Prostate cancer (PCa) is the second most common malignancy in men, and the fifth leading cause of death worldwide. Mesenchymal stromal/stem cells (MSC) have been identified in PCa, although contradictory effects in malignant transformation and tumor progression have been described. Since umbilical cord (UC) MSC and cord blood serum (CBS) are rich in numerous growth and anti-inflammatory factors, UC-MSC secretome and CBS are able to modulate tumor cell proliferation and survival as well as immunity and angiogenesis. In the present study, we address this relationship and investigate the influence of UC-MSC secretome and CBS on two human PCa cell lines (PC3 and LNCaP) and a normal epithelial prostate cell line (HPEpiC). Our results disclosed that upon exposure to UC-MSC-conditioned medium or CBS, both PC3 and LNCaP cells exhibited reduced viability, proliferation, and motility while non-malignant epithelial prostate cells were unaffected. These findings were corroborated by expression analysis of AKT/PI3K signaling pathway, p53 and interleukin genes. UC-MSC and CBS factors decreased the expression of growth-stimulating AKT and PI3K effectors and simultaneously up-regulated the expression of tumor-suppressor p53. Moreover, a more anti-inflammatory expression profile was found in both malignant PCa cell lines. Altogether, these results shed light into possible mechanisms by which UC-MSC and CBS reduce PCa progression, further reinforcing their potential use as novel therapeutic agents in PCa. Prostate anatomical location potentiates several internal and external injuries, such as by infectious agents, carcinogens, urinary reflux, hormonal changes, and also physical trauma , that caThe finding of unique properties of stem cells in modulating immunity, selective migration to inflammatory sites, and growth factors secretion, mesenchymal stem/stromal cells (MSC) have been considered a promising therapeutic avenue, namely in PCa , 10. IndThe composition of UC-MSC secretome is variable and dependent on the microenvironment in which the UC-MSC are found, thus contributing to the multiplicity of effects reported in several studies and experimental conditions , 25. IndAlthough several studies have shown UC marked therapeutic potential in diverse pathologies, from hematology to immunology, and tissue regeneration, the role of some UC components in oncology field is not yet clear. Some reports have shown that the direct interaction between MSC and cancer cells potentiates metastatic capacity by enhancing epithelial–mesenchymal transition, a key event in tumor invasion process , 28. NevEven though the effort to reach new treatment modalities which improve survival in PCa patients with metastasis, considering stem cell as therapy or even as co-adjuvant has not received much attention. Thus, we wanted to investigate the effects of both, CBS, and UC-MSC on cellular proliferation, viability, and function of human PCa. In this study, we evaluated the effect of both CBS and UC-MSC secretome on the behavior of androgen-sensitive human prostate adenocarcinoma (LNCaP) and androgen-nonresponsive grade IV prostate adenocarcinoma with high metastatic potential (PC3) cell lines. In parallel, the effect of both treatments was investigated on a non-malignant human prostate epithelial cell line (HPEpiC). Thus, the aim of the study was to evaluate the functionality and viability of each cell line by characterizing cytotoxicity, proliferation, and migration capacity under CBS and UC-MSC treatments, as well as the evaluation of a panel of survival and inflammatory genes.CBS and UC-MSC derived from UC units were obtained from BebeVida, Ciências para a Vida S.A. The informed consent for the use of samples for research was obtained in accordance with the requirements of the Declaration of Helsinki and local law at the time of collection.CB and UC units were collected by qualified health care professionals in authorized maternity hospitals. Before delivery, mothers signed an informed consent donating these samples for both research and validation purposes.Treponema pallidum (syphilis), cytomegalovirus and Hepatitis A, B, and C viruses, as well as aerobic and anaerobic bacterial and fungi . Only non-reactive units were further processed.Full serological and microbiological analysis of the CB units was carried out to exclude infection. Tests were performed for the presence of human immunodeficiency virus, ™ Basal Medium  supplemented with 10% (v/v) MesenCult™ Stimulatory Supplement  and 1% antibiotic-antifungal . UC-MSC propagated to the 3rd–8th passages were characterized using accepted MSC-positive markers anti-CD73, CD90, and CD105 , negative hematopoietic markers anti-CD45, CD34, CD11b, CD19, and HLA-DR  and cellular viability  by flow cytometry  (data not shown).UC-MSC were isolated from the umbilical cords of healthy neonates using the direct plastic adherence method. Briefly, the UC samples were sheared into long segments and washed to remove residual cord blood. Then, blood vessels and clots were scraped and washed away, and UC tissue was minced and digested using collagenase  and hyaluronidase . Cells were then plated and incubated in a 5% CO2 atmosphere at 37 °C in MesenCultPCa cell lines PC3 and LNCaP were cultured in RPMI-1640 medium  supplemented with 10% of FBS  and 1% penicillin/streptomycin . HPEpiC cell line was cultured in Prostate Epithelial Cell Culture Medium  supplemented with 2% of FBS , 1% of Prostate Epithelial Cell Growth Supplement  and 1% penicillin/streptomycin .Cord blood was transferred into the processing bag set which is placed in the AXP Device and then centrifuged. During centrifugation, component stratification and separation occurs. Red blood cells (RBC) are transferred to a separate sterile bag, the buffy coat which contains the mononuclear cell rich layer is delivered to a separate sterile freezing bag while the plasma remains in the processing bag . AfterwaAfter primary culture, UC-MSC were propagated and allowed to reach confluence. Then, cultures were washed with HBSS  and incubated in serum-free Dulbecco’s modified Eagle’s medium (DMEM). After 24 h, the conditioned medium was harvested from the UC-MSC cultures, spun for 5 min at 300 g and the supernatant was stored at – 20 °C for the subsequent treatments.Treatments were performed for 24 h, using a final concentration of 10%, 20% and 25% of CBS or 25%, 50%, 75% and 100% of UC-MSC secretome. All treatments were performed in serum-free media and accomplished after a starvation period of 4 h.5 cells/mL), LNCaP (2 × 105 cells/mL) and HPEpiC (1 × 105 cells/mL) were cultured and submitted to the different treatments. Then, MTT   was incubated at a final concentration of 0.5 mg/mL with incomplete cell culture medium for 3 h at 37 °C and 5% CO2. The absorbance was then measured using a microplate reader at 570 nm . Results were performed in triplicates and normalized to the control treatment (considered to be 100%).PC3 (1 × 105 cells/mL), LNCaP (2 × 105 cells/mL) and HPEpiC (1 × 105 cells/mL) were seeded and submitted to treatment period with 0.01 mM BrdU for 24 h. Optical density of proliferating cells was measured at 660 nm. Results were performed in triplicates and normalized as a percentage of the control treatment.The BrdU kit from Roche Diagnostics was used to perform the colorimetric bromodeoxyuridine (BrdU) assay , which evaluates the capacity of proliferating cells incorporate the BrdU. Briefly, PC3 . The migrating distance was imaged 24 or 48 h after treatment, and the scratch closure was calculated by measuring the damage width with Image J software .® , according to the manufacturer’s instructions. Briefly, PC3 (4 × 105 cells/mL), LNCaP (8 × 105 cells/mL) and HPEpiC (4 × 105 cells/mL) were seeded, treated, and then subjected to extraction using NZYol. Total RNA was then separated from other organic components with chloroform, precipitated in isopropanol, washed with 75% ethanol, and sequentially solved in DEPC-treated H2O . RNA purity was measured in nanodrop microplate spectrophotometer  by A260/A280 and A260/A230 ratio to verify RNA integrity.Total RNA was extracted using NZYol3 , using One-step NZYSpeedy RT-qPCR Green kit . Accordingly, 10 ng/µL RNA were employed, and the threshold cycle (TC) values from each biological assay were plotted with two experimental replicates for each biological sample, following the manufacturer's procedure. Melting curve analysis was used to monitor the specificity of primers, and results were normalized to GAPDH expression. Table Gene expression was estimated measuring the mRNA from cell extraction by real-time quantitative PCR (RT-qPCR) with qTOWERpost-hoc test was used to evaluate the effects of different concentrations of CBS or UC-MSC secretome in each cell line. For the comparison between two conditions in each treatment, an independent t Student’s test was performed. Normality distribution of data and the homogeneity of variance were assessed by the Shapiro–Wilk and the Levene’s tests, respectively. All data are expressed as a % of control and are presented as mean ± SD. Statistical significance was assumed when p < 0.05.One-way analysis of variance (ANOVA), followed by pairwise comparisons with Bonferroni’s To evaluate the effect of UC-MSC secretome, cells were incubated with different concentrations. Cell viability, proliferation and migration were assessed after treatment in each cell line. Results are depicted in Fig. p < 0.05 vs. control group). Moreover, a reduction of cell viability was observed in the concentration of 75% and 100% in 52.0 ± 11.6% and 50.1 ± 8.6%, respectively (p < 0.0001 vs. control group). For LNCaP cell line, only the concentration of 100% of UC-MSC secretome led to a significant decrease of cell viability in 32.5 ± 15.6% (p < 0.01 vs. control group), with the other concentrations not reflecting a statistical difference compared to the control group.Concerning cell viability, depicted in Fig. p < 0.05 vs. control group), in the concentrations of 75% in 21.3 ± 8.2% (p < 0.01 vs. control group) and in the concentration of 100% in 24.7 ± 7.2% (p < 0.001 vs. control group). In LNCaP cell line, the concentration of 75% leads to a reduction of 28.4 ± 6.8% (p < 0.05 vs. control group) and in the concentration of 100% decreases in 40.5 ± 10.5% (p < 0.001 vs. control group), with no statistical differences in the concentration of 25% and 50%.Regarding cell proliferation represented in Fig. p < 0.05 vs. control group) and in LNCaP, a reduction of 40.9 ± 16.7% was observed (p < 0.001 vs. control group). Figure Since the UC-MSC secretome concentration of 100% shows the higher reduction in viability and proliferation of tumoral cell lines, with no effect on normal prostate epithelial cell line, this concentration was employed to evaluate the effect in the migratory capacity of the three cell lines, depicted in Fig. After the treatment period with different concentrations of CBS, cell viability, proliferation and migration were assessed in each cell line to evaluate the effect of treatment. Results are depicted in Fig. p < 0.001 vs. control group), while the concentration of 10% shows no statistical difference. In PC3 cell line, CBS decreases viability in the concentration of 20% and 25% with a reduction of 30.5 ± 21.7% (p < 0.05 vs. control group) and 43.6 ± 15.4% (p < 0.01 vs. control group), respectively; moreover, no statistical differences were found in the CBS concentration of 10%. In LNCaP cell line, only the concentration of 25% decreased the cell viability with statistical significance in 28.6 ± 15.4% (p < 0.01 vs. control group).Cell viability is depicted in Fig. p < 0.01 vs. control group). In PC3 cell line, every CBS concentration tested leaded to a reduction in cell proliferation: the 10% of CBS decreased cell proliferation in 17.7 ± 9.5% (p < 0.05 vs. control group), the 20% concentration of CBS reduced in 27.1 ± 11.5% (p < 0.01 vs. control group) and the concentration of 25% CBS diminished cell proliferation in 37.6 ± 8.7% (p < 0.001 vs. control group). Identically to PC3 cell line, the LNCaP cell line have presented a reduction of cell proliferation in tested concentrations of CBS: in the concentration of 10% and 20% the reduction of cell proliferation is 15.0 ± 6.3% and 17.6 ± 7.0%, respectively (p < 0.01 vs. control group), and in the concentration of 25%, cell proliferation is reduced in 22.4 ± 4.1% (p < 0.001 vs. control group).In terms of cell proliferation shown in Fig. p < 0.05 vs. control group); in the LNCaP cell line, the migratory capacity decrease in 40.9 3 ± 17.0% compared to the control group (p < 0.001 vs. control group). Figure Based in the fact that 10% of CBS did not have a statistical significance in the HPEpiC cell line viability and proliferation, and once this cell line is a normal prostate epithelial cell line, this concentration was chosen to evaluate the effect of CBS in the migratory capacity of the three cell lines, depicted in Fig. After treatment exposure, total RNA was extracted from each cell line and then used to relative quantification of signaling intermediates for cell proliferation, survival, and growth pathways AKT, PI3K and p53), as well as to quantify pro and anti-inflammatory cytokines . Results are depicted in Fig. K and p53p < 0.05 vs. control group). In terms of PI3K expression, no differences were found in HPEpiC cell line; in LNCaP cell line, a reduction of gene expression was found in UC-MSC treatment in 4.8 ± 2.2% and in both tumor cell lines, a reduction in gene expression was observed in CBS treatment in 4.8 ± 3.1% in PC3 cell line and 6.2 ± 2.3% in LNCaP cell line (p < 0.05 vs. control group). Regarding p53 expression, HPEpiC cell line shows a reduction of its expression under UC-MSC treatment in 32.2 ± 6.9% (p < 0.05 vs. control group) and no statistical differences were founded in CBS treatment; PC3 cell line presented an increase of this gene expression in both treatments, with an increase in 37.3 ± 9.0% in UC-MSC treatment (p < 0.05 vs. control group) and in 45.5 ± 3.5 in CBS treatment (p < 0.01 vs. control group); in LNCaP, an increase of gene expression was observed in UC-MSC treatment in 5.1 ± 4.4 (p < 0.05 vs. control group) and no statistical differences were found in CBS treatment.Concerning AKT expression, although no statistical differences were found in HPEpiC cell line, PC3 shows a reduction of gene expression only in UC-MSC treatment in 5.9 ± 2.6% and LNCaP presents a reduction of gene expression in CBS treatment with a reduction of 4.6 ± 1.5% (p < 0.05 vs. control group). Concerning IL-6 expression, in HPEpiC cell line no statistical differences were reported except upon the CBS treatment, where a reduction of 26.6 ± 11.6% was observed compared to the control group (p < 0.05 vs. control group); in PC3 cell line, both treatments reduced the expression of this gene, with a decrease in 12.3 ± 4.0% in UC-MSC treatment and in 11.4 ± 5.8% in CBS (p < 0.01 vs. control group); the same behavior was observed in LNCaP cell line, where both treatments decreased IL-6 expression in 8.2 ± 2.8% in UC-MSC (p < 0.01 vs. control group) and in 5.8 ± 4.7% in CBS (p < 0.05 vs. control group). Regarding IL-10 expression, no statistical differences were found in HPEpiC cell line; in LNCaP cell line, the UC-MSC treatment enhance this gene expression in 9.2 ± 4.9% and in both PCa cell lines (PC3 and LNCaP), CBS treatment led to an increase of this gene expression in 5.2 ± 4.8% for PC3 and in 7.7 ± 7.5% for LNCaP (p < 0.05 vs. control group).Observing IL-1β expression, although no statistical differences were found in HPEpiC and PC3 cell line, LNCaP shows a reduction of gene expression in both treatments, with a decrease in 4.1 ± 3.4% in UC-MSC treatment and 6.5 ± 4.5% in CBS treatment , even though 25% and 50% of UC-MSC secretome seemed to enhance the viability of the LNCaP cell line, which can derive from the secretome richness in metabolites and favorable factors (such as growth factors) that can activate proliferation pathways in this particular cell lines, overwhelming the anti-tumoral effects. Moreover, the neuroimmune network regulates tumor microenvironment and affect tumorigenesis, the molecules secreted by tumor microenvironment cells may activate signaling pathways with contradictory outcome such as tumor prone microenvironment . Since tMSC are able to modulate the tumor microenvironment by several distinct mechanisms such as direct cell–cell interactions, or in a paracrine manner, through the secretion of cytokines and chemokines among stem cells and cancer cells , 41.Considering the evaluation of the inflammatory cascades, the results showed that the UC-MSC secretome decreased the expression of proinflammatory cytokines while increasing anti-inflammatory cytokines mRNA levels. UC-MSC secretome did not affect the cytokine expression profile in HPEpiC cells and its effect was more noticeable in LNCaP than in PC3 cell line. PC3 cells are generally regarded as more aggressive than the LNCaP cell line, which can explain the differences in cytokine modulation by UC-MSC. These results corroborate the well-established UC-MSC effects towards inflammation. UC-MSC have the ability to modulate the immune system inhibiting immune cells proliferation, such as T cells and B cells; inducing macrophages switch from pro-inflammatory phenotypes to more anti-inflammatory phenotypes and reduce inflammation by secreting IL-10 and IL-4 . UC-MSC Our results disclose that UC-MSC secretome and CBS reduce malignant prostate cell lines aggressiveness. The synergic effects on PCa survival, motility, and immunomodulation, allied to the cell-free nature of MSC secretome and CBS, support the beneficial effects of both CBS and UC-MSC-released factors. Moreover, UC-isolated MSCs have numerous advantages, when compared to those harvested from the bone marrow, including an improved expansion capacity, easier collection procedures, and lower risks of contamination, and they represent a possible source for autologous cell therapy, standing as a novel milestone in the development of active CB and UC-MSC secretome-derived anti-cancer biopharmaceutical formulations.Further studies regarding the impact of the treatments on death signaling pathways of cancer cell lines, as well as the characterization of both UC-MSC secretome and CBS can potentially lead to the discovery of novel therapeutic methodologies to be used as non-cell-based cancer therapies.


Pleural mesothelioma (PM) is a relatively rare malignancy with limited treatment options and dismal prognosis. We have previously found elevated FGF18 expression in PM tissue specimens compared with normal mesothelium. The objective of the current study was to further explore the role of FGF18 in PM and evaluate its suitability as a circulating biomarker.FGF18 mRNA expression was analyzed by real‐time PCR in cell lines and in silico in datasets from the Cancer Genome Atlas (TCGA). Cell lines overexpressing FGF18 were generated by retroviral transduction and cell behavior was investigated by clonogenic growth and transwell assays. Plasma was collected from 40 PM patients, six patients with pleural fibrosis, and 40 healthy controls. Circulating FGF18 was measured by ELISA and correlated to clinicopathological parameters.FGF18 showed high mRNA expression in PM and PM‐derived cell lines. PM patients with high FGF18 mRNA expression showed a trend toward longer overall survival (OS) in the TCGA dataset. In PM cells with low endogenous FGF18 expression, forced overexpression of FGF18 resulted in reduced growth but increased migration. Surprisingly, despite the high FGF18 mRNA levels observed in PM, circulating FGF18 protein was significantly lower in PM patients and patients with pleural fibrosis than in healthy controls. No significant association of circulating FGF18 with OS or other disease parameters of PM patients was observed.FGF18 is not a prognostic biomarker in PM. Its role in PM tumor biology and the clinical significance of decreased plasma FGF18 in PM patients warrant further investigation. We analyzed circulating FGF18 in the plasma of 40 patients with pleural mesothelioma, six patients with pleural fibrosis and 40 healthy controls. FGF18 was significantly decreased in the plasma of patients with pleural mesothelioma and pleural fibrosis compared with healthy controls. Within the pleural mesothelioma cohort, FGF18 was not significantly correlated with overall survival or other disease parameters. Pleural mesothelioma (PM) is a relatively rare malignancy with poor prognosis, limited therapeutic options, and a lack of reliable biomarkers to aid in diagnosis and patient stratification.FGF18 is a secreted glycoprotein and belongs to the fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) family that consists of 18 ligands and 4 transmembrane receptor tyrosine kinases.Physiologically, FGF18 plays a crucial role in the development of skeleton, cartilage, lung, and brain.In the current study, we further explored the role of FGF18 in pleural mesothelioma and evaluated its suitability as a blood‐based biomarker.2 and regularly checked for mycoplasma contamination. Cell line names, cancer type and source for each cell line are listed in Table Cells were cultured in growth medium with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO−dCt × 104 of FGF18 normalized to the housekeeping gene GAPDH as previously published.Cells were grown in flasks to about 80% confluence. Total RNA was extracted with the innuPREP RNA mini kit  according to the manufacturer's instructions. RNA was reverse transcribed with reverse transcriptase . The resulting cDNAs were used as templates for qRT‐PCR analysis with Taqman assays for FGF18 (Hs00826077m1) and GAPDH (Hs99999905m1) both from Thermo Fisher Scientific. Relative gene expression levels were calculated as 2https://doi.org/10.1371/journal.pcbi.1006701].RNASeq expression data for FGF18 as well as clinical data were downloaded into R using the TCGAbiolinks package [A retroviral expression construct for FGF18 was generated by amplifying the full open reading frame of FGF18 with a proofreading polymerase  and primers FGF18‐for: 5′‐TTTTTAATTAACGATGTATTCAGCGCCCTC‐3′ and FGF18‐rev: 5′‐TTTTTAATTAACCTAGGCAGGGTGTGTG‐3′ from cDNA of the PM cell line M38K and ligating the amplicon into the retroviral expression vector pQCXIP (Takara Bio). After sequence verification, viral particles were generated by transient co‐transfection of the FGF18 expression construct or the empty pQCXIP vector (as vector control) with the two helper plasmids VSV‐G and pgag‐pol‐gpt into HEK293 cells. Supernatants containing viral particles were used for target cell transduction and cells with stable integration of the FGF18 construct were selected with puromycin (0.8 μg/mL) as published.3 per well) into 24‐well plates and allowed to grow for up to 14 days until cell clones had formed. Then cells were washed with PBS, fixed with methanol‐glacial acetic acid (3:1) for 20 min and again washed with PBS. Afterward cells were stained with crystal violet  for 1–3 h. To remove the excessive crystal violet, the plates were washed and air‐dried overnight. Images of the stained colonies were taken with a Nikon D90 camera, and afterward the cells were destained with 2% SDS for about 3 h. The solution was transferred into microtiter 96‐well plates and the absorption at 562 nm was photometrically measured with a SynergyHT plate reader.Cells were seeded at low density (104 per well) were seeded into transwell chambers and allowed to transmigrate for 24 h. Cells that had transmigrated to the lower surface of the chamber were fixed with cold methanol for 20 min, whereas cells that remained on the upper surface were removed using a cotton stick. Afterward, cells were washed, stained with crystal violet, washed again, destained with 2% SDS and absorbance of the solution was measured at 562 nm as outlined above for the clonogenic growth assay.For analyzing cell migration, transwell assays using BD Falcon 8 μm pore size cell culture inserts in a 24‐well format were performed. Cells  served as controls. In all analyzed patients, PM diagnosis was histologically proven during clinical routine work‐up. The latest version of the TNM IMIG/IASLC staging systemCirculating FGF18 was measured in plasma samples with the FGF18 ELISA kit from USCN (USC‐SEC907HU). Sample preparation, generation of standard curves, and measurement of samples in duplicates were done following the manufacturer's instructions.n ≥ 3 replicates and unpaired t‐tests were used for comparisons of two groups. Differences were considered statistically significant for p values <0.05. Statistical analyses were performed using the SPSS 28.0 software system (SPSS Inc.) and plots were generated with GraphPad Prism 8.Categorical data are displayed as counts and percentages and metric data are given as median and interquartile range (IQR), or, in case of survival, as median and corresponding 95% confidence interval (CI) if not otherwise indicated. In the plasma sample test cohort as well as in the TCGA dataset, patients were divided into high and low FGF18 level groups by the median value (112.3 pg/mL) of protein and relative gene expression (320.9), respectively, as in previous studies.https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga), where pleural mesothelioma tissue showed the second highest FGF18 expression after ovarian cancer across 32 cancer types when analyzed on the UALCAN portal  and those from biphasic PM (n = 3) and no difference between BAP1+ (n = 6) and BAP1− (n = 2) cell lines were apparent . A comparison of FGF18 mRNA expression with patient survival revealed a trend towards longer OS in patients with high FGF18 .The high FGF18 mRNA expression in PM cell lines as compared with cell lines from other cancer types suggested a potentially important role of FGF18 in PM, and therefore we next retrieved FGF18 gene expression data from the TGCA dataset of PM patients  and the respective empty vector controls . Parental M38K had a higher endogenous FGF18 expression than SPC212 but nevertheless strong overexpression of ectopically expressed FGF18 could be achieved in both cell lines compared with the parental cell lines as well as the respective vector controls  from two different institutions along with 40 healthy controls  and 6 patients with benign pleural disease .n = 40; 112.3 pg/mL, IQR: 79.7–142.5) than in healthy controls . Moreover, the median FGF18 levels were significantly lower in patients with benign fibrosis  than in healthy controls (p = 0.004). No significant difference in FGF18 could be observed between patients with PM and patients with fibrosis (p = 0.974). Receiver operating characteristic (ROC) curve analysis was performed to assess the ability of FGF18 to discriminate between healthy individuals and patients with PM and showed an area under the curve of 0.837  of all cases. Thirteen of 40 patients presented with early‐stage disease (IMIG stage I and II), while 27/40 patients were diagnosed in an advanced stage (IMIG stage III and IV). Treatment strategies included radical surgery as part of multimodality treatment protocols , chemo‐ and/or radiotherapy  and best supportive care . Based on the median FGF18 value, the study cohort was divided into patients with high and low FGF18 levels. Detailed baseline characteristics of both groups are displayed as Table p = 0.234), sex (p = 0.490), histology (p = 0.114), stage (p = 0.567), and treatment (p = 0.329) were observed. However, there was a recognizable, not significant tendency toward higher median plasma FGF18 levels in the nonepithelioid group   and without (n = 15) anamnestically established asbestos exposure  patients were ≥65 years old and 28/40 (70%) were male patients. Epithelioid histology made up 80%  when co) Figure . There w) Figure  or betwee Figure .p = 0.618) . No independent predictors, however, were detected by multivariate analysis. FGF18 was not found to be an independent prognostic factor for OS .Finally, we tested whether levels of circulating FGF18 correlate with patient prognosis. Median OS for the entire cohort was 20.733 months . PM patients with low FGF18 levels had a longer, however not significantly longer overall survival when compared with those with high FGF18 levels  have been proposed for PM.With respect to tumor biology, the effects of FGF18 reported in the literature are tissue‐type specific. Our results show that overexpression of FGF18 in PM cells with very low endogenous FGF18 can result in decreased clonogenicity. This aligns with data from renal cell cancerOverall, our data disprove circulating FGF18 as a prognostic biomarker in PM. The decrease of circulating FGF18 in pleural disease and the role of FGF18 in PM biology should be further evaluated.Conceptualization, B.H., MA.H., M.G.; software, T.M.; investigation, B.M., K.Sc., T.K., C.W., L.R., Y.D., K.Si., A.R.; resources, W.B., B.G.K., K.H., B.D.; data curation, B.M.; writing—original draft preparation, B.M., T.M., M.A.H., M.G.; writing—review and editing, all authors; visualization, B.M., M.G.; supervision, V.L., B.H., M.J.; funding acquisition, B.D. and M.G. All authors have read and agreed to the published version of the manuscript.The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Table S1. Cell lines used in this study.Figure S1. Mesothelioma shows the second highest FGF18 gene expression across 32 cancer types analyzed by the TCGA consortium. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse B cell lymphoma; ESC, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe tumor; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, brain lower grade glioma; OV, ovarian serous cystadenocarcinoma; MESO, mesothelioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PAAD, pancreatic adenocarcinoma; PRAD, prostate adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; READ, rectal adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; LAML, acyte myeloid leukemia; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; STAD, stomach adenocarcinoma; UCEC, uterine corpus endometrial carcinoma; UCS, tterine carcinosarcoma; UVM, uveal melanoma. Image downloaded from http://ualcan.path.uab.edu/cgi-bin/Pan-cancer.pl?genenam=FGF18 on February 02, 2023.Figure S2. FGF18 expression in cell lines from different PM subtypes. FGF18 gene expression levels show no apparent differences between the five cell lines from epithelioid and the three cell lines from biphasic PM (left panel) and the six BAP1+ and the two BAP1− cell lines of the PM cell line panel (right panel).Figure S3. Receiver operating characteristics (ROC) curves for different cohorts. ROC curves showing the sensitivity and specificity of circulating FGF18 to discriminate between healthy individuals and patients with either PM or pleural fibrosis  and between persons with malignant disease (PM) or without malignant disease  .Figure S4. FGF18 levels in the plasma of patients with epithelioid, biphasic, sarcomatoid, and desmoplastic PM. Values are shown as scatter dot plots, medians are shown as horizontal lines. ns, not significant; Mann–Whitney U test. For sarcomatoid and desmoplastic PM, no statistical tests could be performed due to small sample numbers.Click here for additional data file.


Our findings revealed that HCC cell lines had more long-range interactions, both intra- and interchromosomal, compared to human mammary epithelial cells (HMECs). Unexpectedly, HCC cell lines displayed cell line-specific compartmental modifications at the megabase (Mb) scale, which could potentially be leveraged in determining HCC subtypes. At the sub-Mb scale, we observed decreases in intra-TAD  interactions and chromatin loops in HCC cell lines compared to HMECs. Lastly, we discovered a correlation between gene expression and the 3D chromatin architecture of SLC8A1, which encodes a sodium-calcium antiporter whose modulation is known to induce apoptosis by comparison between HCC cell lines and HMECs. Our findings suggest that HCC cell lines have a distinct 3D genome organization that is different from those of normal and other cancer cells based on the analysis of compartments, TADs, and chromatin loops. Overall, we take this as evidence that genome organization plays a crucial role in cancer phenotype determination. Further exploration of epigenetics in HCC will help us to better understand specific gene regulation mechanisms and uncover novel targets for cancer treatment.Liver cancer, particularly hepatocellular carcinoma (HCC), poses a significant global threat to human lives. To advance the development of innovative diagnostic and treatment approaches, it is essential to examine the hidden features of HCC, particularly its 3D genome architecture, which is not well understood. In this study, we investigated the 3D genome organization of four HCC cell lines—Hep3B, Huh1, Huh7, and SNU449—using  In 2022, the American Cancer Society estimated that approximately 800,000 people were diagnosed with liver cancer and 700,000 died from the disease worldwide. Liver cancer is the third leading cause of cancer death, partly reflecting that the lack of accurate liver cancer-specific diagnostic tools has limited early diagnosis ,2. ThereThe most common type of primary liver cancer is hepatocellular carcinoma (HCC), which is a malignant tumor of hepatocytes. The causes of HCC are very diverse; they include chronic infection with hepatitis B virus or hepatitis C virus, alcohol abuse, and metabolic disease . This heThe mammalian genome maintains a highly organized and dynamic 3D form that arises from two kinds of interactions . At the in situ Hi-C and assay for transposase-accessible chromatin sequencing (ATAC-seq) of four HCC cell lines and compared the results to those of a normal epithelial cell line. This comprehensive analysis of the chromatin interactions in HCC extends our knowledge of genome topology and epigenetics in hepatocellular carcinoma.To characterize the 3D chromatin landscape of HCC cell lines, we performed 2.This research used four HCC cell lines: Hep3B, Huh1, Huh7, and SNU449 . The HCCWe harvested cells with 0.05% trypsin-EDTA, resuspended 5 million cells in 5 mL 1× phosphate buffered saline (PBS), added 274 μL of 36.5% formaldehyde , mixed the solution thoroughly by inversion, and incubated the suspended cells at room temperature for 10 min. We added 400 μL of 2.5 M glycine and placed the sample on ice for 15 min. Cells were pelleted by centrifugation at 500 ×g for 5 min, the supernatant was discarded, and the cells were resuspended in 5 mL 1× PBS. The cells were equally distributed to five new tubes (1 million cells/tube) and pelleted by centrifugation, and the supernatant was discarded.in situ Hi-C with 1 million harvested cells. We generated a library using an Arima-HiC kit  and sequenced the library using the 150 bp paired-end method of the Illumina NovaSeq 6000 system .We followed the Arima-HiC protocol  to perform 2, and 10% dimethylformamide) with 2.5 μL Tn5 transposase for 30 min at 37°C. We purified DNA fragments with a QIAquick PCR purification kit  and amplified the library using a KAPA HiFi HotStart ReadyMix  as described in the provided manual, with adjustment of the PCR cycle number. The resulting libraries were purified with a QIAquick PCR purification kit. The purified libraries of HCC cell lines were sequenced via the 150 bp paired-end method of the Illumina NovaSeq 6000 system. The 150 bp paired-end method of the Illumina HiSeq 2500 system was used for HMECs.Harvested nuclei of 50,000 cells were incubated in 25 μL fresh TD buffer  analysis, raw reads were aligned to the human genome (hg19) using STAR (version 2.7.10) with default parameters . CufflinPublicly released total RNA-seq data were downloaded from NCBI Gene Expression Omnibus (GEO) datasets. These files were obtained in fastq format. The total RNA-seq data of Hep3B, Huh1, Huh7, and SNU449 cells  were obtained from GSE97098 . The tothttp://www.ncbi.nnlm.nih.gov/geo/) under accession number GSE226217 (SuperSeries). This SuperSeries (GSE226217) is composed of two SubSeries: GSE226215 (ATAC-seq) and GSE226216 (Hi-C).The Hi-C and ATAC-seq datasets have been deposited in the NCBI GEO; in situ Hi-C in four HCC cell lines and HMECs  were explored. Our results revealed that HCC cell lines had higher observed versus expected (obs/exp) trans-contact ratios  than HMEMarkedly, the contact probabilities of HCC cell lines and HMECs changed as the genomic distance passed 1 Mb  and 1E. From the derivative of the contact probability plot, we could further infer the size of TADs and the linear density of cohesin . The aveHaving observed a discrepancy in genome-wide chromosome contact between HMECs and HCC cell lines, we next defined compartmental domains to explore potential relationships between contact differences and 3D chromatin organization. We also analyzed open chromatin regions via ATAC-seq using two technical replicates.We analyzed compartments through PCA analysis of Hi-C contacts at a 100 kb resolution. Positive PC values were taken as defining A compartments, and negative values were taken as delimiting B compartments . The PC1Regarding compartmentalization changes between HMECs and HCC cell lines, we found that almost 50% of the compartments in HMECs were altered  in HCC cell lines . IntriguNext, we generated heatmaps that sorted bins according to the comparison between HMECs and each HCC cell line . The A-tTo summarize, compartment alterations in HCC cell lines were mostly found in the B compartment of HMECs, which were weakened or changed to A compartment regions in the HCC cell lines. Notably, these B-to-A domain changes were cell line-specific. Our results suggest that compartment analysis could potentially be used to classify the subtype of HCC, which is critical for selecting a cancer treatment strategy.Next, we explored shorter-range neighborhood interactions, namely TADs and chromatin loop interactions, in greater depth. Based on our initial results, we expected smaller TAD sizes and weaker intra-TAD interactions in HCC cell lines compared to HMECs. To analyze this in more detail, we defined TADs by RI analysis using CaTCH . HMECs hAnother critical interaction is the chromatin loop interaction. Chromatin loops are formed by the interaction between the pairs of loci that show significantly higher contact frequencies than their neighbors . We defiTo prevent chromosomal rearrangement from affecting our result, we also analyzed TAD domains and chromatin loops after excluding CNVs from the genome –1F. The We also examined the similarities between chromatin loop interactions. Unexpectedly, only 4.4% of loop interactions were common to all four HCC cell lines and HMECs . Hep3B aFinally, we assessed the relationship between 3D chromatin organization and gene expression. We used the public total RNA-seq results for HMECs, Hep3B, Huh1, Huh7, and SNU449 cells and mapped them to the hg19 genome using STAR . We applSLC8A1  was lower in all tested HCC cell lines compared to HMECs , that expression level reduction and TAD disruption both occurred  compared to HMECs. The HCC cell lines were found to have the following changes relative to HMECs: a higher frequency of long-range (>1 Mb) interactions, such as trans-interactions and intrachromosomal interactions; cell line-specific compartmental changes relative to HMECs; and reductions in the number and strength of TADs and chromatin loops. Using the correlation scores for all 3D chromatin structures, we could divide the four HCC cell lines into two subgroups: one comprising Hep3B and Huh7 cells and one comprising Huh1 and SNU449 cells. This subtyping was supported by the results of our RNA-seq analysis. Finally, we revealed that the gene expression level and 3D chromatin organization are linked in the case of SLC8A1, which encodes a sodium-calcium antiporter that plays a crucial role in inducing apoptosis by increasing the influx of calcium ions [ium ions -48.We previously studied 3D chromatin organization in different breast cancer cells and tissues . Both brAlthough we found some apparent cell line-specific 3D chromatin organizations in HCC, our study has limitations. We used HMECs as a control; as these cells originated from mammary tissue, our findings may not be cancer-specific features but rather liver-specific. Furthermore, we do not report on other epigenetic data, such as histone ChIP-seq results; thus, the results of our Hi-C analysis must be confirmed by additional experiments. Nevertheless, we herein reported distinguishing features of chromatin organization between HCC cell lines and showed that their contact characteristics coincided with those of the previously studied breast cancer cell lines. Finally, the utilized Hi-C analysis pipeline was one that had been previously verified by many studies; thus, our findings should be meaningful in a general sense.in vitro microenvironments for cancer cells, such as with 3D culture techniques [Our results provide new epigenetic perspectives into HCC pathology and cancer biology that can be further explored through future research. There is a long-standing debate over whether cancer cell lines demonstrate phenotypes identical to cancer cells obtained from tissues , and stuchniques . As an e


TNFRSF1A gene was increased in both cell lines; (2) the cell line with increased proliferation, K562, had decreased expression of isoforms 1 and 4 of the TNFRSF1A gene and expression of isoform 2 of TNFRSF1B gene was absent at all; (3) the cell line with decreased proliferation—MCF-7 had significantly increased expression of isoform 2 of TNFRSF1B gene. Thus, we can conclude that TNFα exposure to the K562 and MCF-7 cell lines leads to changes in the expression of TNFα receptor isoforms, which, in turn, can appear via diverse proliferative effects.Alternative splicing is a part of mRNA processing that expands the diversity of proteins encoded by a single gene. Studying the full range of proteins–products of translation of alternatively spliced mRNA is extremely important for understanding the interactions between receptor proteins and ligands since different receptor protein isoforms can provide variation in the activation of signaling pathways. In this study, we investigated the expression of isoforms of TNFR1 and TNFR2 receptors before and after exposure to TNFα in two cell lines that had previously demonstrated diverse effects on cell proliferation under TNFα incubation using RT-qPCR. We found that after incubation with TNFα: (1) expression of isoform 3 of the  It is difficult to overestimate the importance of alternative splicing in protein studies. An overwhelming number of multiexon transcripts undergo alternative splicing to provide protein variety. As a result, several protein variations with different characteristics and functions can be produced from a single gene transcript, increasing the complexity of gene expression regulation ,2. It waBy interacting with its receptors TNFR1 and TNFR2, the tumor necrosis factor α (TNFα) ligand is involved in the inflammatory response activation, cell proliferation, and differentiation . TNF-α rDifferent studies demonstrated that TNFR1 and TNFR2 receptor isoforms have variations in response to TNF-α stimulation and anti-TNF-α therapy ,8,9. HowTNFRSF1A and TNFRSF1B). Only proteins with a signal peptide in their amino acid sequence can become receptor proteins [Previous studies of TNFα-stimulated cell lines performed in our laboratory demonstrated a polar dose-dependent change in the proliferative potential of MCF-7 and K-562 cell lines both towards its increase (MCF-7 line) and towards its decrease (K-562 line) . We suggproteins . Thus, tTNFRSF1A and TNFRSF1B genes, the sequences of which we took from the NCBI database: RefSeq RNA TNFRSF1A—https://www.ncbi.nlm.nih.gov/nuccore?LinkName=gene_nuccore_refseqrna&from_uid=7132 (accessed on 7 March 2023) and RefSeq RNA TNFRSF1B—https://www.ncbi.nlm.nih.gov/nuccore?LinkName=gene_nuccore_refseqrna&from_uid=7133 (accessed on 7 March 2023). We found open reading frames (ORF) in the nucleotide sequences of isoforms in Unipro UGENE and analyzed the ORF data using the InterPro neural network for the presence of the signal peptide using the InterPro web server https://www.ebi.ac.uk/interpro/search/sequence/ (accessed on 7 March 2023). Only isoforms with the signal peptide were selected for further analysis.In this study, we examined mRNA isoforms of TNFRSF1A and TNFRSF1B genes using MUltiple Sequence Comparison by Log-Expectation (MUSCLE) in UniPro UGENE. As TNFRSF1A and TNFRSF1B genes are not well annotated, we selected primers unique to the isoforms (and not to the specific intron or exon) and validated them using the web servers Oligocalculator—http://www.bio.bsu.by/molbiol/oligocalc.html (accessed on 7 March 2023)—and Multiple Primer Analyzer—https://www.thermofisher.com/ru/ru/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html (accessed on 7 March 2023). TNFRSF1A isoform 4 is labeled as a nonsense-mediated decay transcript at NCBI; however, it contains a weak Kozak sequence (TCTCT), an open reading frame, and a signal peptide sequence, so there is a possibility for it to be a regular protein-receptor-coding transcript as well. It was not possible to design primers for TNFRSF1B isoform 1; therefore, we selected primers for the constant region of TNFRSF1B to indirectly assess the effect of other isoforms . The designed primers are presented in We performed multiple sequence alignment of all isoforms’ nucleotide sequences for both We used MCF-7, an epithelial-like cell line derived from invasive adenocarcinoma of human mammary glands, and K-562, an erythromyeloblastoid cell line derived from human myeloid leukemia cells. Previous studies have demonstrated that for the MCF-7 cell line, TNFα stimulation at 5 ng/mL, and for the K562 cell line, TNFα stimulation at 25 ng/mL, resulted in differently directed changes in the proliferation of these cell lines ; so, these are the concentrations used in this present study . The incWe isolated total RNA from cells using the Total RNA Purification Plus Kit ; we also isolated the measured concentration of the RNA on the NanoDrop 2000c. We froze the total RNA at −80 °C until the reverse transcription of the RNA was achieved.n = 4) using RNAscribe RT and oligo-dT primers . We used an input of 100 ng of the RNA. Reverse transcription was conducted as follows: 55 °C for 50 min and 85 °C for 10 min.We performed reverse transcription of the total RNA samples  Mix , 1 μL out of the 20 μL of RT product, Melt curves’ temperatures matched the predicted amplicon melting temperatures, thus ensuring amplicon specificity .TNFRSF1A and TNFRSF1B gene isoforms was calculated using the method of Taylor et al. [The relative expression level of r et al. . In brieT-tests with FDR correction for differential isoform expression testing in GraphPad Prism 9.4 for macOS. We considered q-values < 0.05 significant.We used multiple TNFRSF1A and TNFRSF1B isoforms and found a signal peptide in isoforms 1, 3, and 4 of the TNFRSF1A gene and in isoforms 1, 2, and 3 of the TNFRSF1B gene. We chose all of the above-mentioned isoforms for further experiments as all of them contained an open reading frame and a signal peptide, except isoform 1 of the TNFRSF1B gene . Regarding exon/intron composition, we noted the following: TNFRSF1A isoform 1 contains exons 1–5, exons 7–10, intron 9, and exon 11; TNFRSF1A isoform 3 contains exons 1–10, intron 9, and exon 11; TNFRSF1A isoform 4 contains exon 1, exons 3–5, exons 7–10, intron 9, and exon 11; TNFRSF1B isoform 2 contains exon 1 and exons 4–11; TNFRSF1B isoform 3 contains exons 3–11.We used the InterPro neural network on all TNFRSF1A gene and an increase in the expression of isoform 3 in response to the stimulation  and myeloblast-like (MCF-7). The difference between the cell lines comes from the decreased expression of isoforms 1 and 4 of the TNFRSF1A gene in K562, while no changes were found in MCF-7.An increased expression of isoform 3 of the TNFRSF1B gene, we found significantly increased expression of isoform 2 for the MCF-7 cell line, whereas no expression of this isoform was found at all in the K562 cell line.For the We believe that these differences in the isoforms’ expression profiles of K562 and MCF-7 cell lines may be responsible for the differentially directed functional responses described previously , which h


Juniperus procera (J. procera) leaves. Here, we demonstrate that J. procera leaves’ methanolic extract suppresses cancer cells in colon (HCT116), liver (HepG2), breast (MCF-7), and erythroid (JK-1) cell lines. By applying GC/MS, we were able to determine the components of the J. procera extract that might contribute to cytotoxicity. Molecular docking modules were created that used active components against cyclin-dependent kinase 5 (Cdk5) in colon cancer, aromatase cytochrome P450 in the breast cancer receptor protein, the -N terminal domain in the erythroid cancer receptor of the erythroid spectrin, and topoisomerase in liver cancer. The results demonstrate that, out of the 12 bioactive compounds generated by GC/MS analysis, the active ingredient 2-imino-6-nitro-2H-1-benzopyran-3-carbothiamide proved to be the best-docked chemical with the chosen proteins impacted by DNA conformational changes, cell membrane integrity, and proliferation in molecular docking studies. Notably, we uncovered the capacity of J. procera to induce apoptosis and inhibit cell growth in the HCT116 cell line. Collectively, our data propose that J. procera leaves’ methanolic extract has an anticancer role with the potential to guide future mechanistic studies.Medicinal plants provide a wide range of active compounds that can be exploited to create novel medicines with minimal side effects. The current study aimed to identify the anticancer properties of  Cancer is a condition that can start in any organ or tissue of the body and then spreads to other organs due to the uncontrolled growth of cells. It is the second-most common cause of death, just after heart disease. By 2030, it is anticipated that there will be 26 million cancer patients . MalignaJuniperus procera Hochst. ex Endl. (Cupressaceae), also known as Arar in Arabic and as African pencil cedar in English. J. procera has a pleasant scent since it contains a significant amount of volatile oils -1H-imidazol-4-yl-2-(4-methoxyphenyl)acetamide was obtained from the database and used for the validation of the docking methods. The docked structure had a −7.86 kcal/mol docking score and an RMSD of 1.198.A0. The machine used for this investigation was configured with Windows 10 and an Intel (R) Core (TM) i7-8550U CPU running at 1.80 GHz and 1.99 GHz.The cocrystalline molecule N-1-[50. The flow-cytometry software from Applied Biosystems determined the percentage of cells in each phase, as well as the quantity of necrotic and apoptotic cells, automatically.The results are presented as the mean SDs of the viability of treated cells. The absorbance of treated cells * 100 divided by the absorbance of untreated cells was used to compute the percentage of viability. GraphPad Prism Software  was used to determine the drug ICJ. procera leaf extract is cytotoxic to the cancer cell lines HepG2, MCF-7, HCT116, and JK. Additionally, J. procera leaf extracts cause the death of HCT116 cells by arresting the cell cycle, which is caused by DNA damage and results in cell-stalling at the G1 or G2/M phase. A GC/MS analysis of J. procera leaf extracts showed 12 unique bioactive components. These findings imply that the extract of J. procera leaves contains bioactive substances that may be used as anticancer medications. A decrease in breast cancer cell-proliferation may also result from Hit 1 inhibition of aromatase, a cytochrome P450 enzyme that catalyzes estrogen generation in breast cancer patients with positive estrogenic receptors (MCF-7). Additionally, the function of erythroid spectrin in maintaining the integrity and adherence of cell membranes may be impacted by Hit 1’s reduction in production on the JK cell line. The results of molecular docking suggest that Hit 1’s reduction in proliferation and cell-cycle arrest in liver cancer cells may be caused by topoisomerase inactivation. Purification of the bioactive compound 2-Imino-6-nitro-2H-1-benzopyran-3-carbothiamide from J. procera is required to investigate its cytotoxicity against various cancer cell lines in vitro and in vivo.This study demonstrates that


A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as “possibly carcinogenic”; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied. In 2011, radiofrequency electromagnetic fields (RF-EMFs), ranging from 30 kHz to 300 GHz of the electromagnetic spectrum, were classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans (risk group 2B) [Genotoxic events are considered to be an initial step in carcinogenesis, so during the last decade, many investigations have been carried out on the possible genotoxic effects of RF-EMFs, as summarised by several reviews ,3,4,5. TRecent evidence indicates that carcinogenesis can also be induced by non-genotoxic agents through epigenetic alterations , which cIt was reported that the Earth’s magnetic field is involved in DNA methylation (DNAm) regulation ,9; thereIn the present study, we focused on DNAm, since it is the epigenetic mechanism that has been more extensively studied in relation to environmental exposures . In partWe focused on three types of RE-DNA: (1) long interspersed nuclear elements-1 (LINE-1), (2) Alu short interspersed nuclear elements (SINEs) and (3) the DNA sequences encoding RNA ribosomal (rDNA).LINE-1s are the only active retroelements and constitute approximately 17% of the human genome . A full-Alu elements are primate-specific SINE, which comprise about 11% of the genome. They are 300 base pair sequences and are subdivided into three families  due to different consensus sequences and different times of appearance in the human lineage. Alu elements are nonautonomous retrotransposons; however, they can copy and paste themselves through the enzymatic activity of LINE-1-ORF2 . MethylarDNAs are the most abundantly expressed genes in the eukaryotic genome. In the human genome, rDNAs  are tandemly arrayed in a head-to-tail fashion in nucleolar organiser regions (NORs) on the short arms of five pairs of acrocentric autosomal chromosomes  . Each arSome studies have been conducted to ascertain whether the exposure to extremely low-frequency magnetic fields (ELF-EMF) could affect the DNAm of RE-DNA ,39,40,41One limitation of existing studies exploring the impact of the magnetic field and radiofrequency radiation exposure on epigenetic regulation consists in the large heterogeneity in terms of study models and experimental design adopted .The aim of the present study is to assess this endpoint in human cells exposed to 900 MHz GSM-modulated RF-EMF, which is an important frequency for mobile communication electromagnetic radiation emitted from GSM mobile phones and from their base station antennas. To this purpose, DNA methylation profiles of the LINE-1 5′-UTR promoter, Alu sequences and three regions of rDNA  were investigated in an epithelial human cell line (HeLa) and in two neuroblastoma human cell lines (Be(2)C and SH-SY5Y) exposed to RF-EMF or to sham (control).All three cell lines employed have been used in several studies as models to assess the biological effects of EMF exposure. and its impact on epigenetic regulation and cellular homeostasis ,45,46,47p-value < 0.01)   were considered not reliable and were excluded from the analyses.To measure the DNAm of RE-DNA, we used a bisulfite-targeted sequencing assay as described in Materials and Methods. We checked the assay for bisulfite-PCR bias, a phenomenon associated with the preferential amplification of bisulfite-converted DNA according to its original DNAm status. To this aim, we analysed samples at known DNAm percentages  and then assessed the correlation between observed and expected DNAm values for all CpGs of each RE target . A high  < 0.01) . Similar < 0.01) . The cor < 0.01) . CpGs haTo study the effects of radiofrequency exposure on epigenetic regulation of repetitive elements, HeLa, BE(2)C and SH-SY5Y cell lines were exposed to 900 MHz GSM-modulated RF-EMF at a SAR of 1 W/kg. DNAm of LINE-1, Alu and rDNA elements was evaluated.p-value: 0.033), X183 , X190  and X217  compared to sham-exposed samples. In SH-SY5Y and BE2C cell lines, we did not find significant changes in any of the CpGs assayed in the LINE-1 target, although a trend towards hypermethylation was observed in the SH-SY5Y cell line  and of the body (18S1 and 28S) regions of rDNA was affected by radiofrequency exposure in different ways in the three cell lines examined .p-values ranging from 0.012 to 0.044) , X179  and X190  reaching statistical significance  . A largeificance . On the ificance  and 28S ificance .p-values ranging from 0.004 to 0.045), whereas for RiboProm2, only CpG X159  reached statistical significance  and X205  reaching statistical significance (In the BE(2)C neuroblastoma cell line, we observed hypomethylation on both amplicons targeting the rDNA promoter following RF-EMF exposure. DNAm changes in RiboProm1 amplicon were small but significant for the majority of the target’s CpGs , ×72 , ×83  and ×99  reaching statistical significance  in HeLa cell lines  increase in epihaplotype diversity for the LINE-1-targeting amplicon upon exposure to radiofrequency radiation  increase in epihaplotype diversity for LINE-1 was observed in radiofrequency-exposed SH-SY5Y neuroblastoma cells     and 28S : 0.025)  upon radFinally, for Alu-targeting amplicons, no significant differences in epihaplotype diversity indexes were found in any of the cell lines assayed .In this work, we evaluated the impact of RF-EMF exposure on the DNAm of LINE-1, Alu and rDNA repetitive elements in human neuroblastoma and cervical cancer cell lines. The effects on DNAm were evaluated in terms of both alterations in the average DNAm level and alterations of DNAm profile (epihaplotype) richness and distribution.Our data indicate that RF-EMF exposure can induce alterations in the average DNAm level of LINE-1 CpGs (limited to the HeLa cell line) and of rDNA . No effect was instead found for the methylation of CpGs of the Alu repeat in any of the cell lines. Previously, Alu methylation was evaluated by Benassi et al.  in SH-SYWith regard to LINE-1, we observed an increase in the DNAm of some CpG in HeLa cells; this finding could be consistent with the observation, reported in a previous paper , of a siDifferently, no effect was found on LINE-1 methylation in neuroblastoma cells (SH-SY5Y and BE(2)C cells). Similar investigations were previously carried out in SH-SY5Y and BE(2)C cells exposed to ELF-EMF  in two different studies. In agreement with our results, although concerning a different type of electromagnetic field, no significant DNAm changes were found either in exposed BE(2)C  or in exWith regard to rDNA methylation, alterations were observed in all three cell lines: exposed HeLa cells showed hypomethylation of some CpG on the 3′ end of the 28S amplicon target and exposed SH-SY5Y cells showed hypermethylation of the 5′ of RiboProm2 and 18S targets, while exposed BE(2)C neuroblastoma cells showed hypomethylation of the 5′-end of RiboProm1, the 5′-end of RiboProm2 and the 3′-end of 28S amplicon targets. It is well known that rDNA methylation patterns contribute to the regulation of the expression of ribosomal RNA (rRNA) related to the translational rate, which depends on cell-specific homeostasis requirements . TherefoTo date, DNAm of rDNA has never been analysed in exposed cells to ELF-EMF or to RF-EMF, but it has been investigated in both physiological and pathological conditions including studies focused on cancer ,37, neurIn addition to assessing the impact of radiofrequencies on the average DNAm level, we evaluated their possible effects on the organisation of methylated and unmethylted CpG sites through the analysis of epihaplotype richness and distribution.As far as we know, the analysis of DNAm epihaplotypes, by calculating the Shannon diversity index, has never been performed in cells that are EMF-exposed. Our data indicate that RF-EMF exposure can induce changes in the DNAm epihaplotype diversity. In some cases, such as for LINE-1 DNAm in HeLa and SH-SY5Y cells, epihaplotype diversity increased in exposed samples, whereas in other cases, such as rDNA DNAm in BE(2)C cells, it decreased. Changes in the distribution and richness of epihaplotypes may indicate a dysfunction in the maintenance of epigenetic patterns, which can contribute to epigenetic instability. Changes in the Shannon diversity index have been found in ageing ,57 and iAs a whole, our data indicate that RF-EMF exposure affects the DNAm of tested RE and might induce epigenetic instability. These data add further evidence to our knowledge of the effects of RF-EMF on DNAm ,11. Our A weakness of the present study is the limited number of analysed samples and that the use of cell lines may not exactly reflect what happens in vivo. Analysis of primary tissues and stem cells would provide further knowledge.The issue of what molecular mechanisms underlie the biological effects of EMFs on DNAm regulation is still an open question ,59,60. OMoreover, our results indicate that the effect on DNAm of LINE-1 and rDNA strongly depends on the cellular system considered and on the genetic background, in agreement with the fact that epigenetic modifications are tissue-specific and show inter-individual variability.Previous studies showed that the biological model is relevant in EMF exposure studies. For example, Martin et al.  studied Collectively, these data indicate that many different models should be studied to understand the epigenetic effects of EMF exposure on biological samples.In conclusion, our results suggest that RF-EMF exposure can affect the DNAm of LINE-1 and rDNA sequences, which are highly represented in the human genome, and that the effects depend on cell context (cell genome and cell type). Further investigations on the epigenetic effects of RF-EMF would be useful both for a better understanding of RF-EMF risks and safety assessment.2). Each TEM cell has a squared cross-section (14 cm wide) and can contain 6 4-multiwell plates . The RF-EMF  is uniformly absorbed in the two wells of the two symmetric plates closer to the inner copper septum of the TEM cell, resulting in a SAR of 1 W/Kg. Therefore, only the septum-adjacent wells were used in the experiments in accordance with Del Vecchio et al. [The exposure system was kindly loaned to us by Prof. Laura Calzà (University of Bologna). The exposure system and the dosimetry were previously described in great detail ,76. Brieo et al. ,76. The 2 humified atmosphere at 37 °C. In each assay, cells were plated in 4-well plates   at a density of 80,000 cells/well and allowed to adhere overnight, and then the plates were randomly assigned to each transverse electromagnetic (TEM) chamber and exposed to 900 MHz GSM-modulated RF-EMF at SAR of 1 W/kg or to sham.HeLa cells, neuroblastoma BE(2)C cells and neuroblastoma SH-SY5Y cells were kindly provided by Prof. Perini . All cell lines were grown in DMEM medium , containing 10% heat-inactivated fetal bovine serum , 100 UI/mL penicillin  and 100 μg/mL streptomycin (Sigma), under a 5% COGenomic DNA was extracted by using the QIAmp DNA Mini Kit  according to the manufacturer’s instructions and quantified using the Qubit dsDNA BR Assay Kit . Then, 500 ng was treated with sodium bisulfite using the EZ DNA Methylation Direct Kit  as indicated by the manufacturer. Each sample was sequenced in triplicate. DNA methylation calibration curves were prepared by combining Universal Methylated and Universal Unmethylated DNA samples  to generate standards at 0, 25, 50, 75 and 100% DNAm levels. Each point of the curve was sequenced in triplicateTM adapter sequences, respectively, TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG and GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG. In addition, a random nucleotide spacer (N) was included between Illumina adapters and primers to increase sequencing variability.DNAm of RE-DNA was analysed using a targeted bisulfite sequencing approach. Primers for rDNA distal (RiboProm1) and proximal (RiboProm2) promoters as well as for 5′UTR of LINE-1 were previously published , whereas2  and 200 μM dNTP . Thermal cycler conditions were set as follows: 1× cycle at 95 °C for 1′ 40 ″; 1× cycle at 98 °C for 1′; 1× cycle at 58 °C for 2′; 1× cycle at 72 °C for 1′; 36 cycles at 98 °C for 10″, 58 °C for 40″ and 72 °C for 20″; 1× cycle at 72 °C for 5′; and hold at 4 °C. Amplification products were pooled sample-wise and purified using MagSi-NGS plus beads . Afterward, 10 uL of pooled and purified samples were indexed using Illumina Nextera XT Index Set A  as indicated in the Nextera Library Prep Guide. Finally, indexed libraries were purified yet again and normalised to 4 nM. Sequencing was performed on an Illumina Miseq Platform with Micro v2 300PE chemistry .Sequencing libraries were generated using a two-step PCR approach. Briefly, 5 ng of bisulfite-converted DNA was amplified using Phusion U  added with 1M Betaine , 150 nM forward and reverse primers, 1.75 mM MgClhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 25 May 2023)). Adapter sequences were trimmed off using cutadapt [PEAR [seqtk. All processing tools were compiled in anaconda2 environments.Pair-end reads were quality-checked using FastQC . Subsequently, it recovers all reads with at least 80% alignment identity with bisulfite-specific primers provided (--primTresh 0.8) and retains all reads whose length differs less than 20% from that of a reference sequence provided (--threshLen 0.2). Afterwards, each read is aligned to a reference sequence provided using blastn. Alignment parameters were modified from default in order to switch off automatic low-complexity, repetitive sequence masking (--dust no). This allows us to limit read loss, as bisulfite-conversion and subsequent amplification reduce sequence complexity by converting unmethylated Cytosines (C) into Thymines (T). For each aligned read, AmpliMethProfiler generates a DNAm profile by assigning binary values to CpG positions that were found methylated (1) or unmethylated (0). If a CpG position contains a nucleotide other than T or C, AmpliMethProfiler assigns an unknown (2) value. Samples with coverage lower than 400 reads were excluded from the analysis. After filtering, sequencing coverage varied between 1252 and 9432 (3858 ± 2140) for 18S, 1316 and 11,481 (5147 ± 2462) for 28S, 809 and 7512 (3027 ± 1240) for Alu, 822 and 7838 (3454 ± 1477) for LINE-1, 973 and 7580 (3995 ± 1539) for RiboProm1 and 864 and 8366 (3918 ± 1736) for RiboProm2.To extract CpG DNAm profiles for each target sequence, the  applied . This piFinally, average DNAm levels of each CpG unit are calculated as a percentage of methylated Cs on the total number of methylated and unmethylated Cs. For each cell line, average DNAm was compared between the treated and untreated groups by Student t-test, using the ANOVA function in R (v3.6).filter_otus_from_otu_table.py –n 2). Epihaplotype composition and distribution within each sample were then evaluated using a qiime-based pipeline implemented within the AmpliMethProfiler environment . Epihaplotype diversity was calculated through the Shannon alpha-diversity index. For each cell line, samples were grouped as either treated or control, and then observations were rarefied by randomly resampling the pool of epihaplotypes at increasing depth . Shannon indexes were calculated at a depth equal to the number of reads of the sample with the lowest coverage. However, it emerged that none of the Shannon index rarefaction curves generated was able to reach plateau at the selected depth. Therefore, in order to increase the number of reads on which observations were rarefied, we included an additional filter and removed all samples with a maximum coverage lower than 60% of the median coverage for each amplicon. Once the dataset was filtered, for every target amplicon, Shannon indexes were compared between samples exposed to radiofrequency radiation and to sham using Student’s t-test.AmpliMethProfiler provides information on the DNAm status of each CpG site at the single DNA molecule level. The specific combination of methylated and unmethylated CpGs on a single strand of DNA defines an epihaplotype. To study epihaplotype organisation, a pipeline of analysis was developed using AmpliMethProfiler built-in functions. As the number of possible methylated and unmethylated CpG combinations largely exceeds sequencing depth, singletons, namely epihaplotypes occurring only once in the entire sample, were removed from epihaplotype count tables (


Capsicum annuum L.) fruits from diverse varieties with different capsaicin contents  against several tumor cell lines  has been investigated. The results showed that the capsaicin content in pepper fruits did not correspond with their anti-proliferative activity against tumor cell lines. By contrast, the greatest activity was promoted by the pepper tissues which contained the lowest capsaicin amount. This indicates that other compounds different from capsaicin have this anti-tumor potentiality in pepper fruits. Based on this, green fruits from the Alegría riojana variety, which has negligible capsaicin levels, was used to study the effect on the oxidative and redox metabolism of tumor cell lines from liver (Hep-G2) and pancreas (MIA PaCa-2). Different parameters from both lines treated with crude pepper fruit extracts were determined including protein nitration and protein S-nitrosation  promoted by nitric oxide), the antioxidant capacity, as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), among others. In addition, the activity of the NADPH-generating enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and NADP-isocitrate dehydrogenase (NADP-ICDH) was followed. Our data revealed that the treatment of both cell lines with pepper fruit extracts altered their antioxidant capacity, enhanced their catalase activity, and considerably reduced the activity of the NADPH-generating enzymes. As a consequence, less H2O2 and NADPH seem to be available to cells, thus avoiding cell proliferation and possibly triggering cell death in both cell lines.Cancer is considered one of the main causes of human death worldwide, being characterized by an alteration of the oxidative metabolism. Many natural compounds from plant origin with anti-tumor attributes have been described. Among them, capsaicin, which is the molecule responsible for the pungency in hot pepper fruits, has been reported to show antioxidant, anti-inflammatory, and analgesic activities, as well as anti-proliferative properties against cancer. Thus, in this work, the potential anti-proliferative activity of pepper ( Nowadays, cancer is considered one of the main causes of human death worldwide, as a consequence of the interaction of both environmental and genetic factors. Globally, cancer is a polyhedral pathology that implies both genetic alteration through mutations and uncontrolled cell proliferation . It has 2O2. Thus, Saha and colleagues -4-methyl-octanamide (DMBMO), as an internal standard. In our experimental conditions, an XBridge 2.1 × 10 mm pre-column and an XBridge 2.1 × 100 mm C18 3.5 µm column  were connected to an Alliance 2695 HPLC system coupled to a Micromass Quattro micro API triple quadrupole mass spectrometer (Waters). The retention time for capsaicin in our experimental conditions was 1.88 min, and the concentration of capsaicin was expressed as μg g−1 of fresh weight . I. I51]. IIn order to seek the specific mechanisms where this anti-proliferative activity resides, green pericarps from the cultivar Alegría riojana  were set, and the IC50 index for Hep-G2 and MIA PaCa-2 tumor cells was determined. Thus, for Hep-G2, it was found that 15.48 mg of pepper fruit fresh weight provoked 50% lethality in 10,000 cells A, whereaThen, the potential effect of crude pepper extracts on the tumor stage of cells was assayed by using an antibody against ERK1/2, two related protein-serine/threonine kinases which are used as markers of tumor events through their participation in the Ras-Raf-MEK-ERK signal transduction cascade . Thus, a2-Tyr), nitro-tryptophan (NO2-Trp), nitroso-cysteine (CysNO), and glutathione. As observed, several proteins seem to be post-translationally modified by the treatment of cells with pepper fruit extracts. Thus, the expression of Tyr-nitrated proteins studied in Hep-G2 and MIA PaCa-2 tumor cell lines showed few differences in both cell lines after incubation with crude extracts from pepper fruits  such as tyrosine (Tyr) and tryptophan (Trp) nitration, as well as oxidative PTMs (oxiPTMs), including S-nitrosation and S-glutathionylation, were analyzed to follow the nitro-oxidative status in tumor cell lines incubated with crude extracts from the Alegría riojana cultivar. r fruits . As depir fruits  were detr fruits  were obs2-Trp, with no intensity changes after the incubation with pepper fruit extracts. Likewise, nine cross-reacting bands , superoxide dismutase (SOD), and glutathione peroxidase (GPX). Additionally, the total antioxidant capacity (TAC) was evaluated in both lines. Thus, as shown in 2O2 that is converted into H2O and O2, was increased after the treatment with pepper fruits . Catalase activity, a peroxisomal enzyme associated with the scavenging of Hr fruits B. Thus, −, whereas CuZnSODs are sensitive. In Hep-G2, MnSOD II and CuZnSOD II were only visible, whilst in MIA PaCa-2 cells, the four isozymes could be detected, with CuZnSOD I being the most abundant. None of these SOD isozymes were influenced by the treatment of cells with crude extracts from pepper fruits.The SOD isoenzymatic pattern was also investigated as a consequence of the incubation of both cell lines with pepper fruit extracts. Overall, four SOD isozymes were identified: two manganese-containing SODs (MnSOD I and MnSOD II) and two copper zinc-containing SODs (CuZnSOD I and CuZnSOD II), according to their sensitivity to cyanide . MnSODs The metabolism of NADPH has been reported to be of relevance in tumor cell lines. Thus, the behavior of the three main NADPH enzymatic sources was studied in this work. The activity of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) were significantly reduced after the incubation of both Hep-G2 and MIA PaCa-2 cell lines with crude extracts from Alegría riojana pepper fruits. The incubation with pepper fruit extract inhibited G6PDH activity up to 63% in Hep-G2 cell lines, and 40% in MIA PaCa-2 cell lines A. A simiCapsicum (pepper) species, and where the pungency trait from those products resides [Plants have always been considered as health sources for humans, due to their great amount of exclusive secondary metabolites, which positively interact with our physiological functions ,60,61,62 resides ,63. It h resides ,44,64,65 resides ,45,46,47 resides ,48. AccoAs a first unexpected discovery, it was found that the pepper fruits/tissues which contain the highest capsaicin levels  did not Once preliminary and discriminating assays were performed, green fruits from the Alegría riojana variety were set to investigate their effect on the ROS and NADPH metabolism of tumor cells from the Hep-G2 and MIA PaCa-2 lines. Apparently, the progression of cancer in Hep-G2 cells does not appear to be affected by the treatment with the crude pepper extract, since no changes were observed in the ERK1/ERK2 levels. ERK1 and ERK2 are related protein-serine/threonine kinases that take part in the signal transduction cascade associated with a great number of processes, among which cell adhesion, cell cycle progression, cell migration, cell survival, differentiation, metabolism, proliferation, and transcription are the best known . Further2- group to either tyrosines (NO2-Tyr) or tryptophan (NO2-Trp) protein residues, whereas S-nitrosation occurs by the direct addition of NO to some cysteine residues in a reversible way [Cancer processes are commonly linked to altered oxidative metabolism in the cells, with oxidative damages occurring through the initiation, development, metastasis, and progression of the pathology ,4,5,6,7.ible way . The resible way ,71.2•−) and converts them into H2O2 plus O2; MnSOD II, and CuZnSOD II were shared by both organs, although the activity in MIA PaCa-2 was almost negligible. On the contrary, the most prominent SOD isozymes present in the pancreas cells were undetected in hepatoma cells. This is an interesting issue that deserves attention, since this may indicate different regulation at the organ level, and perhaps developmental level, and this could be essential for the progress of a localized cancer to metastasis. Thus, it has been reported that in colorectal adenocarcinoma and its liver metastases, the differences in mRNA and protein levels of SOD isoenzymes indicate that SOD takes part in the adaptation of tumor cells to oxidative stress. This situation helps keep a certain level of ROS, which is necessary for appropriate cell proliferation. Furthermore, the expression of SOD isoenzymes seemed to be regulated both at transcriptional and post-transcriptional levels [Since cancer causes alteration in the oxidative metabolism, a broader analysis of how the treatment with pepper fruit extracts influenced the oxidative and redox metabolism of the two cell lines was performed in this work. Thus, it was observed that the treatment provoked significant changes in the total antioxidant capacity of both cell lines, although with an opposite trend. We further studied the isozyme profiles of the superoxide dismutase and the glutathione peroxidase systems. In both types of cells, a different isoenzyme pattern was observed for SOD depending on the organ, either the liver or pancreas. SOD scavenges superoxide radicals targeted metabolomics and biological assays can contribute to identifying those molecules from pepper fruits with anti-proliferative activity. Those compounds seem to be responsible for the increased catalase activity and lowered NADPH-generating enzyme activities, which seems to lead to lower hydrogen peroxide and NADPH levels, thus avoiding cell proliferation and triggering cell death in both cell lines. However, more investigation is necessary to decipher the intimate mechanism exerted by crude pepper fruit extracts. Additionally, the use of purified compounds, once they have been identified from pepper fruits, will allow us to envisage new cancer therapies, thus providing a panel of nutraceutical plant products.


Standard platinum-based therapy for ovarian cancer is inefficient against ovarian clear cell carcinoma (OCCC). OCCC is a distinct subtype of epithelial ovarian cancer. OCCC constitutes 25% of ovarian cancers in East Asia  and 6–10% in Europe and North America. The cancer is characterized by frequent inactivation of ARID1A and 10% of cases of endometriosis progression to OCCC. The aim of this study was to identify drugs that are either FDA-approved or in clinical trials for the treatment of OCCC.High throughput screening of 166 compounds that are either FDA-approved, in clinical trials or are in pre-clinical studies identified several cytotoxic compounds against OCCC. ARID1A knockdown cells were more sensitive to inhibitors of either mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738) or MDM2 (RG7388) compared to control cells. Also, compounds targeting BH3 domain (AZD4320) and SRC (AZD0530) displayed preferential cytotoxicity against ARID1A mutant cell lines. In addition, WEE1 inhibitor (AZD1775) showed broad cytotoxicity toward OCCC cell lines, irrespective of ARID1A status.In a selection of 166 compounds we showed that inhibitors of ATR and WEE1 were cytotoxic against a panel of OCCC cell lines. These two drugs are already in other clinical trials, making them ideal candidates for treatment of OCCC.The online version contains supplementary material available at 10.1186/s13048-023-01160-y. Ovarian clear cell carcinoma (OCCC) patients show low response rates 10–25%) to standard ovarian cancer therapy (carboplatin and paclitaxel). This therapy is effective (~ 80% response rate) in other subtypes  of ovarian cancer 0–25% to . MoreoveWe established ARID1A isogenic OCCC cell lines and performed high throughput drug screening using 166 compounds . We identified 7 inhibitors of OCCC targeting WEE1 (AZD1775), mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738), MDM2 (RG7388), BH3 domain (AZD4320) and SRC (AZD0530). We found that the combination of WEE1 (AZD1775) and ATR (AZD6738) inhibitors synergistically killed the OCCC cells. These two drugs are in clinical trials for other cancer-types, making them ideal candidates for treatment of OCCC.OVCA429 OCCC cell line , 19–24 wCells were harvested in the presence of lysis buffer  plus protease inhibitor cOmplete cocktail (Sigma). Protein lysates were separated by SDS polyacrylamide gel electrophoresis followed by Western blotting with different antibodies. Expression levels of individual protein were quantified using ImageJ . RepreseRNA extraction from cells were performed by using RNeasy mini kit (Qiagen). After digestion with DNase (ThermoFisher Scientific), cDNA was synthesized using MAXIMA H(-) MASTERMIX (ThermoFisher Scientific). Real-time PCR was performed using POWRUP SYBR MASTER MIX (ThermoFisher Scientific) in CFX96 Real-time system (Bio-Rad).Human OCCC cell lines  were from in-house collection of ovarian cancer cell lines purchased or acquired from various sources described in Supplementary Table Lentiviral vector pLKO.1 was used to select stable silencing (shRNA) of ARID1A in 3 ARID1A wild type cell lines  by puromycin. Two shRNA target sequences for ARID1A were Sh1 (GAAAGCGAGGGCCCCGCCGT) and sh5 (GCTTCGGGCAACCCTACGGC) as well as control scrambled shRNA (shC) (GAACCTATTCCCGCAATCTAA). Isogenic silencing of ARID1A in OVCA429, ES2, JHOC5 was verified by real-time PCR and Western blots  were treated with drugs in 96-well plates for 72 h; and cell viability was measured using MTT -2,5-diphenyltetrazolium bromide). All experiments were performed three times in triplicates. IC50 values were generated using Graphpad Prism software.Data were analyzed by either Student’s t-test or two-way Anova. Generation of hierarchical clustering of heatmap was performed using ClustVis .OCCC  were grown for 14 days in top layer of 0.3% agar with a bottom layer of 0.6% agar in 24-well plates. Drugs were mixed with top layer of soft agar at day 1. Colonies were fixed with formaldehyde and stained with crystal violet. Each assay was performed in triplicates and repeated once. ImageJ2 was used to quantify number of colonies , 31. RepGDSC (The Genomics of Drug Sensitivity of Cancer database) contains drug response data and genomic markers of sensitivity from ~ 1,000 human cancer cell lines screened with 180 to 400 compounds . This daPropidium iodide-stained cells was used to measure DNA contents after drug treatment. Briefly, after drug treatment, cells were fixed with ethanol, treated with RNaseA, and stained with propidium iodide. APC Annexin V Apoptosis Detection Kit (Biolegend) was used to identify apoptotic cells following manufacturer’s instructions. Data were acquired on LSRII flow cytometer and analyzed with FlowJo software. Representative results are shown from 2 independent experiments. Two-tailed t test was performed. *, p < 0.05, compared to control; #, p < 0.05 combination treatment compared to single agent.p < 0.05) different cytotoxicity between KD and WTC  and ARID1A knockdown (KD). Thirty-eight compounds showed significantly   . After single agent treatment of AZD1775, OCCC cells increased in G1 phase (≤ 23%) Fig.  A. CellsProtein expression levels of molecules involved in cell cycle arrest and apoptosis were measured after drug treatment. Inhibitors of either WEE1 (AZD1775) or ATR AZD6738) alone increased levels of cleaved caspase-3 in OCCC cell lines  and their combination further significantly enhanced expression levels of active caspase-3  targeting both BCL2 and BCL-XL and was selectively more active in Mut compared to WT OCCC cell lines. This inhibitor induced tumor regression and remission in venetoclax-resistant acute myeloid leukemia (AML)  and B-ceActivated SRC is associated with an unfavorable survival in endometriosis-associated OCCC . In our Another frequently activated kinase is PIK3CA (activation mutations found in approximately 50% of OCCC). A prior study identified mTORC1/2 inhibition as treatment for OCCC by analysis of kinase mutations (518 kinases) and copy number alterations . We idenMutations or copy number alterations in DNA repair/cell cycle arrest/apoptosis occur in 82% of OCCC samples . DNA damOCCC has low response rates versus standard ovarian cancer therapy. High throughput drug screening of ARID1A isogenic OCCC cell lines with 166 compounds  identified 7 inhibitors targeting BH3 domain (AZD4320), WEE1 (AZD1775), mTOR (PP242), dual mTOR/PI3K (GDC0941), ATR (AZD6738), MDM2 (RG7388), or SRC (AZD0530). We further revealed combination of AZD6738 and AZD1775 synergistically induced growth inhibition of OCCC cells. These two drugs are already in other clinical trials, making them ideal candidates for treatment of OCCC. These results highlighted drug screening will contribute to discovery of novel therapeutic targets and alternative treatments for the improvement of OCCC patient outcomes.Below is the link to the electronic supplementary material.Supplementary Material 1