which describes the movement of albumin consequent to water flow. The volume response of glomerular capillaries to an oncotic gradient that was generated by defined concentrations of albumin was measured, as described previously . BrieflyThe transepithelial albumin permeability of human podocyte cells was assessed by measuring FITC-labeled BSA diffusion through a podocyte monolayer, as previously described , with miX-axis at a 1 μm distance is expressed as a percentage of the mean value of the fluorescence intensity that was calculated for the total X-axis. The cell membrane was positioned at point 0 on the plot.To visualize the F-actin network in human podocytes that were cultured on human fibronectin-coated coverslips, Alexa Fluor 633 Phalloidin  was used. NIS-Elements General Analysis 3 software (Nikon) was used to generate fluorescence intensity profiles from the basal membrane to the nucleus. The normalization of fluorescent profiles of different cells was performed. The fluorescence intensity on the post hoc test, unpaired t-test) or nonparametric tests . Values of p ≤ 0.05 were considered statistically significant. The results are expressed as mean ± SEM.The data were analyzed using Prism 5.03 software . Datasets were tested for a Gaussian distribution using the Shapiro–Wilk test. Depending on the results of this test, the statistical significance of the data was analyzed using parametric tests  followed by Bonferroni’s multiple-comparison In conclusion, we observed significantly higher serum levels of soluble Klotho and a decrease in its urinary excretion, which may be considered early biomarkers of DN. We found that Klotho improved the function of renal tissue through effects on the restoration of FGFRs, improved glycolysis, and lowered albumin permeability under hyperglycemic conditions. Our findings indicate that Klotho should be investigated further with regard to its potential role in reducing the pathological effects of DN.


BRCA1 or BRCA2 deficiency and in their BRCA1/2-proficient counterparts. Patients with tumors positive for MET had worse overall survival  compared to patients with MET-negative tumors. The prognostic role of MET was even more prominent in the subgroup of patients with SOX2-negative tumors (p = 0.0081). No synergistic effects of the combined treatment with PARP and MET inhibitors were found in the cell lines examined. We conclude that MET expression could be used as a marker for OS in HGSC and that stemness should be taken into consideration when evaluating the mechanisms of this effect.Overexpression of the receptor tyrosine kinase MET has been linked to poor survival in several cancer types, and MET has been suggested to interact with stem cell networks. In vitro studies have further suggested a possible benefit of a combined treatment using PARP and MET inhibitors. We used a tissue microarray (TMA) with 130 samples of advanced-stage high-grade serous fallopian tube/ovarian cancer (HGSC) to investigate the prognostic value of MET protein expression alone and in combination with the stem cell factor SOX2. The possible synergistic effects of a PARP and MET inhibitor treatment were evaluated in two cell lines with  BRCA1/2 mutation is present (either germline or somatic) [BRCA1/2 mutation status, again requiring at least a partial response to re-treatment with platinum [In 2020, 314,000 women worldwide were diagnosed with ovarian or fallopian tube cancer. The disease accounted for 3.4% of all female cancer cases and 4.7% of all cancer-related deaths in women [platinum ,6. To daBRCA1 and BRCA2, but PARPi also show effect in ovarian cancer patients without BRCA1/2 mutations. Overall survival (OS) rates have been shown to increase in both BRCA1/2-mutant and -wildtype carriers following PARPi treatment, provided that the tumors are still platinum-sensitive [The response to PARPi is facilitated through binding of the drug to PARP proteins, thereby disrupting the repair of single-strand breaks. This in turn leads to an accumulation of double-strand breaks in cells with a defective homologous repair (HR) machinery and eventually results in cell death. HR deficiency is most commonly caused by mutations in important DNA repair genes such as ensitive ,6,7.BRCA1/2, loss of PARP1 expression, or increased expression of ATP-binding cassette (ABC) transporters, effectively pumping the PARPi out of the cells. Another previously investigated mechanism involves tumors overexpressing the tyrosine kinase receptor MET, which is a common feature in many cancer forms [Nevertheless, many patients develop resistance to PARPi , and thier forms . High MEer forms , triple-er forms , and noner forms .Overcoming treatment resistance in an era of targeted treatments is an unmet clinical need. Several studies report that MET can phosphorylate PARP1, thereby preventing binding of the inhibitor and thus making cells PARPi-resistant ,14. The Another possible mechanism underlying the development of resistance and relapse is that tumors may harbor cancer stem cells capable of evading chemotherapy treatment . An incrSOX2 and POU5F1 (encoding OCT4) mRNA levels, an enrichment of ALDH-, CD133-, and CD117-positive cells, and a significant increase in sphere-forming capacity following PARPi treatment of ovarian cancer cell lines [Interestingly, connecting these two possible resistance mechanisms, overexpression of MET has been linked to increased levels of the stem cell markers ALDH1A3 and CD133 in breast cancer . Nozaki ll lines . Finallyll lines .BRCA1 or BRCA2 and in their counterparts with restored BRCA1/2 function to emulate platinum-resistant disease. The purpose was to explore if the capacity to activate a cancer stem cell network might be a clue to the ability of BRCA1/2-proficient cells to evade PARPi treatment.The aim of this study was to assess whether MET overexpression or co-expression of MET and SOX2 showed any prognostic potential in a consecutive and well-annotated contemporary cohort of advanced HGSC. Based on the links between MET and cancer stem cells in multiple tumor types and the potential role of MET in treatment resistance, we also wanted to investigate the role of stem cell factors in the proposed synergistic effect of METi and PARPi in HGSC cell lines with mutations in either Ethical approval for this study was granted by the Lund University ethics committee (Sweden), waiving the requirement for informed consent EPN 2014/717). A consecutive cohort of 156 HGSC cases, Cohort 1, was collected at the Department of Obstetrics and Gynecology at Skåne University Hospital (Sweden) between 2011 and 2015. All cases were reviewed by a gynecologic pathologist according to the World Health Organization Classification 2014  and stag14/717. ATMA sections, 3 µm in thickness, were stained with a primary antibody anti-c-Met  using the fully automated DISCOVERY ULTRA (Ventana). Antigen retrieval was performed using CC1  for 64 min at 95 °C, and the slides were incubated with ready-to-use primary antibody for 32 min at 37 °C. The TMA was scored using PathXL (Philips) by two independent examiners (MB and LT). In case of discordance, a consensus for each core was reached through joint reevaluation and, when needed, a trained gynecologic pathologist was consulted (AM). For each patient, a mean of the cores was calculated, and expression of MET was grouped as membranous staining in 0%, <5%, 5–9%, 10–49%, or 50–100% positive cells, with either weak or moderate intensity (representative images of intensity levels are shown in mRNA data from 489 HGSC patients included in the 2009 TCGA study  was retrBRCA1/2 mutations (germline or somatic), and those positive received olaparib treatment in line with clinical guidelines at the time. Pathological BRCA1 or BRCA2 mutations were found in 10/25 patients. Ethical approval for this cohort was granted by the Lund University ethics committee (EPN 2016/508). Written informed consent was obtained from patients receiving the olaparib treatment, while the consent requirement was waived for the remaining participants.An additional, partially overlapping cohort of 25 HGSC patients was used for global copy number analyses (referred to as Cohort 2). This cohort was collected between 2015 and 2016 and consists of patients presenting with relapsed high-grade serous ovarian, tubal, or peritoneal carcinomas who had at least partial response to second-line platinum treatment. Patients were screened for ® FFPE Assay Kit (Affymetrix) was used with 80 ng DNA as input. Segmentation, calculation of ploidy, normal cell contamination, and allele-specific copy number analyses were performed using ASCAT [For this cohort, DNA was extracted from FFPE tumor tissue using the Qiagen Allprep kit for FFPE tissue (Qiagen). Sections of the tissue were stained for validation of tumor content. SNP array analysis was performed at Eurofins Genomics Europe Genotyping A/S. The OncoScanng ASCAT   were cultured in RPMI-1640 HyClone with 10% FBS, 2 mM glutamine, 2 mM sodium pyruvate, and 1% penicillin–streptomycin . The UWB1.289 cell line is derived from an ovarian tumor of papillary serous histology with a BRCA1 mutation and defective DNA repair. UWB1.289+BRCA1 cells are from the same line, but in this version, a BRCA1-containing vector was stably transfected. UWB1.289 and UWB1.289+BRCA1 (ATCC) were cultured in 50% MEGM  and 50% RPMI-1640 HyClone (Nordic Biolabs). Media were supplemented with 3% FBS, and for UWB1.289+BRCA1, 200 ug/mL G-418  was additionally added. Mycoplasma testing and cell line authentication for all four lines was performed by GATC .Four HGSC cell lines were cultured. The PEO1 and PEO4 cell lines are derived from ascites from a patient with poorly differentiated serous adenocarcinoma. PEO1 was collected at first relapse when the tumor was considered platinum-sensitive, and PEO4 was collected after the second relapse, when the tumor no longer responded to platinum . A BRCA2w/v trichloroacetic acid , and air-dried. Cells were stained with 0.4% (m/v) sulforhodamine B (Sigma Aldrich), dissolved in 1% acetic acid for 30 min, and subsequently washed with 1% acetic acid. Plates were air-dried, and the dye was dissolved in 10 mM TRIS Base  before the absorbance was measured.Cells were seeded in 96-well plates, and single-drug treatment was performed with a range of concentrations for carboplatin, olaparib, and crizotinib, all purchased from Selleckchem. For combination treatment assays with olaparib and crizotinib, several doses for each drug were used, and the concentrations were chosen specifically for each cell line, ranging between approximately IC5 and IC60, as determined from the single-drug treatment growth assays. Cells were incubated with drugs for 6 days, fixed with 17% Copy number and mRNA expression data for the cell lines PEO1 and PEO4 were retrieved from CCLE , 21Q1 Public). Data had been log2-transformed using a pseudo-count of 1. The panel of 40 HR-associated genes described earlier  was used® (Bio-Rad), and intensity was normalized to stain-free blots.UWB1.289 and UWB1.289+BRCA1 cells were collected by scraping on ice, and Western blot analyses were performed using TGX stain-free gels (Bio-Rad Laboratories). Following blocking in 5% milk on a shaker at 4 °C overnight, membranes were incubated with primary antibodies against SOX2 , Nanog , OCT4 , RAD51 , BRCA1 , and Met  on a shaker overnight at 4 °C. The primary antibody HSP90 , used to ensure equal protein loading on gels, was applied to the membranes for 2 h at room temperature. The membranes were washed and incubated with HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Detection was performed with Luminata Forte Western HRD Substrate  or SuperSignal West Femto Maximum Sensitivity Substrate , using Bio-Rad’s Stain-Free imaging technology . Quantification of Western blot results was performed using ImageLabt-test alternatively linear regression was used, as indicated in the Results section. For ordinal, non-parametric data  Wilcoxon rank–sum test was used, and for non-ordinal variables  Fisher’s exact test was used. To evaluate survival outcome, Cox regression was used both in a univariate model comparing MET-positive/MET-negative patients, and in a multivariate model where also age, stage, and residual tumor left after surgery were included. Age was used as a continuous variable, and stage and residual tumor as binary variables (III/IV and yes/no). Spearman rank correlation (rho) was used to evaluate co-expression of MET and SOX2 at different levels of expression. For Cox regression, when including an interaction term for MET and SOX2, MET-negative and SOX2-negative were marked as reference. All calculations, visualizations, and statistical analyses were performed using R statistical environment version 4.0.5 [To analyze the difference in the mean of continuous variables, Student’s on 4.0.5 .BRCA1/2 mutation status, and amount of tumor remaining after debulking surgery . MET-positive staining (defined as ≥5% cells with membrane staining) was found for 31 out of 130 patients. The groups of patients with MET-positive vs. MET-negative tumors were comparable in age,  surgery . Patientp = 0.62). When combining data for MET and SOX2 expression, we found that the subgroup of patients with tumors negative for SOX2 expression separated from those positive for SOX2. In the SOX2-negative subgroup, patients with MET-positive tumors had a significantly worse outcome than those negative for MET , indicating the significant impact of these markers on outcome. There was no significant difference in MET expression between primary and metastatic tissue .Kaplan–Meier analyses of OS  and PFS  revealed shorter survival for patients with MET-positive tumors compared to those with MET-negative tumors . SimilarMET mRNA expression alone and in combination with four closely connected transcription factors, described as stem cell markers  using a cut-off of z-score > 0 to define the expression of the individual markers. None of these markers were independently connected to OS .To further investigate the role of SOX2 and other stem cell markers in relation to MET expression, we retrieved a TCGA data set consisting of 489 patients, 461 of whom had both clinical and mRNA data available . We evaled to OS , but wheBRCA1/2-mutated and -wildtype samples, separately, are shown in BRCA1/2-mutant and -wildtype groups, but no significant difference was found . OS was longer for patients with a BRCA1/2 mutation, however, not to a significant degree , however, not to a significant degree (Student’s t-test p = 0.10).HRD scores were calculated in 25 HGSC tumors with available copy number data (Cohort 2). HRD scores in this selected cohort ranged from 26 to 85, with a median of 57. Clinico-pathological information is shown in BRCA1/2 status, HRD scores, and survival outcomes are displayed in SOX2 and HRD score . MET amplification and expression were rare in this cohort , a PARPi (olaparib), and a METi (crizotinib). The cell lines PEO1 and PEO4 were obtained from the same patient before and after platinum resistance was established. UWB1.289 and UWB1.289+BRCA1 originate from the same patient, and UWB1.289 is the parental cell line from which the UWB1.289+BRCA1 line was generated upon stable transfection with a plasmid containing a full-length functional BRCA2-deficient PEO1 and BRCA2-proficient PEO4 cells. By investigating the expression of HR-associated core genes, we found that these genes in general exhibited higher expression in the BRCA2-mutant PEO1 cell line compared to PEO4. For 20 genes, expression was higher in PEO1 cells, and for 6 genes, expression was higher in PEO4 cells. Copy numbers, however, were comparable between the cell lines. mRNA expression levels differed markedly between the cell lines for the stem cell factor POU5F1 (encoding OCT4) as well as the stemness-associated genes ALDH1A3 and PROM1 (encoding CD133), with higher levels in PEO1 than in PEO4 cells. Copy number and mRNA data for stem cell factors, as well as the crizotinib targets ALK, MET, and ROS1, DNA repair markers RAD51 and BRCA1/2, and genes involved in the MET signaling pathway, are displayed in Copy number and mRNA expression data for PEO1 and PEO4, available from the CCLE database, were examined for a panel of 40 HR-associated genes  as well BRCA1-proficient cell line were not available in the CCLE database. Results are displayed in BRCA1-proficient cell line. Nanog levels were below the level of detection by Western blot in both lines.Western blot analyses for the presence of MET, the stem cell factors SOX2, Nanog, and OCT4, as well as the HR markers RAD51 and BRCA1 were performed for the UWB1.289 and UWB1.289+BRCA1 cell lines, as mRNA data for the MET was discovered to be a potent oncogene in the 1990s when studies reported that when co-expressed with its ligand, hepatocyte growth factor (HGF), it promoted metastases in an animal model [MET did not have an effect on survival in the SOX2-negative subgroup; however, in the POU5F1-negative subgroup, we saw similar effects as in the TMA cohort. The SOX2 and OCT4 proteins interact closely by forming dimers with the ability to bind specifically to genes involved in cell fate and pluripotency [SOX2, POU5F1, Nanog, and KLF4, we observed that MET expression affected OS in patients whose tumors expressed none or only one of the factors. This indicates that these factors may complement each other and suggests that the tumor relies on other mechanisms for progression when an insufficient number of stem cell factors are present. Further studies are required to confirm these theories. In this context, Robinson et al. recently reported a stronger role for SOX2 compared to OCT4 or Nanog for tumor relapse potential following chemotherapy in ovarian cancer patients and that HGSC cell lines displayed greater chemoresistance and increased expression of SOX2, OCT4, and Nanog when grown in 3D conditions compared to 2D [al model . Overexpal model . The proal model . We set ipotency . Both coipotency ,38. The ed to 2D .BRCA1/2 mutations. This may be due to generally high HRD scores in this cohort, since all patients were platinum-sensitive and had at least a partial response to re-treatment with platinum. The HRD scores for the whole cohort were nonetheless comparable to the range of scores previously reported in HGSC [BRCA1/2 mutations had a longer OS, which could be expected as they received olaparib treatment and, as a group, also generally respond better to platinum treatment [To explore potential links between PARPi response, copy number aberrations, and cancer stem cell factors, we compared the copy number landscape and HRD scores in a cohort consisting of patients receiving either olaparib maintenance or surveillance (Cohort 2). We found, surprisingly, small differences between the groups with and without  in HGSC . Using t in HGSC , 84% of reatment .SOX2 was common, as has previously been reported in several cancer forms, including HGSC [MECOM, PIK3CA, ECT2, and PRKCI). Whether SOX2 should be considered a passenger or a driver gene is not fully determined. In this study, SOX2 protein levels appeared to correlate with SOX2 amplification, supporting the notion that it plays a role in cancer development and that overexpression of SOX2 to some extent is dependent on amplification.Amplification of ing HGSC . The comBRCA2 mutation, and PEO4 has a reversal mutation restoring BRCA2 function. This result contradicts earlier reports, which stated that the PEO1 line was more sensitive [BRCA2 function. PEO1 also expressed higher levels of several key stem cell factors, which could indicate a more stem cell-like phenotype. As cancer stem cells have been reported to express higher levels of drug transporter pumps, have a more intact DNA repair system, and are more prone to epithelial-to-mesenchymal transition (EMT) [Comparing growth inhibition in cell line pairs, we found that the post-treatment platinum-resistant PEO4 cells were more sensitive to olaparib treatment than PEO1 cells, although PEO1 cells harbor a ensitive ,42. We fon (EMT) , a higheon (EMT) , and appBRCA1-proficient line compared to the BRCA1-deficient line. This may indicate that the platinum and PARPi resistance acquired through the restoration of the BRCA1 function is connected to the activation of stem cell networks. Of note, the recently concluded PRIMA trial reported benefit in all patient groups receiving niraparib as first-line maintenance treatment, provided complete or partial response to upfront platinum therapy irrespective of the HRD status [In order to compare gene activity in the UWB1.289 and UWB1.289+BRCA1 cell lines, Western blot analyses were performed on selected proteins, as the UWB1.289+BRCA1 cell line was not included in the CCLE DepMap mRNA data set. The Western blot analyses revealed markedly higher protein expression of SOX2 in the D status , clearlySynergistic effects of olaparib and the c-MET inhibitor crizotinib have been reported in earlier studies, including ours ,16,17, bWe conclude that MET expression could be used as a prognostic tool in HGSC, but measures of stemness should be taken into consideration when further evaluating the mechanism/s underlying the driving effect of this receptor tyrosine kinase in cancer. Combining MET and PARP inhibitors did not increase cancer cell death in vitro in this study; nevertheless, MET inhibition could still be a viable treatment option. It should, however, be noted that patients with tumors expressing stem cell factors might not benefit from the treatment. Including stem cell marker screening in future clinical trials could help pinpoint the subgroup of patients eligible for METi treatment.


The incidence of depression among humans is growing worldwide, and so is the use of antidepressants. However, our fundamental understanding regarding the mechanisms by which these drugs function and their off-target effects against human sexuality remains poorly defined. The present study aimed to determine their differential toxicity on mouse spermatogenic cells and provide mechanistic data of cell-specific response to antidepressant and neuroleptic drug treatment. To directly test reprotoxicity, the spermatogenic cells (GC-1 spg and GC-2 spd cells) were incubated for 48 and 96 h with amitriptyline (hydrochloride) (AMI), escitalopram (ESC), fluoxetine (hydrochloride) (FLU), imipramine (hydrochloride) (IMI), mirtazapine (MIR), olanzapine (OLZ), reboxetine (mesylate) (REB), and venlafaxine (hydrochloride) (VEN), and several cellular and biochemical features were assessed. Obtained results reveal that all investigated substances showed considerable reprotoxic potency leading to micronuclei formation, which, in turn, resulted in upregulation of telomeric binding factor (TRF1/TRF2) protein expression. The TRF-based response was strictly dependent on p53/p21 signaling and was followed by irreversible G2/M cell cycle arrest and finally initiation of apoptotic cell death. In conclusion, our findings suggest that antidepressants promote a telomere-focused DNA damage response in germ cell lines, which broadens the established view of antidepressants’ and neuroleptic drugs’ toxicity and points to the need for further research in this topic with the use of in vivo models and human samples. According to the World Health Organization (WHO), depressive disorders are the greatest burden of disease in developed countries, affect millions of people, and are the leading cause of disability around the world . DepressPsychiatrists routinely use antidepressants to treat depression, making their application significant and growing worldwide . In addiThe exact mechanism of the effect of antidepressants on human sexual function is unknown, but most of them modulate the concentration of central neurotransmitters such as serotonin, norepinephrine, and dopamine. Serotonin inhibits the production of nitric oxide, which physiologically plays a key role in the sexual response, and its elevated levels impair sexual function. Furthermore, norepinephrine and dopamine are also involved in the cycle of sexual response related to desire and arousal. Many antidepressants can also suppress normal sexual function through cholinergic and alpha-1 adrenergic receptors ,16. ImpoThe purpose of this study was to determine the effect of selected antidepressants and neuroleptic drugs on mouse spermatogenic cells (GC-1 spg and GC-2 spd cell lines) and provide mechanistic data of cell-specific responses to antidepressant treatment. This preliminary study becomes an important aspect in terms of effectiveness for treating depression and will provide clinically relevant data associated with side effects of drugs and male fertility reduction.p < 0.05, p < 0.001). Interestingly, at the same time, the GC-2 spd cell line was characterized by a significant decrease in ATP level  (p < 0.001). The most pronounced decrease was noted for cells treated with MIR and VEN (both cell lines tested) after 96 h incubation (p < 0.001). For the rest of the drugs analyzed, a statistically significant decrease in the ATP was also observed after 96 h incubation. The exceptions were two drugs—ESC and OLZ (GC-1 spg)—where the ATP level remained unaffected  (GC-1 spg) and VEN (p < 0.01) (GC-2 spd). In turn, for ESC and REB, the S phase of the cycle was reduced (p < 0.05) (GC-2 spd only). Along with longer exposure to antidepressants, a significant reduction of the G0-G1 phase (p < 0.01–0.001) with a simultaneous extension of the S (p < 0.05) and G2/M phases (p < 0.05–0.01) (GC-1 spg) was observed for most experimental sets. A similar trend was not confirmed for the GC-2 spd cell line (p < 0.05–0.001). The second line, in general, also showed an increase in the synthesis level of these proteins after 48 and 96 h (p < 0.05–0.001) of incubation with drugs  caused a reduction in cell population in G0-G1 phase for FLU . The highest number was noted for ESC, FLU, and VEN in all experimental sets. Only MIR and in part REB drugs were characterized by the percentage of TUNEL + cells at the control level A,C. In aPersistent stress can lead to further damage to the nucleic acids. We noted a significant increase in micronuclei production after 48 h (except IMI) and 96 h incubation for both cell lines tested. Interestingly, after 96 h exposure, we observed a slight decrease in the amount of MN compared to 48 h, and this trend was observed for all experimental sets A–D. SimuNext, we assessed the interaction between nuclear mitotic apparatus protein (NuMa) and α-tubulin. We noted a decrease in NuMa/α-tubulin ratio intensity in most experimental sets of the GC-1 spg cells (48 and 96 h) except AMI and ESC (48 h) A,B. A siFinally, we have conclusively confirmed cell death resulting from exposure to antidepressants. We observed a significant decrease in cell viability after 48 h incubation with drugs for the GC-1 spg . The effect was even more pronounced after 96 h of exposure A,B. In t2+ reuptake, which leads to their overload and cell death  × 100%.The immunostaining was done accordingly to Mytych et al. 2017 . Brieflyp-value of <0.05 and considered as statistically significant . (*) indicates a comparison between non-treated and antidepressants-treated cells, and (^) indicates a comparison between the same drugs in different periods .All statistical analyses were carried out using Microsoft Excel and GraphPad Prism 6. The results are presented as the mean ± standard deviation of at least three independent experiments. The data were normalized separately for 48 h and 96 h experimental set-ups, with the control group being considered as 100%. The calculations were performed with a one-way analysis of variance and Dunnett’s posttest. The significance level was set at In conclusion, antidepressants are characterized by a delayed onset of therapeutic action, which requires long-term use. The use of this type of drug may be associated with side effects. Few reports also indicate that this may result in delayed various disorders of life functions, including reproductive functions and processes. Here, for the first time to our knowledge, we provide molecular evidence of possibly irreversible disorders in the course of spermatogenesis by germ cell damage.


Rhabdomyosarcomas (RMS) are soft tissue sarcomas predominant in the pediatric age range. Current standard multi-modal treatments can result in severe life-long side-effects across all subtypes and dismal outcomes in RMS patients classified as high-risk. There is an urgent need to find novel, more targeted therapies for these children. High expression of a number of histone demethylases is shown to support proliferation of RMS cells and here we direct our attention on KDM4B which has become a recent focus for drug discovery efforts. We show that KDM4B regulates cell cycle genes in the context of RMS cells and demonstrate that long-term silencing of KDM4B is functionally compensated for by another KDM4 family member, KDM4A. Upfront reduction of both KDM4A and KDM4B kills RMS cells demonstrating that therapeutics designed to target both histone demethylase family members is desirable to circumvent functional redundancy.CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach.Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including  Epigenetic marks on chromatin and the enzymes that modulate them are emerging as key mechanisms underlying cancer tumorigenesis and progression. Histone modifications are central to controlling chromatin architecture, gene transcription and DNA repair, and a number of proteins controlling the reading, writing and erasing of such marks are aberrant in cancer with targeted epigenetic modulators against them undergoing clinical trials .Histone demethylases (HDMs) are a family of epigenetic modulators responsible for the removal of methyl groups from specific residues on histone tails and have been shown to play a role in stem cell identity and differentiation ,3. The KKDM4 family members have also been shown to be genetically aberrant and overexpressed in certain malignancies ,8. MoreoCDK6 and CCNA2 via H3K9me3 at their promoters. However, after sustained silencing of KDM4B, we also show that recovery of cell growth is attributable to KDM4A recruitment to the promoters of CDK6 and CCNA2. Furthermore, upfront knockdown of both KDM4B and KDM4A led to cell apoptosis, not seen with silencing of either demethylase alone. Taken together, our results support therapeutic potential for RMS patients by KDM4 family inhibition.Rhabdomyosarcomas (RMS) are a type of soft tissue sarcoma that resemble developing skeletal muscle and are a major cause of cancer-related deaths in children and adolescents. Despite an overall increase in survival rate over the past few decades, drug resistance is a major factor in treatment failure . A numbe2.RMS cell lines RH30, RD, RMS-YM, RH4, JR-1, RMS-01, RH41, HFF-1 have been described previously . Cell liCell lines were authenticated using the Geneprint 10 kit according to manufacturer’s instructions  and subsequent fragment length analysis was carried out by Eurofins Genomics . The resulting short tandem repeat (STR) typing results were compared with public databases.For siRNA silencing experiments 3 siRNAs were used for each histone methyltransferase family member. siRNAs were synthesised by Sigma and RMS cell lines were transfected using Lipofectamine RNAiMAX  with siRNAs at a final concentration of 10 nM. The data was subsequently processed and analysed using python 3 using pandas, numpy, matplotlib, seaborn libraries B. For su® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) at 96 h. GI50s were calculated in GraphPad Prism 7  using the mean of six replicates. Cells for dose-response curves using were plated in 96-well plates and JIB-04  was added at increasing dose increments from 0 (DMSO (Sigma-Aldrich) only) to 10 µM and growth measured using using CellTiter 96Cells were fixed in 2% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100, blocked with 10% normal donkey serum with 1% BSA, and incubated with primary antibody O/N. Alexa Fluor 555-conjugated secondary antibody was used. Nuclei were stained with DAPI before fluorescence imaging . Antibodies listed in 2O2 at room temperature for 5 min. The sections were blocked with 4% BSA and incubated with KDM4B antibody at 4 °C overnight. Then, the samples were probed with secondary Ab , K5007, DAKO, Denmark), followed by incubation with peroxidase substrate solution  and counterstained with hematoxylin.The immunohistochemistry was performed on tissue microarrays containing samples from 219 RMS patients as well as normal tissues. Tissue microarrays were deparaffinized, hydrated through graded alcohol series and rinsed in water. After antigen retrieval by HIER, peroxide blocking was performed with 3% HThe immunoreactions were evaluated independently by two pathologists blinded to the clinicopathologic information. KDM4B immunostaining was evaluated based on the percentage of positively stained tumor cells. Additionally, the staining intensities were classified into the following four categories: 0 (no staining); 1 (weak staining); 2 (moderate staining), 3 (strong staining) in at least 10% of cells. Gene expression data described previously ,18 was a® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) and CyQuant Proliferation Assay kits  according to the manufacturer’s instructions. For cell cycle analysis cells were harvested after siRNA silencing, washed in 1XPBS fixed in 70% ethanol and stained for 30 min with propidium iodide (Invitrogen). Cells were then analyzed for to cell cycle distribution on an LSR II . Apoptosis assays were carried out after siRNA transfection using Caspase Glo assay (Promega). Cell proliferation assays were carried out using CellTiter 96RNA for RNA-sequencing was extracted 48 and 72 h post-transfection with either a non-targeting control siRNA or one of 2 KDM4B siRNAs (two replicates of each). RNA was sent to Oxford Gene Technology  for library preparation and sequencing. Gene expression data was obtained and analysed for differential gene expression between control siRNAs and KDM4B siRNAs.The diff files from the RNASeq were processed using R  using dplyr, ggplot2, gplots, RColorBrewer packages to manipulate the data and generate the scatterplots and heatmaps. The full data set was clustered using the R hclust hierarchical clustering function. Using this the data was split into 10 clusters for uploading to genego.com for GO analysis. Further analysis of the clusters was analysed in Python 3.Protein expression of KDM4 family members and cell cycle proteins was carried out by Western blotting. Protein from transfected cells was extracted using cell lysis buffer . Proteins were resolved on 3–8% Bis-Tris acrylamide gels and transferred onto PVDF membranes using the X-Cell II and iBlot systems according to manufacturer’s instructions (Invitrogen). Antibodies can be found in Chromatin immunoprecipitations (ChIP) were carried out using the SimpleChIP Enzymatic Chromatin IP kit in accordance with the manufacturer’s instructions . Eluted chromatin underwent qPCR analysis using primers specific to promoter regions of CDK6 and CCNA2. Primary antibodies can be found in Stable KDM4B and control shRNAs were packaged into virus in HEK293T cells as described previously . HEK293Tt-test (two-tailed) in GraphPad Prism 8.0.2 softwaree. Data is presented as the mean ± standard deviation. Significance is indicated as p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), while NS indicates a non-significant p-value.Each examination was performed in at least triplicate. For in vitro experiments, statistical differences were analyzed using Welch’s 50 and compared these with known sensitive (MCF-7) and insensitive (HT-29) cell lines [As we had previously shown that a number of demethylase gene family members are overexpressed in RMS relative to normal skeletal muscle samples  we deterll lines . All RMSz-score of >−0.5 at either time point in each of the two cell lines. Overlaying the hits from the screen with those determined to be overexpressed in RMS patient samples, both fusion positive and negative  and alveolar RMS , at two time-points using three siRNAs for each HDM . Briefly 0.0001, ) identif 0.0001,  and the p < 0.0001, one-way analysis of variance) and a panel of normal tissues , with no statistical difference in expression between fusion gene positive and negative RMS  (p = 0.001) , all of which have long-term follow-up clinical information available. 40% of RMS were positive for KDM4B staining (>10% cells stained), comparing to negative staining in normal muscle (< 0.001) , consist< 0.001) . Based o= 0.001) , consistA role for KDM4B in cell proliferation was assessed after RNAi-mediated silencing of KDM4B using KDM4B-specific siRNAs in 6 RMS cell lines representing both ERMS and ARMS and compared with HFF-1 cells. KDM4B silencing resulted in a reduction in cell proliferation in both ARMS and ERMS cell lines of up to 70% by 96 h post transfection . To confTo determine which genes may be responsible for the phenotypic effects seen in ARMS cell lines, we investigated the transcriptional effect of silencing KDM4B in the ARMS line RH30 by RNA-sequencing at 48 and 72 h post-transfection. Gene Set Enrichment Analysis (GSEA) revealed both overlapping and distinct genes that were down-regulated at 48 h and 72 h post-transfection –D. Gene Of the genes that were down-regulated at either 48 h and/or 72 h after KDM4B RNAi we noted that a number of these are known to be involved in cell cycle checkpoint control at different phases of the cell cycle, including CDK6, a G1-S checkpoint gene, and CCNA2, a G2-M checkpoint gene. To confirm that KDM4B is regulating these genes in RMS we performed KDM4B silencing in four RMS cell lines and confirmed a reduction in CDK6 and Cyclin A2 (CCNA2) expression by Western blot in all cell lines . In the p < 0.03, Day 17, p = NS, To determine the long-term effects of sustained KDM4B-silencing in RMS cells we tested 6 different shRNAs directed against KDM4B . We measAs functional redundancy has been postulated to exist between KDM4 sub-family members ,3, we soDespite a lack of upregulation in expression of other KDM4 family members, there remains the possibility of functional redundancy in regulating genomic targets of KDM4B in its absence. To test this hypothesis and determine whether the lack of reduction in expression of KDM4B-controlled cell cycle genes could be compensated for by the presence of KDM4A on these promoters we analyzed KDM4A binding on the promoter of CDK6 and CCNA2. In RH30 cells transduced with control shRNA, KDM4A does not bind to these promoters. However, after KDM4B silencing by shRNA, we observed an enrichment of KDM4A at these promoters, suggesting that KDM4A is now regulating expression of CDK6 C and CCNAs sustained silencing of KDM4B led to an increase in KDM4A at the CDK6 promoter we hypothesized that simultaneous targeting of both KDM4A and KDM4B may result in a stronger phenotype and thus mitigate the effects of stable silencing of KDM4B alone. siRNA-mediated dual targeting of KDM4A and KDM4B lead to a significantly greater decrease in RMS cell proliferation than with either KDM4A or KDM4B alone A,C. FurtIn this study, we demonstrate for the first time the role of KDM4B in controlling cell proliferation and cell cycle progression in RMS. Moreover, in this context, we also show the existence of functional redundancy between KDM4B and KDM4A in cell cycle regulation. Building on our previous study showing that HDMs are highly expressed in RMS compared to normal tissues, here we demonstrate that a number of HDMs affect RMS cell growth and show that silencing of KDM4 family members results in a reduction in cell proliferation in RMS cell lines including the H3K9me3 demethylase KDM4B. CDK6 in bladder and lung cancer cells [CDK6 in RMS and may therefore be a more widespread driver of proliferation in tumors. This phenotype is enhanced through the associated expression of several other cell cycle checkpoint genes known to be expressed at different phases of the cell cycle. Our RNA-sequencing data revealed that cell cycle genes, including CDK6, CCNA2, SKP2 and MYC were all down regulated after KDM4B silencing. SKP2 has been shown to be highly expressed in RMS and is a downstream target of the PAX3-FOXO1 fusion protein [SKP2 in RMS via H3K9me3 regulation. Interestingly, silencing of KDM4B in ERMS cell lines resulted in cell cycle arrest, compared to ARMS cell lines in which silencing of KDM4B results in a slowing down cell cycle progression. KDM4B has been shown to regulate different genes dependent on cell context . As ARMiewed in  it is liHere we show that KDM4B plays a role in the proliferative capacity of RMS cells. Indeed, in stem cells expression of KDM4 proteins is required for maintaining the undifferentiated, proliferative state and regulating differentiation ,3,5,6. RRNA-sequencing following KDM4B silencing also showed that the majority of genes that changed were down-regulated, consistent with transcriptional activation via H3K9me3 demethylation by KDM4B. However, around 25% of genes were upregulated after KDM4B silencing, suggesting that these genes are regulated by the H3K36me3, a hallmark of active transcription, implicating a demethylating, repressive role of KDM4B on these genes. Mutations in histone H3 lysine 36 to methionine (H3K36M) have been shown to prevent differentiation in mesenchymal progenitor cells (MPCs) and have been linked to sarcomagenesis in mouse models . SilenciAlthough short-term silencing of KDM4B by siRNA in RMS cells led to a reduction in proliferation, long-term stable silencing by shRNA did not. This is in contrast to reports in neuroblastoma, where stable silencing of KDM4B led to a reduction in cell proliferation and a concomitant induction of differentiation in these cells . In RMS Over the past few decades drug discovery efforts have commonly sought a single molecule that selectively targets a single protein. However, functional redundancy between protein family members and/or signaling pathways have been shown to be potential mechanisms of resistance to therapies in the treatment of cancer ,29, and There has been a recent drive to find KDM4 inhibitors, although very few of these studies have been able to derive selective compounds without poly-pharmacology with other KDM families, largely due to the fact that they are either cofactor mimics or substrate competitive, both of which are often shared across histone demethylase families. However, some selectivity has been achieved using cyclic peptides, which even allow for some selectivity over subfamily members . Based oOverall, the data we have presented highlights the importance of understanding the underlying functional redundancy that may exist between histone demethylase family members in order to effectively target these epigenetic proteins for the effective treatment of cancer. Our discovery that KDM4A is able to compensate for down-regulated KDM4B by assuming the latter’s H3K9me3 demethylating role to drive transcription of KDM4B-regulated genes highlights the potential for sub-family targeting of these proteins rather than striving for single-target therapies. KDM4A itself has been associated with mechanisms that may lead to resistance , thus itFurthermore, our data show that effectively targeting both KDM4A and KDM4B together leads to apoptosis in RMS cells and thus suggests that effective dual targeting of these proteins could lead to a more effective therapy, whilst maintaining the benefits of more targeted therapies with reduced toxicities. A number of histone demethylases are highly expressed in rhabdomyosarcoma and impact on cell proliferation, including KDM4B. We determine that KDM4B regulates the expression of cell cycle genes in rhabdomyosarcoma. However, after clonal selection of KDM4B-silenced cells, KDM4A is able to functionally compensate for the regulatory effects of KDM4B on the promoters of the cell cycle genes CDK4 and CCNA2. We conclude that, in order to mitigate for this functional redundancy and enhance therapeutic potential for rhabdomyosarcoma patients, a dual targeting approach to pharmacological inhibition of these histone modifying enzymes will be necessary.


Pyrazoles are an interesting class of compounds showing potent anticancer activities. Our previous studies have demonstrated the potent anticancer activity of pyrazole analogues. Therefore, we focused on developing anticancer agents through structure optimization of the pyrazolyl lead molecule.  The pyrazole derivatives were prepared by the appropriate synthetic protocols. The antiproliferative activities were evaluated using a sulforhodamine B assay against three cancer cell lines. In vitro and in silico molecular docking studies employing xanthine oxidase were used to explore the mechanism by which pyrazole derivatives exert anticancer effects. 50 4.2 μM), with a potent xanthine oxidase inhibitory activity (IC50 0.83 μM).  One of the pyrazole derivatives demonstrated the greatest promise as an anticancer agent against the human colon cancer cell line (IC In summary, our findings suggest that these pyrazolyl analogues containing a pyridine nucleus could serve as a promising lead molecule in the development of novel anticancer agents. Cancer is one of the four major types of noncommunicable diseases, along with cardiovascular disease, diabetes, and chronic respiratory diseases . It is a50 value in the range of 0.2–3.4 μM  × 100, where Ac indicates the absorbance of the control sample and As is the absorbance of the treated sample. Readings were taken in triplicate and represented as IC50 ± SD. IC50 values were calculated using GraphPad Prism 8 software.These compounds were further evaluated for their XO inhibitory potential by measuring the formation of uric acid concentration spectrophotometrically at 292 nm using a method previously described . Solutiohttp://www.hyper.com). AutoDock tools (ADT) version 1.5.6 (http://www.autodock.scrips.edu) was used to prepare the molecular docking. The active site was considered as a rigid molecule, while the ligands were treated as being flexible. Using default parameters, grid-based docking studies were carried out and docking was performed on all compounds using the standard ligand oxipurinol. The best binding conformation was selected from the docking log (.dlg) file for each ligand, and further interaction analysis was performed using PyMOL and Discovery Studio Visualizer 4.0.Molecular docking studies were performed using AutoDock v. 4.2.2 to identify appropriate binding modes and conformation of the ligand molecules. The crystal structure of xanthine dehydrogenase  was retrieved from the RCSB Protein Data Bank as a PDB format . The str50 0.2–3.4 μM) as a potential anticancer lead molecule, we focused our attention on optimizing the anticancer potential of arylhydrazono-pyrazole derivatives with different substitutions at the phenyl ring , hepatocellular carcinoma (HepG2), and colorectal carcinoma (HCT-116), by the sulforhodamine B (SRB) assay. The SRB assay is the “gold-standard” assay and is extensively used in the high-throughput screening program at the National Cancer Institute (NCI), USA. The SRB uses an aminoxanthene dye, which readily stains the cells through binding with the basic amino acids of cellular proteins. The SRB assay estimates the dye released from the stained cells after washing as stoichiometric proportions of cellular mass. In 50) values. The IC50 values were determined by interpolation from dose-response curves. From the results, it was observed that most of the newly synthesized pyrazoles showed excellent to moderate antiproliferative activity against the three cell lines, especially against colorectal carcinoma (HCT-116). Compound 1 effectively inhibited cell growth at IC50 values of 17.8 ± 0.5, 4.4 ± 0.4, and 4.2 ± 0.2 μM against the breast cancer (MCF-7), hepatocellular carcinoma (HepG2), and colorectal carcinoma (HCT-116) cell lines, respectively. The anticancer activity of compound 1 was comparable to the standard drug doxorubicin against the HepG2 (3.9 ± 0.06) and HCT-116 (4.4 ± 0.04) cell lines, but 3.7 times less potent in the MCF-7 (4.7 ± 0.08) cell lines. Compound 2 was found to be moderately effective, with IC50 values of 94.2 ± 0.3, 34.6 ± 2.6, and 17.3 ± 0.5 μM against the MCF-7, HepG2, and HCT-116 cell lines, respectively. Our previous studies have shown that unsubstituted phenyl derivative of aryldiazenyl pyrazole had a poor antiproliferative activity (IC50 > 100 μM) against all three cell lines [Based on the encouraging results of 1-isonicotinoyl-3-methyl-4-[2-(4-nitrophenyl)hydrazono]-2-pyrazolin-5-one  exhibited significant XO inhibition, 18.03 times more potent than the standard drug allopurinol (IC50 14.97 ± 1.61 μM) (2 was equipotent (IC50 14.50 ± 2.25 μM) to allopurinol. These results confirm the XO inhibitory action of these compounds and suggest the possible influence of XO inhibition in the anticancer activity of pyrazoles. However, further kinetic studies are required to establish a more detailed mode of enzyme inhibition of these compounds.Pyrazolyl analogues have been reported as promising anticancer agents acting through xanthine oxidase (XO) inhibition . The pro1.61 μM) , whereas4+) of the molybdopterin cofactor, which is essential for the mechanism-based inhibition. Furthermore, it forms hydrogen bonds with Glu802, Arg880, and Glu1261 in the active site of the enzyme  using AutoDock. Both the enzymes XO and XDH are interconvertible forms of the same enzyme, known as xanthine oxidoreductase (XOR), with only slight differences in the FAD binding domain. The crystal structure of bovine milk XDH bound with oxipurinol was used in the current study and retrieved from the raw PDB structure 3BDJ, which has 90% similarity to the human liver enzyme . Oxipurie enzyme . These be enzyme . The keye enzyme . The res1 and 2 were prepared by using the method described earlier [50 > 100 μM), and compound 1 was about >22 folds more active than its unsubstituted derivative in the HepG2 and HCT-116 cell lines. These findings suggested that the electron withdrawing groups at the phenyl ring might be important for high potency. Furthermore, HCT-116 was the most sensitive cell line against these pyrazolyl analogues.After obtaining interesting results with arylhydrazono-pyrazole analogues, different substitutions were made at the phenyl ring for optimizing the template. The analogues  earlier . Their a1 emerged as a promising anticancer agent effective against the colorectal carcinoma cell lines (IC50 4.2 μM) acting through xanthine oxidase inhibition (IC50 0.83 μM). Results of in vitro and in silico xanthine oxidase inhibitory activities revealed that the anticancer activity of these pyrazolyl analogues might be due to XO inhibition.In conclusion, compound


Type II germ cell tumors (GCT) are the most common solid cancers in males of age 15 to 35 years. Treatment of these tumors includes cisplatin-based therapy achieving high cure rates, but also leading to late toxicities. As mainly young men are suffering from GCTs, late toxicities play a major role regarding life expectancy, and the development of therapy resistance emphasizes the need for alternative therapeutic options. GCTs are highly susceptible to interference with the epigenetic landscape; therefore, this study focuses on screening of drugs against epigenetic factors as a treatment option for GCTs.We present seven different epigenetic inhibitors efficiently decreasing cell viability in GCT cell lines including cisplatin-resistant subclones at low concentrations by targeting epigenetic modifiers and interactors, like histone deacetylases (Quisinostat), histone demethylases (JIB-04), histone methyltransferases (Chaetocin), epigenetic readers  and polycomb-repressive complexes . Mass spectrometry-based analyses of the histone modification landscape revealed effects beyond the expected mode-of-action of each drug, suggesting a wider spectrum of activity than initially assumed. Moreover, we characterized the effects of each drug on the transcriptome of GCT cells by RNA sequencing and found common deregulations in gene expression of ion transporters and DNA-binding factors. A kinase array revealed deregulations of signaling pathways, like cAMP, JAK-STAT and WNT.Our study identified seven drugs against epigenetic modifiers to treat cisplatin-resistant GCTs. Further, we extensively analyzed off-target effects and modes-of-action, which are important for risk assessment of the individual drugs.The online version contains supplementary material available at 10.1186/s13148-021-01223-1. According to the World Health Organization (WHO) classification system, germ cell tumors (GCT) of the testis can be subdivided into two main groups . One groEven though cure rates are up to 90%, mostly due to cisplatin based therapy, the incidences keep rising . AdditioCompared to most other cancer types, GCTs harbor a very low mutational burden , 8. HencPreviously, we described the cytotoxic effects of the histone deacetylase (HDAC) inhibitor Romidepsin , 18 and bona fide epigenetic targets in GCTs, we re-analyzed microarray expression data of different epigenetic writers, erasers and readers in the GCT entities GCNIS, SE, EC and teratoma  among the different GCT entities  at the BRD4 promoter, indicative of an active DNA demethylation process (ADD) [6-methyladenosine (m6A)-marked enhancer RNAs (eRNAs) have been identified to recruit the nuclear m6A reader YTHDC1, resulting in the formation of m6A-eRNA/YTHDC1 condensates, which promote BRD4/BRD4 co-activator formation [BRD4 was further regulated by miRNA binding to the 3′ UTR of BRD4, as well as post-translationally by acetylation, phosphorylation, ubiquitination and proline hydroxylation [Of note, mRNA levels of ss (ADD) . Moreoveormation . Furtherxylation . Additioxylation , 38. In 50 values  in a nanomolar range, while GSK343, PBIT, PRT4165 and LP99 showed EC50 concentrations up to 50 µM. Additionally, VZ185, a recently published PROTAC [50 > 50 µM), Selisistat, Nicotinamide, GSK2879552, Bizine, BI-9564 and Aurintricarboxylic Acid were excluded from further analyses. Generally, all drugs were less effective in targeting JAR cells (CC) as well as in the MPAF fibroblast cells  , while JAR and JEG-3 were less responsive to the epi-drugs  , JEG-3, BeWo (CC) and GCT72, 1411H (YST), their cisplatin-resistant sublines (-R), as well as another fibroblast cell line (HVHF2) and Sertoli cells FS1   and HDAC1 (Quisinostat target) were observed in 17% and 13% of the samples, respectively. All target genes were expressed in seminomatous and non-seminomatous GCT (with lowest mRNA levels of SUV39H1 in YST), further corroborating the potential use of the selected drugs  were analyzed by screening the 'Testicular Germ Cell Tumors' cohort of ‘The Cancer Genome Atlas’ TCGA , 42, 43), 4342, 4EZH2 (GSK343), which was marginally expressed in PCa and RCC. Based on these data, the three most potent drugs Quisinostat, JIB-04 and Chaetocin were tested in UC, PCa and RCC cell lines . Similar to the observations found in GCTs, JIB-04 and Chaetocin were highly effective in these urological entities, thereby highlighting the pan-urologic applicability of these epi-drugs.In addition, other urological malignancies , prostate cancer (PCa) and renal cell carcinoma (RCC)) were screened for the expression of the target molecules of Quisinostat, JIB-04, PBIT, Chaetocin, MZ-1, LP99, PRT4165 and GSK343 ) induced apoptosis in the majority of the tested GCT cell lines, with TCam-2 and 2102EP being affected the most , PBIT was excluded from further molecular analysis due to its higher ECTo screen for the effects of each epi-drug on the epigenetic landscape, mass spectrometry analyses of histone modifications were performed in the EC cell line 2102EP after treatment with Quisinostat, JIB-04, Chaetocin, MZ-1, LP99, PRT4165 or GSK343, allowing to not only confirm known changes in the epigenetic landscape caused by each epi-drug, but also to identify epigenetic effects beyond the postulated modes of action  analysis was performed in 2102EP EC cells treated with Quisinostat, JIB-04, Chaetocin, MZ-1, LP99, PRT4165 or GSK343  and DDIT4 (DNA damage-inducible transcript 4) and downregulation of EIF3CL (eukaryotic translation initiation factor 3 subunit C-like)  as well as ‘cell differentiation regulators’, while downregulated genes were involved in ‘regulation of respiratory gas’ and ‘response to organic substance’  and ‘negative regulation of transcription’    and the ‘extracellular matrix,’ while 7 downregulated genes were found to be related to ‘cytoplasm’. Furthermore, the STRING algorithm  predicted interaction between the  development- and pluripotency-associated genes DPPA3 and DPPA5   in cell lines of different GCT subtypes   showed similar deregulations in gene expression as found in the RNA seq analysis .A qRT-PCR analysis was performed to validate selected deregulations in gene expression after inhibitor treatment (EC72) Fig. B. In theA proteome profiler phospho-kinase array was used to detect phosphorylation of well-known kinase targets Fig. C. Thus, 22) [PRKCB, which is targetable by Enzastaurin  , which is targetable by kb NB 142–70 , upregulation of PRKCB (Quisinostat), PRKD1 (Chaetocin), HDAC9 and CSF1R (GSK343) was observed  . Quisinoal) Fig. A. Chaeto–70 Fig. A. For GS124 Fig. A. GW-258The combination of Quisinostat and Enzastaurin as well as Chaetocin and kb NB 142–70 significantly reduced cell viability in JAR (30% and 5%) and GCT72 (10% and 10%) cells compared to the mono treatments, while having little to no effect on TCam-2 and 2102EP cells. The two combinations (Panobinostat and Sunitinib) with the PRC2i GSK343 showed promising results in all cell lines, significantly reducing cell viability compared to the mono treatments of the drugs. The combinatorial effect for GSK343 was strongest in TCam-2 (30%) and 2102EP (13%) upon treatment with 20 nM Panobinostat. The combination of GSK343 with Sunitinib was also effective in these two cell lines, since the mono treatments did not reduce cell viability. he combination led to a reduction of cell viability by 60% (TCam-2) and 80% (2102EP).In this study, 16 different and selective epi-drugs were analyzed for their potential to treat (cisplatin-resistant) GCTs. Of these, seven were selected for further analyses due to their cytotoxicity at low nano-  to micromolar  concentrations  upregulated by the epi-drugs . Thus, drugs targeting these genes are putative combination partners for co-application with the corresponding epi-drug. Indeed, our analyses demonstrated that a combinatory application  reduced the viability considerably compared to mono application in a cell line-dependent manner, suggesting that these drugs are promising combination partners for related epi-drugs. Nevertheless, the suitability as a therapeutic option for GCTs and other urological malignancies needs to be extensively analyzed in future studies.We identified four druggable genes . Regarding H2AK119ub after PRT4165 treatment , Ismail et al.  observedmiR-497-5p and its target gene SALL4. Specifically, DNA hypermethylation of the miR-497-5p coding sequence resulted in silencing of its expression, eventually leading to enhanced SALL4 transcription in the CC cell line JEG-3 [SALL4-overexpressing JEG-3 CC cells [Most of the tested epi-drugs in this study were less sensitive in CC cell lines. CC is not only a cancer of the testis, but is also one of the most aggressive gestational trophoblastic neoplasia due to its propensity to invade the vascular system . Specifine JEG-3 . SubsequCC cells . Though,How can we explain the detected off-target effects on the epigenetic landscape? Expression of alternative target molecules of the same enzyme class as the primary target of each epi-drug is quite low in GCT tissues and cell lines  –70. To aIn addition to the changes in histone modifications, epi-drug treatment (except HDACi Quisinostat) affected the DNA-methylation status, i.e. decreased 5mC. Passive DNA-demethylation occurs over several cycles of replication; therefore, the 5mC decrease after 24 h is more likely due to active DNA demethylation (ADD). We observed a tendency for  decreased 5mC, which was accompanied by 5hmC accumulation. 5hmC formation is the first step of ADD and mediated by the TET enzymes (TET1—3) . Thus, tFurther analyses are necessary to validate ADD via the oxidation pathway, which is preferentially utilized in GCTs .CACNA1A/B/C/G; Additional file 2+ uptake through calcium channels being linked to membrane depolarization and cell death in adrenal chromaffin cells [KCN genes), which could also result in pro-apoptotic release of potassium ions. Chaetocin treated cells showed downregulation of potassium and sodium transporters (ATP1A2), eventually leading to a reduction of K+ and accumulation of Na+, which have been associated with apoptosis in lymphoma cells [Quisinostat treatment caused deregulations in expression of genes associated with ion-transport and -binding factors. Downregulation of transporters regulating the ion uptake and upregulation of channels mediating gradient-dependent flow of potassium and chloride ions have been shown to be pro-apoptotic via inducing water loss of the cells . In turnin cells . In addima cells .DDIT4,DNA damage-inducible transcript 4 protein) was also upregulated in H358  [ HOX genes, also showing a tendency towards downregulation in our study , thereby inducing apoptosis and/or cell cycle arrest. Cell lines from different pan-urologic cancer entities were highly sensitive toward the epi-drugs within a therapeutic window, mostly ruling out effects on healthy cells, e.g. fibroblasts. Additionally, we provide a detailed analysis of drug related (off-target) effects on the histone modification landscape. Further, we gained mechanistical insights by characterizing each epi-drug on transcriptional level and analyzing related signaling pathways. Our data provide a valuable source of information on tested epi-drugs, which might be of benefit for drug combination attempts and patient/therapy risk assessment.2, while bladder, prostate and renal carcinoma cell lines were grown at 37 °C and 5% CO2. Short tandem repeat (STR) profiles of all cell lines are checked on a regular basis and are available upon reasonable request. All cell lines are scrutinized for mycoplasma contamination on a regular basis using PCR strategy. Briefly, medium from cultured cells was boiled at 95 °C for 5 minutes (min) and 2 µl used in the PCR. The primers were used as a mix of ten primers. Sequences are given in Additional file All GCT cell lines were grown as described previously , 91. Cel3 cells  per 96-well were seeded and different drug concentrations  assays were performed as described previously . In summ50 concentrations for each cell line. For Annexin V/PI staining, cells were washed with Annexin V Binding Buffer before being resuspended in 70 µl Annexin V Binding Buffer and 2.5 μl Annexin V FITC-conjugate  and 15 µg PI  and incubated in the dark at room temperature for 15 min. 500 µl of Annexin V Binding Buffer was added before measurement. For the cell cycle analysis, cells were washed with PBS , fixed by ice cold 70% ethanol and stained with 2 µg/ml PI and 200 µg/ml RNaseA  in PBS shortly before every measurement. At least 5 × 104 cells were counted and analyzed using the MACSQuant software v 2.13.0.Flow cytometry analyses of apoptosis rates and cell cycle distribution was performed by Annexin V/propidium iodide (PI) or PI staining, respectively, using MACSQuant Analyzer 10  after 16 h of treatment with the corresponding ECDNA was isolated using the QIAamp DNA Mini Kit  according to the manual. RNA from cell lines was isolated using the RNAeasy Mini Kit  according to the manual. Proteins were extracted using RIPA buffer  containing protease inhibitor tablets . Histones were extracted using the Histone Extraction Kit according to the manufacturer protocol . Protein concentrations were assessed by the BCA Protein Assay Reagent Kit . RNA concentrations as well as 260/280 nm, 260/230 nm purity ratios were determined by NanoDrop measurement .2O to 200 ng, 100 ng and 50 ng in 200 µl per slot. Slot-Blotter device  was assembled as according to the manual. DNA was blotted onto a positively charged nylon membrane  using vacuum application. Three identical membranes were prepared. The membranes were air-dried for 15—20 min and UV-crosslinked (20 s (s), 1200 mJ/cm2). One membrane was stained with methylene blue  in 0.3 M sodium acetate (pH 5.2)) to validate DNA loading across the samples. The remaining two membranes were blocked in 5% milk powder in PBST for 1 h at RT. Membranes were incubated with 5mC or 5hmC antibody overnight at 4 °C. After washing, membranes were incubated with secondary antibody for 2 h at RT. Signals were detected using the ChemiDoc Imaging Systems . See Additional file Purity of DNA was verified using NanoDrop 260/280 ratio and agarose gel electrophoresis. DNA was diluted in HWestern Blots were performed as described previously . In totaFor the detection of phosphorylated kinase targets, the Proteome Profiler Human Phospho-Kinase Array Kit  was used according to the manufacturer’s instructions. The ChemiDoc Imaging System  was used for detection. 2102EP cells were harvested after 16 h of treatment with the epi-drugs. Pixel density was analyzed using Fiji ImageJ 1.53c .Quantitative RT-PCR (qRT-PCR) was performed as described previously . A total50 concentrations for Quisinostat, JIB-04, Chaetocin, MZ-1, LP99, PRT4165, GSK343 and DMSO as solvent control. The cells were harvested via trypsinization after 16 h. The mass spectrometric analysis of histone modifications was outsourced to the Mod Spec service by Active Motif . Histone extraction and mass spectrometric analysis was performed as described previously [2102EP cells were treated with the ECeviously . Raw dathttps://www.ncbi.nlm.nih.gov/geo/, GSE189472) [p values were corrected for multiple testing by false discovery rate (FDR). Accompanying PCA and heatmap were generated using Qlucore Omics Explorer 3.7 . Values of solvent controls wereaveraged from the studies GSE189472, GSE190792 and GSE190022 [Transcriptome analysis has been performed as previously described at the core facility ‘Genomics & Transcriptomics’  and has been publicly shared via GEO (E189472) , 95. RNAE189472) , 95. Stan = 4), GCNIS (n = 3), SE (n = 4), EC (n = 3), TE (n = 3) and mixed non-seminomas (n = 4). All tumors were classified according to the WHO classification of tumors based on their histology and assessment of tumor or GCNIS amount. GCT cell line datasets included TCam-2 (n = 5), 2102EP (n = 5), NCCIT (n = 4) and JAR (n = 2). Data are available via GEO . Expression intensities < 10 (Affymetrix) and < 8 (Illumina) were considered as not expressed based on expression intensities of SOX2 and SOX17 in SE  and EC  cell lines or tissues.Experimental setup and microarray data of GCT tissues (Affymetrix expression array) and cell lines (Illumina HT-12v4 expression microarray) have already been published , 90, 96.https://david.ncifcrf.gov/) for functional annotations [https://string-db.org) [https://www.cbioportal.org/) [http://www.interactivenn.net/) [http://mkweb.bcgsc.ca/tableviewer/) [https://www.wordclouds.com/) by Zygomatic . PINAv3.0 (https://omics.bjcancer.org/pina/) was used to identify drugs suitable to target molecules induced by epi-drugs [https://app.biorender.com/) .Analysis of RNA seq data was performed using the online tool DAVID Bioinformatics Resources 6.8  , 100. Thal.org/) , 45. Thenn.net/)  and the viewer/) . The funpi-drugs . Proposet test after confirming equality of two variances according to the F test has been performed to analyze differences between groups .A two-tailed Student’s Additional file 1: Figure S1. Identification of 16 potential epigenetic targets for GCT therapy. A Re-analysis of gene expression of epigenetic modifiers in GCT tissue samples , SE (n = 4), EC (n = 3), TE (n = 3) and mixed non-seminomas (n = 4)). B, C A Venn diagramof commonly expressed epigenetic modifiers in GCT tissues (B) and cell lines (C) highlighted 80 commonly expressed genes in GCT tissues and 33 in GCT cell lines . D Re-analysis of the 80 commonly expressed epigenetic modifiers of GCT tissues in GCT cell lines , 2102EP (n = 5), NCCIT (n = 4) and JAR (n = 2)). E Final list of 35 target genes (including BRD4 and EZH2) as well as corresponding drugs. F Western blot analysis verifying protein levels of BRD4, VHL and EZH2 in GCT cell lines. β-Actin was used as loading control. Expression microarray data were re-analyzed in context of this study [is study , 90, 96.Additional file 2: Figure S2. XTT data for 16 epigenetic inhibitors in three GCT cell lines and MPAF fibroblast cells (n = 4). Cells were treated once with indicated epi-drugs. Cell viability was detected after 24, 48, 72 and 96 h. The red line indicates 50% cell viability. Asterisks indicate significant changes between treatment and solvent control  .Additional file 3: Figure S3. XTT data for eight epigenetic inhibitors in 14 GCT cell lines, fibroblast cells MPAF and HVHF2, and Sertoli cells FS1 (n = 4). Cells were treated once with indicated epi-drugs. Cell viability was detected after 24, 48, 72 and 96 h. The red line indicates 50% cell viability. Asterisks indicate significant changes between treatment and solvent control  .Additional file 4: Figure S4. Epi-drugs decrease cell viability in GCTs and other urological entities. A EC50 values for each GCT cell line, fibroblasts and Sertoli cells and epi-drug treatment were calculated from data shown in Fig. S3. B Analysis of the mutational and gene expression alteration spectrum of the top eight target genes using the cBioPortal tool on the 'Testicular Germ Cell Tumors' cohort of TCGA. C–E XTT data for Quisinostat, JIB-04 and Chaetocin treatment in three cell lines each for UC (C), PCa (D) and RCC (E). The red line indicates 50% cell viability. Asterisks indicate significant changes between treatment and solvent control  . F EC50 values from (C), (D) and (E) calculated using GraphPad Prism v8. EC50: half-maximal effective concentration where the cell viability has 50% compared to the solvent control. SE, seminoma; EC, embryonal carcinoma; YST, yolk-sac tumor; CC, choriocarcinoma; SC, Sertoli cells; UC, urothelial carcinoma; PCa, prostate cancer; RCC, renal cell carcinoma.Additional file 5: Figure S5. Raw data of flow cytometry analysis of Annexin V/PI (apoptosis) or PI staining only (cell cycle) of indicated cell lines (columns) after 16 h of treatment with indicated epi-drugs (rows).Additional file 6: Figure S6. Mass spectrometry analysis of histone modifications arranged by frequency. A–C Circos diagrams are illustrating the difference in peptide frequency of given histone modifications of treated 2102EP cells compared to the solvent control. Histone modifications with a general peptide frequency of 10–100% (A), 1–10% (B) and 0–1% (C) are depicted separately for better comparison. Only differences in modification abundancy > 1 (10–100%), > 0.1 (1–10%) or > 0.01 (0.1–1%) were considered. D Depiction of the information given by the circos diagram rings. E Western blot analysis of histone extracts from PRT4165 or solvent control DMSO treated 2102EP cells after 24 h and 48 h. Coomassie staining is shown as loading control. F Western blot analysis of selected histone modifications in GCT cell lines. Cells were treated with indicated epi-drugs for 16 h. Total H3/H4 was used as loading control. ↑: upregulation, ↓: downregulation.Additional file 7: Figure S7. A Unsupervised clustering heatmap of RNA seq data . 2102EP were treated with indicated epi-drugs for 16 h. B STRING analysis of upregulated genes in Quisinostat treated (16 h) 2102EP cells.Additional file 8: Figure S8. STRING analysis of deregulated genes after 16 h of indicated epi-drug treatments in 2102EP cells . A LP99 upregulated genes, B LP99 downregulated genes, C PRT4165 upregulated genes, D PRT4165 downregulated genes, E GSK343 upregulated genes, F GSK343 downregulated genes.Additional file 9: Figure S9. A Proteome Profiler Human Phospho-Kinase Array raw data. 2102EP were treated with indicated epi-drugs for 16 h (n = 2). B Membrane layout of kinase array with corresponding spotted antibodies given in C. Detectable dots (marked in red) were used for quantification with ImageJ as shown in D. Gray bar indicates cut-off value of 2500.Additional file 10: Figure S10. A XTT cell viability raw data of co-treatments. Cells were pretreated with the 0.5 × EC50 concentration of each epi-drug for 24 h, followed by treatment with different concentrations of the co-drug for 24 h and 48 h. B, C Epi-drug side target gene expression in GCT tissues (B) , GCNIS (n = 3), SE (n = 4), EC (n = 3), TE (n = 3) and mixed non-seminomas (n = 4) and cell lines (C) (TCam-2 (n = 5), 2102EP (n = 5), NCCIT (n = 4) and JAR (n = 2). Expression microarray data were re-analyzed in context of this study [50 values from published data for Quisinostat [50, half-maximal inhibitory concentration; HDAC, histone deacetylase; BET, bromodomain and extra-terminal motif; PRC, poly repressive complex.is study , 90, 96.sinostat , JIB-04 sinostat , Chaetocsinostat , MZ-1 [1sinostat , LP99 [3sinostat , PRT4165sinostat  and GSK3sinostat . GCT, geAdditional file 11: Table S1. A Raw data of mass spectrometry analysis of histone modifications given as peptide frequency (n = 3). B Corresponding bar charts to (A) scaled to histone peptide frequency of 100%, 10% and 1%. C–I RNA seq data given as fold change compared to solvent control (log2) as well as DAVID functional annotation analysis for C Quisinostat, D JIB-04, E Chaetocin, F MZ-1, G LP99, H PRT4165 and I GSK343.Additional file 12: Table S2. A Studied cell lines including appropriate culture conditions. B Sequences of used oligonucleotides. C Epi-drug solvents and suppliers. D Used antibodies.


Tongue cancer is one of the most aggressive forms of oral squamous cell carcinoma which needs more investigations. Herein, we aimed to establish and characterize a tongue cancer cell line. Tumor tissue was obtained from a 70-year-old woman with tongue cancer. The established cell line named as EZB-ICR and characterized for doubling time, expression of specific markers, HPV corporation and migration status using flow cytometry, immunofluorescence staining, multiplex PCR, and migration assay. EZB-ICR was negative for expression of mesenchymal specific markers, cytokeratin19, pan-cytokeratin, vimentin and EPCAM, but was positive for S100 and Nestin. No appearance of human papilloma virus DNA was seen. The doubling time of EZB-ICR was 31 hours and migration assay confirmed its metastatic nature. To the best of our knowledge, EZB-ICR is the first tongue human cancer cell line established in Iran, and its features make it appropriate for cancer-based in vitro studies and contribute to more studies on tongue cancer. Head and neck cancer is the world’s fifth most common type of cancer worldwide. Oral squamous cell carcinoma (OSCC) is a prevalent cancer consisted of more than 90% of oral malignancies and in spite of recent advancements in diagnostic approaches and treatments, 5 year survival showed no difference in recent years . The most common oral squamous cell carcinoma is found in tongue and mouth floor . The age range of these patients is 20–44 years old and consuming tobacco and alcohol are among the most important risk factors. Furthermore, genetic, environmental or viral factors may lead to the pathogenesis of tongue cancer .The feature of tongue cancer is completely different from malignancies of other parts of oral cavity because tongue has a significant lymphatic structure and as a result shows aggressive metastatic pattern . Based on the American Cancer Society, 5-year relative survival rate for tongue cancer is as follows: 81% for localized (no sign the cancer has spread outside), 68% for regional (spread to nearby structures or lymph nodes), and 39% for distant (spread to distant parts of the body) type of tongue cancer . Although tongue cancer is increasing throughout the world, no effective treatment is exist and common therapies such as surgery, radiation therapy, chemotherapy and combination therapies are not enough . Thus more in vitro and in vivo investigations using tongue cancer cell lines are undoubtedly needed to establish new therapeutic intervention for tongue cancer. Here, we established a new tongue cancer cell line named EZB-ICR and characterized it for the expression of various markers, growth condition, HPV status and metastatic pattern.Cell line establishment2. The media was changed every 48 hours. In this experimental study, tumor tissue sample was obtained from a 70 years old woman patient with tongue cancer. The fragments were washed with sterile phosphate buffered saline (PBS) containing 1% pencillin/streptomycin. Next, the segments were cultured as a transplantation method in complete DMEM  and 1% penicillin/streptomycin) at 37°C in a humidified atmosphere containing 5% COGrowth curve and proliferation assay4 cells were seeded on six well tissue culture plates and the number of the cells was calculated each day for 7 days; this experiment was repeated three times. Growth curve and doubling time were measured by online doubling time calculating software (http://www.doubling-time.com).For evaluation of doubling time and growth rate of the cell line established, 2.5 × 10Flow cytometryFor phenotypical characterization, the cells were trypsinized and washed with PBS. Then the cells were stained with the appropriate flourchrome labeled monoclonal antibodies against mesenchymal lineage markers such as anti-CD34, CD19, CD166, CD105, CD44, CD10, CD146, CD29, CD45, CD73, CD90, CD24, CD69 and also some chemokine/chemokine receptors like MCP1, IP-10, RANTES, EPCAM and CXCR4, CCR5. The cells were fixed by 1% cell fix for 10 minutes and then washed with PBS. Next, 500ml of 0.2% Saponin was added, and incubated for 15 minutes and washed again. Consequently, cells were stained with above mentioned antibodies  and finally were evaluated using FACSculibure flow cytometer and analyzed by FlowJo software version 10.Immunofluorescent assay4 cells were seeded on chamber slides for 24 hours. Then, the slides were washed by PBS and fixed with cold methanol at room temperature, and blocked with 1% bovine serum albumin (BSA) in PBS containing 1% Triton X-100 (PBST). Subsequently, slides were incubated with primary monoclonal antibodies overnight at 4°C in 1.5% BSA in PBST. Then, slides were washed with PBST and incubated with secondary fluorescent antibodies  for 2 hours at room temperature. After that, the slides were mounted by solution containing propidium iodide (PI) and observed by fluorescent microscope. Our negative controls were the slides without antibodies and those treated with secondary antibody. Nestin, cytokeratin 19, vimentin, pancytokeratin and S100 were assessed with immunofluorescent assay using appropriate antibodies . 3 × 10Migration assay3 cells were cultured in the upper 8 μm transwell cell culture chambers in the FBS deficient condition media and the lower wells was filled out by 650 μl of DMEM media containing 10% FBS. The cells were incubated at 37 °C in 5% CO2 humidified chamber for 24 hours. Then, the non-migratory cells were eliminated from the upper side by cotton swab and the cells which were transferred from upper to lower chamber were fixed and stained with 10% ethanol and 0.5% Crystal Violet. At the end, the migrated cells were counted by phase contrast microscope. This assay was performed in triplicate for each cell line including EZB-ICR, KB  and MDA-MB-231 (as an invasive cell line). The motility of cells was investigated by migration assay via transwell inserts. 5×10Colony formation unit assay (CFU)th and 10th days of culture, cells were washed with PBS and stained by 0.5% Crystal Violet in methanol for 15 minutes at room temperature, finally flasks were washed by PBS and colonies were calculated by following formulation: colony number/cells of seeding ×100.The EZB-ICR cells were harvested with trypsin–EDTA and around 1,000 single cells were seeded in 25mm tissue culture flask in DMEM containing 10% FBS and 1% penicillin/streptomycin. On the 5Human Papilloma Virus (HPV) molecular genotyping (32 genotypes)Total DNA of EZB-ICR cells was extracted by fully automated instrument, part of SPF10 region of the HPV genome was amplified using Multiplex PCR method and the genotype of HPV was determined by various specific probes using reverse hybridization.Establishing and characterizing tongue cancer cell lineEZB-ICR cell line was generated from a 70-year-old woman patient that used waterpipe (hubble-bubble) sometimes. Examination diagnosed aggressive squamous cell carcinoma in right oral tongue lesion in 2016. The tumor mass size was 2.7x1.8x 0.7 cm at right lateroinferior site with ulcerative surface and also, base, apex and medial margins grossly not involved and inferior margin grossly seems to be involved. The patient underwent a hemiglossectomy surgery. The cells were firstly extracted as explants of chopped tumor mass and the cells were grown out after 3 weeks. EZB-ICR cell line was passaged frequently until 72 passages .Doubling timeDoubling time of EZB-ICR cell line was assessed at passage 25 in the course of the exponential growth phase of the cell line by calculating the number of cells for 7 days. Accordingly, the doubling time of EZB-ICR was about 31 hours .Flow cytometryFlow cytometry examination indicated that EZB-ICR cell line was negative for mesenchymal stem cell markers such as CD166, CD44, CD90,CD146 and also markers such as CD10, CD45, CD34, CD105, CD106, CD14,CD24, CD29, CCR5, CD69, CXCR4, IP10, MCP-1, RANTES and EPCAM as depicted in ImmunostainingAccording to immunofluorescent staining assay, EZB-ICR cell line was strongly positive for the expression of S100 and nestin but it was negative for cytokeratin19, pan-cytokeratin and vimentin .HPV molecular genotypingEZB-ICR cell line was negative for 32 genotypes of HPV. These genotypes were divided to 4 subgroups which included high risk , partially high risk , Low risk  and finally unclassified HPV genotypes . Migration assayMetastatic ability of EZB-ICR cell line was evaluated by migration assay and compared with MDA-MB-231 breast cancer cell line as an invasive cell line and KB cell line which originally from epidermal carcinoma of the mouth. EZB-ICR showed higher capability of migration compared to MDA-MB-231 and in particular KB cell lines .Colony formation assayThe colony formation assay was applied to recognize the ability of the EZB -ICR cell line to grow into a colony forming shape. In this regard the number of colonies after 5 and 10 days were respectively 63 and 45% .Establishment of cell line is a profitable option for further understanding the pathways which contribute to the development of models for cancer metastasis and invasion. In addition, cell lines are required for evaluating the effects of numerous novel drugs and finding better treatments . Several studies identified that the wide variety of patients who suffered from tongue cancer were smokers and most cell lines were determined from them, whereas few cell lines recognized from tumors of nonsmokers .In the present study, we established and characterized the tumorigenic and metastatic EZB-ICR tongue cell line. EZB-ICR cell line did not express any surface mesenchymal markers. Also, it was not positive for the expression of cytokeratin19, pan-cytokeratin and vimentin. In contrast, this cell line was positive for the expression of S100 as well as nestin.In vivo and in vitro experimental studies showed the over-expression of one subgroup of S100, S100A4, related to potential metastatic and poor clinical outcomes in different kinds of cancers . Experimental studies demonstrated the expression of nestin in several tumor cells like gastrointestinal stromal tumors, malignant melanoma, OSCC, pancreatic, prostate and breast cancers . High expression of nestin correlates with consistent growth and poor treatment prognosis in most solid tumors . As our established cell line expresses both S100 and nestin, these markers may be considered as tongue tumor markers for diagnostic and therapeutic options.It has been recently demonstrated that S100 plays significant roles in biological processes such as proliferation, apoptosis and cell survival associated with both normal development and tumorigenesis . Wanga et al., (2017) established a tongue cancer cell line from a nonsmoker 44-year-old woman (UCSF-OT-1109). Experimental studies demonstrated that never-smoking patients had aggressive patterns and their survival were even less than smoker patients . EZB-ICR and UCSF-OT-1109 both were isolated from nonsmoker patients and showed similar morphology. Although the connection of HPV infection to OSCC has been hypothesized, it did not emerge in neither EZB-ICR nor UCSF-OT-1109 . In contrast to EZB-ICR that did not express CD44 and EpCAM, UCSF-OT-1109 was positive for both markers .Migration index showed that EZB-ICR has strong motility in comparison with MDA-MB-231 and KB cell lines. So, this behavior could answer important questions regarding the metastatic pattern of the EZB-ICR cell line.To the best of our knowledge, EZB -ICR is the first human tongue cancer cell line that registered in the Cell Bank of Pasteur Institute of Iran. Furthermore, the EZB-ICR has enabled us to figure out more about the characteristics of tongue cancer, consequently, enhance our information in regard to finding novel therapeutic possibilities. The key important limitation in this study was that the number of tongue cancer cell lines in Iran is rare so we did not have a chance to compare EZB-ICR with other tongue cancer cell lines.