It is assumed that combining PARP inhibitors with radiotherapy could be beneficial for cancer treatment. In this study, melanoma cells were treated with Niraparib and Talazoparib in combination with ionizing radiation (IR). (2) Methods: The effects of Talazoparib and Niraparib in combination with IR on cell death, clonogenicity and cell cycle arrest were studied in healthy primary fibroblasts and primary melanoma cells. (3) Results: The melanoma cells had a higher PARP1 and PARP2 content than the healthy fibroblasts, and further increased their PARP2 content after the combination therapy. PARP inhibitors both sensitized fibroblasts and melanoma cells to IR. A clear supra-additive effect of KI+IR treatment was detected in two melanoma cell lines analyzing the surviving fraction. The cell death rate increased in the healthy fibroblasts, but to a larger extent in melanoma cells after combined treatment. Finally, a lower percentage of cells in the radiosensitive G2/M phase is present in the healthy fibroblasts compared to the melanoma cells. (4) Conclusions: Both PARP inhibitors sensitize melanoma cells to IR. Healthy tissue seems to be less affected than melanoma cells. However, the great heterogeneity of the results suggests prior testing of the tumor cells in order to personalize the treatment. Niraparib MK-4827) inhibits PARP1 and PARP2 7 inhibit. The FDAPoly (ADP-ribose) polymerases (PARP) play an essential role in different cellular processes, including several pathways of DNA repair ,6,7. PARFurthermore, PARPi treatment could be extended to cancer treatment in combination with DNA-damaging treatments . The pri2 atmosphere.The primary melanoma cell cultures ANST, ARPA, RERO, LIWE, ICNI and HV18MK were used for the experiments. The primary cells were established from human melanomas (primary tumors) in the Department of Dermatology of the University Hospital Erlangen. All tumor samples were obtained after the informed consent of the patients. The ethical approval was obtained from the Ethics Committee of the medical faculty of the Friedrich-Alexander-Universität Erlangen-Nürnberg (204_17 BC). The tissue samples were digested with collagenase and hyaluronidase  as well as with DNAse  resulting in single-cell suspensions . ThereafTalazoparib and Niraparib  were stored at −80°C after producing aliquots of 1000 nM stock solution. For each experiment, a portion was thawed immediately before use. The concentrations of Talazoparib and Niraparib were chosen in accordance with the achievable plasma concentrations in humans. The maximum plasma concentration (cmax) of Talazoparib after repeated daily dosing of 1 mg is 16.4 ng/mL . Due to Cells were irradiated with a dose of 2 Gy ionizing radiation (IR) by an Isovolt TitanX-ray generator . For the combined treatment with an inhibitor plus IR, the irradiation was performed 3 h after the inhibitor treatment .The relative PARP expression in melanoma cells compared to healthy fibroblasts was analyzed using the Western Blot assay. In a second pass, the melanoma cells were treated with a combination of the inhibitor Talazoparib respectively Niraparib and irradiation. The change of the PARP expression was determined 48 h after this treatment.The cytoplasmic and the nuclear proteins of the cells were isolated separately. The nuclear fractions were used for the further procedures. The proteins were denatured at 95 °C and each sample was diluted to the same protein concentration of 1775 µg/mL. Subsequently, gel electrophoresis and Western blots were performed ,25. The Healthy fibroblast and melanoma cells were seeded in a 20% FBS medium to generate approximately a 50% confluence. Afterwards, the medium was replaced by a 2%-FBS medium. This reduced cell culture medium was used to not artificially enhance the effect of the inhibitor. The cells were divided into four groups: the first group stayed untreated, being used as a control group, the second one was treated with IR, the third one with an inhibitor  and the fourth one with IR plus inhibitor . All groups were incubated for 48 h. Subsequently, the adherent cells, as well as the non-adherent cells, were harvested. Each sample was bisected, the first half being used in a DNA cytometry test by Hoechst 33342 staining and the second half in an apoptosis/necrosis assay. The first half was placed in a mixture of cold 70% ethanol with FBS-reduced medium and was incubated at 4 °C for at least 12 h for fixation. Then these cells were stained with Hoechst 33342  . The secCells from healthy fibroblast and melanoma cell cultures were seeded in Petri dishes. After 14 hours of seeding, the cells were treated equivalently to the flow cytometry experiments. After 10 days, the cells were stained with methylene blue  for 30 min at room temperature. Stained colonies of at least 50 cells were visually selected and manually counted. The results of the cell line experiments are depicted in semi-logarithmic plots.For proficiency analysis of homologous recombination, cells were seeded on cover slides to 90% confluence . The celp-value of ≤0.05 was determined to be statistically significant.Flow cytometry experiments and colony formation experiments were repeated at least three times. All results were analyzed by a two-sided Mann–Whitney-U-test using GraphPad Prism version 7.04 . A Standard biosecurity and institutional safety procedures were followed.Ethical approval was obtained in the Department of Dermatology, Universitätsklinikum Erlangen following approval by the institutional review board . The patients provided written informed consent.The baseline PARP1 and PARP2 levels were higher in the four melanoma cell lines ANST, ARPA, RERO and LIWE compared to the two healthy skin fibroblast cell cultures SBLF7 and SBLF9 A. In theTwo characteristic examples of the Kaluza analysis of our apoptosis and necrosis experiments are displayed A,B. Mostp = 0.036 respectively p = 0.048). In the cancer cell lines (p ≤ 0.05) (p ≤ 0.05) (p ≤ 0.05) (p ≤ 0.05). After combined Talazoparib plus IR treatment, however, cell death decreased slightly in the RERO cell culture compared to the untreated control group as well as to the IR mono-treatment (p ≤ 0.05). In addition, the combined treatment of Niraparib plus IR increased cell death compared to the IR mono-treatment, varying between a 2% increase in HV18MK and a 64% increase ICNI (p ≤ 0.05).In the subsequent analysis, apoptosis and necrosis are summarized as cell death. In the healthy fibroblast cell lines SBLF7 respectively SBLF9 C,D, neitll lines E–J, the  ≤ 0.05) F,H,I. Th ≤ 0.05) E,G,J. Ta ≤ 0.05) F,I,J,H.  ≤ 0.05) I,F,J,H. reatment G. In allThe difference between the sum of the particular mono-treatments and the corresponding combined treatment was calculated to obtain PARPi and IR interactions. The underlying values of the logarithmic graphs in Cell cycle arrest in the G2/mitosis (G2/M) phase was studied using Hoechst 33342 DNA staining. The DNA content was graphically visualized A,B. The p ≤ 0.05). Niraparib mono-treatment did increase the G2/M fraction, whereas the combined treatment tended to reduce this effect.In the healthy fibroblasts SBLF7 and SBLF9, the G2/M phase cells accounted for 3% C,D. IR tp ≤ 0.05). Compared to the IR mono-treatment, the combined treatment with one inhibitor plus IR did increase the G2/M arrest values in all melanoma cell cultures, varying between 7% in ANST and 34% in ICNI (p ≤ 0.05). There are large differences in G2/M arrest between the different cell lines. Additionally, we analyzed the subG1 phase for comparison with our cell death FACS analysis . For the fibroblasts and the melanoma cell cultures ANST, RERO and LIWE, there were minor supra-additive effects and in the case of ANST, there were none for Talazoparib.The curves of the combined treated cell lines were shifted parallel to display the interaction between the inhibitors and irradiation. The differences of the parallel displaced combined treatment to IR treatment alone are listed in Additionally, we analyzed the homologous recombination (HR) status of the cell lines . The heaDue to a growing interest in PARPi for cancer therapy, the development of such substances and the further extension of their application have progressed in recent years . Since iA comparison of cell death and clonogenic survival can be made based on the Annexin/7-AAD stain and colony formation assays. Clonogenic survival considers additional types of cell death such as mitosis-coupled cell death and the additional loss of clonogenic survival through senescence. Talazoparib treatment had no effect on cell death of the healthy skin fibroblasts. In contrast, Niraparib treatment, with a markedly higher concentration than Talazoparib, according to the higher concentration in clinical use, had a clear effect: cell death increased compared to the untreated control group and compared to the IR mono-treatment. However, in the clonogenic survival assays, both drugs indicated a very pronounced effect in the healthy fibroblasts, which further increased by additional IR. The cause for the strong effects in clonogenic survival is probably the induction of senescence, which is very common in fibroblasts . SimilarRegarding cell death, two remarkable supra-additive effects were found after the combination of Niraparib and IR. On the contrary, two sub-additive effects were noticeable in the SBLF9 and ANST. Thus, the PARP inhibitor Niraparib in particular seems to interact with IR treatment exceptionally divergent. Since clonogenic survival is the gold standard in radiation biology it is of remarkable interest that 13 out of 14 KI+IR combinations were able to decrease survival in a supra-additive manner. This gives clear hints to the benefit of concomitant radiotherapy and targeted therapy of the DNA repair system. Overall there were clear differences between cell death and loss of clonogenicity. The loss of clonogenicity is probably the more relevant variable to make a prediction for use in radiochemotherapy ,33. It pPrevious studies reported a G2/M cell cycle arrest after PARPi ,35,36. AThe effects of the PARPi Talazoparib onto the cell lines partially differed significantly from the effects of Niraparib onto identical cell lines. The comparison of cell death analysis and the colony formation assay sheds light on the assumption that Talazoparib triggers senescence and clonogenicity loss in cells rather than cell death. On the other hand, the application of Niraparib indicates increased cell death. An additional analysis of the subG1 phase showed corresponding results . The emeAnother possible cause for these differences in the cell lines could be variations in the function of the HR. Therefore, we analyzed the homologous recombination (HR) status of our cell lines to get a better understanding of the inter-cellular differences . An HR pNevertheless, when thinking about future clinical development of such combination therapy, it is of great importance to consider the possibility of side effects. It is known that the ability of kinase inhibitors to radiosensitize cells carries the risk of side effects. In 2012, the first patient suffered from radio-dermatitis after concomitant radiotherapy and treatment with the BRAF inhibitor Vemurafenib. Hecht et al.  describeSeveral ongoing trials of Talazoparib  and Niraparib  in combination with radiation may also yield interesting results regarding potential side effects. The ongoing clinical trials confirm the relevance of the consideration, whether combining Talazoparib respectively Niraparib with radiation could potentially benefit cancer treatment.We found that most of the melanoma cell lines studied were sensitive to the combined treatment with inhibitors and irradiation with regard to cell survival: four out of six cell lines were sensitive to Talazoparib plus IR treatment and six out of six cell lines were sensitive to Niraparib plus IR treatment. These results were even more pronounced when considering clonogenic survival: four out of five cell lines were sensitive to both inhibitors in combination with irradiation treatment. In addition, we found a clear potential for radiosensitization of melanoma cells with both inhibitors in combination with irradiation: in six out of six cell lines, the proportion of cells in the G2/M phase increased. This radiosensitization could increase the efficiency in clinical radiotherapy of melanoma patients. In addition, the less radiation-sensitive healthy tissue would be spared during therapy. However, we could not establish a general benefit of the combined therapy, since individual melanoma cell lines are resistant to the therapy. Therefore, an individualization of the therapy with prior testing of the tumor tissue should be the first step towards efficient combination therapy.


Acute myeloid leukemia (AML) is a hematological malignancy with a dismal prognosis. For over four decades, AML has primarily been treated by cytarabine combined with an anthracycline. Although a significant proportion of patients achieve remission with this regimen, roughly 40% of children and 70% of adults relapse. Over 90% of patients with resistant or relapsed AML die within 3 years. Thus, relapsed and resistant disease following treatment with standard therapy are the most common clinical failures that occur in treating this disease. In this study, we evaluated the relationship between AML cell line global metabolomes and variation in chemosensitivity.We performed global metabolomics on seven AML cell lines with varying chemosensitivity to cytarabine and the anthracycline doxorubicin  using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Univariate and multivariate analyses were performed on the metabolite peak intensity values from UHPLC-MS using MetaboAnalyst to identify cellular metabolites associated with drug chemosensitivity.vs. sensitive cell lines, including D-raffinose, guanosine, inosine, guanine, aldopentose, two xenobiotics  and glucosamine/mannosamine. Pathway analysis associated these metabolites with the purine metabolic pathway.A total of 1,624 metabolic features were detected across the leukemic cell lines. Of these, 187 were annotated to known metabolites. With respect to doxorubicin, we observed significantly greater abundance of a carboxylic acid (1-aminocyclopropane-1-carboxylate) and several amino acids in resistant cell lines. Pathway analysis found enrichment of several amino acid biosynthesis and metabolic pathways. For cytarabine resistance, nine annotated metabolites were significantly different in resistance Overall, our results demonstrate that metabolomics differences contribute toward drug resistance. In addition, it could potentially identify predictive biomarkers for chemosensitivity to various anti-leukemic drugs. Our results provide opportunity to further explore these metabolites in patient samples for association with clinical response. Acute myeloid leukemia (AML) is a hematological disease resulting from proliferation and expansion of malignant myeloid cells. Successful treatment of AML requires the achievement of complete remission. Therefore, chemotherapeutic regimens for treatment of AML are very aggressive. The most commonly used induction treatment regimen for AML is the 7 + 3 model, named for the schedule of a 7-day infusion cycle of cytarabine combined with a 3-day intravenous push of an anthracycline agent, most often doxorubicin, daunorubicin, or idarubicin . The 7 +Changes to cellular metabolism have been associated with cancer development and tumor growth for multiple cancer types. The growing field of metabolomics and its approaches allows full metabolic profile evaluation in AML patients and leukemic cell cultures to explore the relationship between metabolism and resistance. Metabolomics studies of leukemia cell lines have been targeted to an identify metabolic pathways of resistance in multiple forms of leukemia –8. One s2 concentration of 5%. MTT assay was used to measure cytotoxicity as described previously (Seven AML cell lines were used for metabolomics profiling. AML cell lines HL-60 (ATCC CCL-240), MV-4-11 (ATCC CRL-9591), AML-193 (ATCC CRL-9589), Kasumi-1 (ATCC CRL-2724), KG-1 (ATCC CCL-246), and THP-1 (ATCC TIB-202) were purchased from the American Type Culture Collection . ME-1 (ACC-537) cell line was purchased from DSMZ . All cell lines were cultured using appropriate growth media recommended by the cell line supplier. Cell cultures were incubated at 37°C with a COeviously . Briefly6) were 3x washed with 40 mM ammonium formate solution followed by addition of 50 μL of a 5 mM ammonium acetate. Bead Beater  was used to homogenize the cells at 1,800 rpm for 30 s, followed by incubation at 4°C for 15 min. Homogenization was repeated two more times with incubation at 4°C for 15 min. Two microliters of internal standard solution  and 1 mL of 80% methanol in water solution were added to each sample. Samples were mixed using the Bead Beater at the previous settings and incubated at 4°C for 10 min. Following centrifuging at 200,000xg for 10 min at 4°C, 1 mL supernatant were collected and dried using nitrogen gas . Samples were reconstituted using 100 μL reconstitution solution  on ice for 15 min, following centrifugation, supernatants loaded for ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS).Cells  with the assistance of the Southeast Center for Integrated Metabolomics (SECIM) Core Lab 1 at the University of Florida. All samples were run in positive and negative ionization through heated electrospray as separate injections. Mass spectra analysis had a mass resolution of 35,000 at m/z 200. Separation was achieved on an ACE 18-pfp 100 × 2.1 mm, 2 μm column. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The flow rate was 350 μL/min, and column temperature was set to 25°C. Two microliters was injected for positive ionization and 4 μL for negative ionization.Mass spectra output files were converted to mzxml file format using MS Convert software (ProteoWizard 3.0). MZmine (freeware) was used to identify features, deisotope, align features and perform gap filling to fill in any features that may have been missed in the first alignment algorithm. All adducts and complexes were identified and removed from the data set. Positive and negative ionization datasets were merged to be used for statistical analyses. Peak intensity values for each cell line were grouped according to assigned cytarabine and doxorubicin sensitivity groups (sensitive or resistant) in separate datasets for categorical analysis. Missing value estimation was performed by removing metabolites missing over 30% of values among the samples and replacing the remaining missing values with a small value . Data processing included normalization by sum, log transformation, and Pareto scaling.Processed and normalized metabolomics data were then analyzed using categorical and continuous forms of the chemosensitivity variables, chemosensitivity group assignment, and cell viability AUC, respectively. Categorical statistical analysis was performed on the MetaboAnalyst 4.0 web-based analysis platform  using unPeak intensity values for metabolites with significantly different abundance AML cell lines were imported into MetaboAnalyst pathway analysis function, which performed pathway enrichment and topology analysis. Metabolite identifiers were converted as necessary according to synonyms listed in the human metabolome database (HMDB). The pathway impact measurement represented the sum of importance measures, generated by topology analysis, of significant metabolites normalized by importance measures of all metabolites in the associated pathway. The datasets generated and/or analyzed during the current study are available from the corresponding author on request. Additionally, we also cross-referenced the significant metabolites identified in the study with drug candidate analysis using Human Metabolome Database (HMDB) (hmdb.ca) database.in vitro cytotoxicity determined using MTT assays. For doxorubicin the HL-60, MV-4-11, Kasumi-1, and KG-1 (AUC below 1000) were classified as sensitive and AML-193, ME-1, and THP-1 were classified as resistant  values for cytarabine and doxorubicin obtained from Global metabolomics profiling of the AML cell lines was conducted using UHPLC-MS with the assistance of the SECIM Core Lab at the University of Florida. Global metabolomes were generated with the application of both positive and negative ionization settings separately. A total of 1,624 metabolic features were detected across the leukemic cell lines of which 187 were annotated to known metabolites as per the SECIM metabolite library.We utilized MetaboAnalyst software  to perfoThe pathway analysis used a pathway library in the MetaboAnalyst platform, which contained 80 known human metabolic pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database –14. A faFor cytarabine chemosensitivity, a correlation analysis was conducted to test the relationship of the abundance of known metabolites in the AML cell lines with the cytarabine cytotoxicity AUC data. A total of nine known metabolites were found to be significant, with all nine metabolites positively associated with cytotoxicity AUC (p<0.05). These included three nucleosides , the carbohydrate aldopentose, two xenobiotics , and the amino sugar glucosamine/mannosamine Table 3.In this study, we evaluated the global metabolic profiles of seven AML cell lines with varying sensitivity to two of the most commonly used chemotherapeutic agents used for treatment of AML: cytarabine and doxorubicin. Though the chemosensitivity grouping according to cytarabine or doxorubicin was not able to define the global metabolome for the AML cell lines in unsupervised multivariate analysis, we identified metabolites and metabolic pathways that showed significant differential abundance based on differences in chemosensitivity to both cytarabine and doxorubicin.With respect to cytarabine chemosensitivity, we identified a greater abundance of multiple nucleosides in AML cell lines that are more resistant to cytarabine relative to those that are sensitive. Pathway analysis further related these nucleosides to a significantly impacted purine metabolism pathway in the cytarabine resistant cell lines. An increase in nucleosides may be related to the pathway of cytarabine activation, and specifically indicate an established mechanism for cytarabine resistance , 20. CytWith respect to doxorubicin, several amino acids showed a significantly higher intracellular abundance in doxorubicin resistant cell lines. The importance of amino acids for cancer cell proliferation and survival is well established , 24 withThe increased abundance of asparagine in the doxorubicin resistant cells seen in our results shows a classic example of altered amino acid metabolism seen in acute leukemia. Acute lymphoblastic leukemia (ALL) cells have an increased dependence on exogenous asparagine as an essential amino acid due to decreased activity of asparagine synthetase. This results in a powerful therapeutic opportunity targeted by asparaginase, which depletes the circulating asparagine and starves the leukemic cells of the asparagine they require for survival . While tOf particular interest were also metabolites with a role in altered lipid and fatty acid metabolism associated with respect to doxorubicin chemosensitivity (low levels of LysoPC 16:1 and high levels of glycerol in doxorubicin resistant AML cell lines). Alterations to lipid metabolism are a common feature across multiple cancer types –46, and Overall, our study demonstrates that significantly different patterns of metabolite abundance can be found when comparing AML cell lines based on sensitivity to chemotherapeutic agents commonly used for AML treatment. These metabolites can be linked to several metabolic pathways known to be related to AML disease progression and chemotherapy resistance. AML cell lines that are resistant to cytarabine therapy show a significant alteration to purine metabolism, with a higher abundance of nucleosides related to this pathway. Resistance to doxorubicin in AML cell lines was found to be related to more widespread changes in metabolic pathways, including lipid catabolism, increased fatty acid oxidation, increased amino acid uptake, increased glycine and serine metabolism, and increased glutathione synthesis.While the results of cell line metabolomics analysis are promising, they require a follow-up study on a larger scale. Ideally, a large patient cohort study integrating metabolomics data in cell samples with additional patient omics data, such as genomics, transcriptomics, and proteomics, could clarify the full picture of what alterations can contribute to the chemoresistant phenotype in leukemic cells. However, this study helps support our understanding of what metabolic processes are linked to resistance to commonly used chemotherapeutic agents. Expanding our knowledge of this relationship can potentially improve our systems of selecting initial therapies and may lead to new targets for improving AML treatment.The data supporting the conclusion of this article are available upon request from the authors.BS and JKL contributed to study design. BS, NB, MS, JG-C did experiments and generated data. BS, JG-C, SP, TG and JKL performed data analysis and interpretation. BS and JKL drafted the manuscript. All authors provided critical review of the manuscript and have approved it for publication. All authors contributed to the article and approved the submitted version.This research was supported by NIH under the award numbers: NCI-R01CA132946.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose: To investigate the toxicity of the low-molecular-weight components (LMWCs) in ophthalmic silicone oils (SilOils) on retinal cell lines. Methods: The toxicity of six types of LMWCs were studied and compared with conventional SilOil 1000 cSt. In vitro cytotoxic tests of LMWCs, in both liquid and emulsified forms, on three retinal cell lines (Müller cells (rMC-1), photoreceptor cells (661W) and retinal pigment epithelial cells (ARPE-19)) were conducted using a transwell cell culturing system. The morphology and viability of cells were assessed by light microscopy and Cell Counting Kit-8 (CCK-8) assay at different time points . The ARPE-19 apoptotic pathway was investigated by Mitochondrial Membrane Potential/Annexin V Apoptosis Kit at different time points . Results: Apart from dodecamethylpentasiloxane (L5), all liquid LMWCs showed varying degrees of acute cytotoxicity on retinal cell lines within 72 h. Emulsified LMWCs showed comparable cytotoxicity with liquid LMWCs on retinal cell lines. Cyclic LMWCs, octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) had significantly higher cytotoxicity when compared with their linear counterparts decamethyltetrasiloxane (L4) and L5 with similar molecular formula. Using ARPE-19 cells as an example, we showed that LMWCs induce the apoptosis of retinal cells. Conclusions: Most LMWCs, in both liquid and emulsified forms, can induce acute cytotoxicity. In addition, cyclic LMWCs are suspected to have higher cytotoxicity than their linear counterparts. Therefore, LMWCs are suspected to be the main cause of the long-term toxicity of ophthalmic SilOil, due to their toxicity and propensity to cause ophthalmic SilOil to emulsify. The amount of LMWCs should be considered as the paramount parameter when referring to the quality of SilOil. Since the early years of vitrectomy surgery in the 1980s, SilOil became an important tool in the management of complicated vitreoretinal diseases. However, its use has been reduced due to a series of complications including emulsification, cataract, secondary glaucoma, keratopathy, and inflammation ,3 and enA recent in vitro study evaluated the short-term cytotoxicity of LMWCs to human ocular cell lines . HoweverTo address these limitations , in thisFour linear LMWCs, namely methyldisiloxane (L2) , octamethyltrisiloxane (L3) , decamethyltetrasiloxane (L4) , dodecamethylpentasiloxane (L5)  and two cyclic LMWCs, octamethylcyclotetrasiloxane (D4)  and decamethylcyclopentasiloxane (D5)  were tested in this study. Silicone oil with a viscosity of 1000 cSt is one of the most commonly used SilOils in ophthalmic surgeries . TherefoThree eye cell lines, including photoreceptor cells (661W),retinal Müller cells (rMC-1) and retinal pigment epithelial cells (ARPE-19) were used. The 661W cells were immortalised murine cone cells used at passage number 26–30 . rMC-1 i®, IKA) under 30,000 rpm/min for 30 s to form SilOil emulsions. Since only a small portion of SilOil clinically emulsifies inside the eye, the 1:99 ratio of LMWCs to aqueous phase was used to mimic the in vivo scenario with 1% of the SilOil content being emulsified within the eye. In reality, this is probably more significantly severe than would occur in most clinical situations. Both the droplet number and size distribution profile of each LMWC emulsion were characterised using a Coulter counter Multisizer®4 . A stable emulsion should have both its droplet number and size distribution remain unchanged during the experimental period .Our previously published methodology was used to produce SilOil emulsions . Mixture2 at 37 °C for 2 h before use. After sufficient rinsing with PBS, cells were seeded on the transwell membrane. The cell seeding density of the three cell types for different experimental time points was optimised to prevent the over-confluence of cell growth in the media control group. The results of the optimisation are summarised in The cells were cultured as monolayers on 12 mm-diameter semipermeable, 0.4 µm-pore-size, 10 µm-thick polyethylene terephthalate (PET) membranes . The apical side of the transwell membrane was coated with laminin (source) in a concentration of 2 µg/cm2 at 37 °C. This achieved direct contact between the test agents and cells that were on the transwell membrane. Glass coverslips were placed on top of the transwell to prevent the evaporation of test samples during the incubation period. The cell counting kit (CCK-8)  was used to evaluate the cell viability of the experimental groups relative to the control groups. Cell morphology was also compared before and after incubation. All the results collected in this part of study are from three independent experiments with two replicates each.All six kinds of LMWCs were included. Cells incubated with culture media and 1000 cSt. SilOil were treated as control groups. After 1 day of incubation, the media on the apical side were removed. Then, 300 µL of LMWCs in either liquid or emulsified form were added to the apical side of the transwell system and cultured with the cells for 6, 24 and 72 h in a humidified chamber with 5% CO®488 dye can identify apoptotic cells by binding to PS exposed on the outer leaflet of the plasma membrane to show green fluorescence signal. In addition, the MitoTracker® Red dye can indicate the status of the mitochondrial membrane potential by showing red fluorescence signal. After staining ARPE-19 cells with these two dyes in the provided binding buffer, the apoptotic cells showed green fluorescence with decreased red fluorescence, and living cells showed very little green fluorescence and bright red fluorescence under the 530 nm (for green fluorescence) and 630 nm (red fluorescence) bandpass filters. The experiment for the ARPE-19 cell line was conducted one time with duplicated samples as demonstration.Mitochondrial Membrane Potential/Annexin V Apoptosis Kit  was used to test the two hallmarks of apoptosis–phosphatidylserine (PS) externalisation and changes in mitochondrial membrane potential, on ARPE-19 cells. The annexin V labelled with Alexa Fluor p-value of <0.05 was considered to be statistically significant.One-way analysis of variance (ANOVA) followed by a Bonferroni test was used to evaluate the statistical differences of cell viability among different testing groups. The results of all individual comparisons are summarised in The various emulsion samples were stable during the 72 h period after generation by homogenisation. They had a relatively stable total number of droplets a, as welp < 0.05). The cell viability rapidly dropped down to 30% within 6 h and further decreased over time (p > 0.05 when compared with the controls), they exhibited significant toxicity after 24 h of incubation (p < 0.05) (p < 0.05), while they showed acceptable compatibility after 72 h of incubation. In contrast, L5 showed no significant adverse effects on the retinal cell lines during the observation period as compared with both control groups (p > 0.05).LMWCs L2, L3 and D4 all displayed acute toxicity to three retinal cell lines rMC-1, 661W and ARPE-19 when compared with the controls (ver time a–c. The  < 0.05) a,b. For l groups d–f, in tl groups a–c (p > p < 0.05) (p < 0.05 compared with controls) (p > 0.05). In contrast, they showed significant toxicity to ARPE-19 during the observation period as compared with the control group (media) (p < 0.05). In general, higher cytotoxicity was observed with emulsified LMWCs in ARPE-19 as compared with the other two retinal cell lines  a–c. Thisontrols) a–c. In aontrols) d,e, in tontrols) a,b (p >  (media) f, in ter (media) c . The number of cells with bright red fluorescence decreased in the D4, D5, L4, L5 and SilOil1000 emulsion groups a–c. ThesSilOil has been used as a long-term endo-ocular tamponade agent since 1962, and is still currently indispensable in the treatment of complicated vitreoretinal diseases . HoweverThe retinal ganglion cell (RGC) layer is the innermost layer of the neural retina, which makes it psychologically closest in position to SilOil. Therefore, RGC is the retinal cell type that is the most likely to be impaired due to the closet contact with SilOil. However, the RGC-5 cell line has been abandoned in vision science research due to reports of their lack of RGC-specific genes and proteins  and the In our study, most LMWCs showed acute cytotoxicity with a varying degree on retinal cell lines within 72 h. The cell viability varied with the molecular weight of the LMWC tested, in which LMWCs with smaller molecular weights had more significant adverse effects on viability. The exact mechanism behind the difference in cytotoxicity among various LMWCs is still not well understood. The potential reason could be that LMWCs with a smaller size have a greater ability to pass through the lipid bilayer of cell membrane, leading to an inflammatory response . In addiAlthough the underlying mechanism of retinal toxicity remains unclear, the emulsification of SilOil was believed to cause inflammatory response and retinal degeneration ,14. As lIn addition, the emulsion of ophthalmic SilOil—which is significantly more viscous in nature —exhibited a great variation in cytotoxicity to three retinal cell lines. Our results show that the emulsion of SilOil 1000 cSt had no significant cytotoxicity on two retinal cell lines during the experimental period, including the rMC-1 and 661W. This is consistent with the results of another in vitro study, in which emulsions of SilOil 5000 cSt did not induce a severe inflammatory response in neutrophils or stimulate phagocytosis by monocytes when compared with other less viscous ophthalmic liquids . However® Red dye in the living cells. In our study, the bright green fluorescence with different proportion was observed in the cells in both liquid and emulsified LMWC groups, indicating that the apoptotic process was involved in the ARPE-19 cell death pathway after LMWC treatment. This is consistent with the results of another in vitro study in which D4 induced the death of Jurkat cells with several apoptotic processes including the cleavage of caspase-3 and DNA fragmentation [To determine the cell death pathway upon both liquid and emulsified LMWC exposure, specific characteristics associated with apoptosis were investigated. The phosphatidylserine (PS) is a type of amino phospholipid which is exclusively located in the inner leaflet in most viable cells. The migration of PS into cell surface has been considered the universal feature of apoptotic cells . In addientation . HoweverBased on these results, we therefore suggest that the toxicity of SilOil could result from the presence of LMWCs in SilOil and is further aggravated by the emulsification of both LMWCs and long-chain SilOils. In addition, cyclic LMWCs have been quantified as the most abundant impurities in SilOils . TherefoThere are several limitations to this study. The cytotoxicity of LMWCs was only studied in the short-term . Given the routine longer-term use of SilOil intraocularly, longer-term investigations are required. Furthermore, only three isolated retinal cell lines were evaluated to more comprehensively evaluate LMWC toxicity, further long-term studies, including ex vivo and in vivo animal studies, will be necessary. In addition, only the ARPE-19 cells death pathway was investigated for demonstration to further investigate the underlying mechanism of cell death induced by LMWCs, distinct biochemical signalling associated with the apoptosis of retinal cell lines needs to be comprehensively studied.In this study, we showed the short-term cytotoxicity of LMWCs in both liquid and emulsified forms on retinal cell lines in vitro. The molecular weight and molecular geometry may contribute to the difference in cytotoxicity between the various LMWCs tested. The toxicity of SilOil could result from the presence of LMWCs in SilOil and may be further aggravated by the emulsification of LMWCs and long-chain SilOils. In addition, the emulsions of LMWCs could be the main cause of retinal toxicity observed in clinical cases that were related to SilOil emulsification. The apoptotic process may be partially involved in the retinal cell death pathway induced by both liquid and emulsified LMWCs. In agreement with Steel et al., , manufac


Triple-negative breast cancer (TNBC) is a highly diverse group of cancers with limited treatment options, responsible for about 15% of all breast cancers. TNBC cells differ from each other in many ways such as gene expression, metabolic activity, tumorigenicity, and invasiveness. Recently, many research and clinical efforts have focused on metabolically targeted therapy for TNBC. Metabolic characterization of TNBC cell lines can facilitate the assessment of therapeutic effects and assist in metabolic drug development. Herein, we used optical redox imaging (ORI) techniques to characterize TNBC subtypes metabolically. We found that various TNBC cell lines had differing redox statuses (levels of reduced nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD), and the redox ratio (FAD/(NADH+FAD)). We then metabolically perturbed the cells with mitochondrial inhibitors and an uncoupler and performed ORI accordingly. As expected, we observed that these TNBC cell lines had similar response patterns to the metabolic perturbations. However, they exhibited differing redox plasticity. These results suggest that subtypes of TNBC cells are different metabolically and that ORI can serve as a sensitive technique for the metabolic profiling of TNBC cells. This heThe mitochondrial redox status provides critical information about the energy-linked biological processes in cells and tissues for various types of cancer, including TNBC . Nicotin40.240.2.1®, Cat No. 11875085) supplemented with 10% fetal bovine serum (Corning®). Cells were maintained at 37 °C and 5% CO2 and passaged at 80% confluency using 0.25% trypsin-EDTA. Oligomycin (oligo), trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), rotenone (rot), and antimycin A (aa) (Sigma®) were reconstituted in DMSO (Sigma®) and used at the following final concentrations: oligo (2 μg/mL), FCCP (0.5 μM), rot (1 μM), and aa (1.25 μg/mL).TNBC cell lines MDA-MB-231, MDA-MB-436, MDA-MB-468, and HCC1806 were cultured in RPMI 1640 (Gibco40.2.2®) and incubated at 37 °C and 5% CO2 overnight. Approximately 1 hour before imaging, the medium was replaced with 1 mL live cell imaging solution (Life Technology®) supplemented with 11 mM glucose and 2 mM glutamine (Sigma®), the same as in the RPMI 1640 medium. A DeltaVision widefield microscope was used to collect NADH and FAD fluorescence signals. The excitation band-pass filters for NADH and FAD channels were 360/40 nm and 470/40 nm, respectively; the emission filters of NADH and FAD channels were 455/50 nm and 520/40 nm, respectively. Nine to fifteen dishes were imaged on different days for each cell line with three fields of view (FOV) for each dish with an exposure time of 3 seconds for each channel. For mitochondrial redox plasticity experiments, perturbation drugs were added sequentially in the order of oligo, FCCP, and a mixture of rot and aa (rot/aa), allowing the cells to be pushed to the two redox extremes and the consequent calculation of the cell lines’ redox plasticity. The dishes were imaged 3–5 minutes after each drug administration. A customized MATLAB® program was used to extract NADH and FAD signals and the pixel-to-pixel based FAD/(NADH+FAD) ratio, which were first averaged across FOVs and then across individual dishes to obtain the group mean and standard error of the mean (SEM). Heteroscedastic two-tailed t-tests were used for statistical analyses of redox differences among cell lines and the differences before and after a specific treatment. P < 0.05 was considered statistically significant.As detailed previously , 80,000 40.340.3.1p < 0.05) between all cell lines except between MDA-MB-436 and MDA-MB-468. NADH intensity was similar in MDA-MB-231, MDA-MB-436, and HCC1806. Furthermore, we found that MDA-MB-231 exhibits the highest redox ratio of 0.65 ± 0.02, followed by MDA-MB-436, MDA-MB-468, and HCC1806, with the redox ratios of 0.55 ± 0.01, 0.49 ± 0.01, and 0.47 ± 0.01, respectively. The TNBC cell lines significantly differ in their redox ratios, with an exception between MDA-MB-468 and HCC1806, both of which are classified as the basal-like subtype (p > 0.05).We first imaged MDA-MB-231, MDA-MB-436, MDA-MB-468, and HCC1806 cells under normal conditions . Our qua40.3.22 to NAD+ and FAD, respectively, resulting in decreased NADH and increased FAD. Conversely, rot and aa are inhibitors of complexes I and III, respectively, fully inhibiting respiration, resulting in a maximal NADH signal and decreased FAD. Our results show that FCCP and rot/aa treatments effectively caused a decrease and increase in NADH intensity, respectively, in all four cell lines and an increase and decrease of FAD in three lines except MDA-MB-231 (To show that ORI can reflect the metabolic phenotypes of TNBC cell lines, we sequentially administered oligo, FCCP, and rot/aa and obtained the NADH and FAD fluorescence signals accordingly. Oligo is an inhibitor of the ATP synthase in the ETC, causing a buildup of NADH. FCCP is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential, causing unregulated oxidation of NADH and FADHA-MB-231 . Further40.3.3By taking the difference between the average fluorescence values after FCCP and rot/aa application, we quantified the changes, i.e., the plasticity in NADH, FAD, and the redox ratio, in the TNBC cell lines . Our resTo our best knowledge, this is the first systematic investigation that reports how ORI differentiates the redox plasticity of TNBC cells. Changes in the redox indices caused by therapeutic interventions could reflect compromised mitochondrial redox plasticity. This information would be helpful in assessing drug effects in vitro, such as screening drugs for developing therapeutic interventions that target the metabolic activities in TNBC cells. Our previous studies have linked the redox ratio of the more oxidized tumor subpopulations to breast tumor and melanoma metastatic risk , 10. TheIn short, the four TNBC lines can be clearly differentiated by ORI, and the underlying mechanisms remain to be elucidated.


Sebaceous carcinoma and sweat gland carcinoma  are rare malignant skin adnexal tumors that differentiate toward sebaceous gland and eccrine and apocrine glands, respectively. Owing to the rarity of these carcinomas, standard treatments for advanced disease have not been established. Because the prognosis of patients with systemic metastasis is poor, a new treatment for these diseases is eagerly desired. Trophoblast cell surface antigen 2 (TROP2) and sacituzumab govitecan, an antibody–drug conjugate of TROP2, have attracted attention in the treatment of various solid tumors. In the current study, we immunohistochemically investigated TROP2 expression in 14 sebaceous carcinoma and 18 sweat gland carcinoma samples and found strong and relatively homogeneous TROP2 staining in both cancer types. The mean Histoscore, a semi-quantitative scoring ranging from 0 (negative) to 300, was 265.5 in sebaceous carcinoma and 260.0 in sweat gland carcinoma. These observations directly suggest that both sebaceous carcinoma and sweat gland carcinoma could be potentially treated with TROP2-targeted antibody–drug conjugates such as sacituzumab govitecan. Sebaceous carcinoma is a malignant tumor consisting of basaloid or squamous atypical cells with sebaceous differentiation. Sebaceous carcinoma has been classified into two types (ocular and extraocular). The former type occurs on the eyelids and originates from the Meibomian glands and glands of Zeis. The latter type most commonly arises in the head and neck, but it can also occur in the anogenital area and other locations. Histopathologically, sebaceous carcinoma is composed of irregular lobules consisting of many undifferentiated cells and distinct sebaceous cells with foamy cytoplasm. These cells are atypical, displaying nuclear and nucleolar pleomorphism .Sweat gland carcinomas  comprise a heterogeneous group of carcinomas that are divided into 17 subgroups in the latest (4th) version of the World Health Organization (WHO) classification of skin tumors . Owing tTrophoblast cell surface antigen 2 (TROP2) is a surface glycoprotein originally identified in human placental trophoblasts ,4,5. TROWe previously reported TROP2 expression in normal skin and skin appendages  and deteThis study was conducted in accordance with the concepts enshrined in the Declaration of Helsinki, and approved by the Kyushu University Institutional Ethics Committee . Written informed consent was obtained from the patients prior to their inclusion in the study.We retrieved data from 14 patients with sebaceous carcinoma (14 samples) and from 13 patients with sweat gland carcinoma (18 samples) who were treated at the Department of Dermatology, Kyushu University  between December 2008 and December 2018. At least three experienced dermatopathologists confirmed the diagnosis. To confirm tumor differentiation, an immunohistochemistry panel was used when necessary. The diagnosis was made in accordance with the latest version of the WHO classification of skin tumors [Immunohistochemical staining was performed as reported previously with slight modification ,27,28. SThe immunohistochemical results were evaluated by a semi-quantitative approach using the Histoscore (H-score) . The intWe collected 14 sebaceous carcinoma samples from 14 patients and 18 sweat gland carcinoma samples from 13 patients. The demographic data of patients with sebaceous carcinoma are presented in Representative histopathological images of TROP2 are presented in Representative images of TROP2 staining are presented in Cutaneous adnexal tumors comprise a large and heterogeneous group of neoplasms that differentiate toward one or more skin appendages or recapitulate events occurring during embryo development. They include tumors with predominant apocrine, eccrine, follicular, sebaceous, or multilineage differentiation. Malignant adnexal tumors are less common than their benign counterparts ,2. OwingThe first-line treatment for localized sebaceous carcinoma and sweat gland carcinoma is complete surgical removal, which leads to a relatively good prognosis ,37,38,39TROP2 has recently attracted attention as a potential target for anticancer therapy. It is involved in a variety of cell signaling pathways , and higIn conclusion, we observed TROP2 overexpression in sebaceous and sweat gland carcinomas. Most tumor cells exhibited high TROP2 expression, suggesting that TROP2-targeted therapies could be new treatment options for unresectable sebaceous carcinoma and sweat gland carcinoma.


PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CAE545K into the LAM1 PTC cell line, which carries a BRAFV600E mutation. In culture, the engineered cell line (LAM1:PIK3CAE545K) proliferated faster and demonstrated increased clonogenic potential relative to the parental line carrying an empty vector (LAM1EV). Both the LAM1EV and LAM1:PIK3CAE545K edited lines were implanted into hind flanks of athymic nude mice for in vivo determination of disease progression. While tumour weights and volumes were not significantly higher in LAM1:PIK3CAE545K mice, there was a decrease in expression of thyroid differentiation markers TTF-1, thyroglobulin, PAX8 and B-catenin, suggesting that introduction of PIK3CAE545K led to dedifferentiation in vivo. Collectively, this study provides evidence of a role for PIK3CAE545K in driving disease progression from a well-differentiated to an undifferentiated thyroid cancer; however, over-expression was not a determinant of an accelerated growth phenotype in ATC.Anaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While The online version contains supplementary material available at 10.1186/s40463-022-00558-w. Well-differentiated subtypes of thyroid cancer  have an excellent prognoses, typically with greater than 95% five-year survival rates . In contBRAFV600E and the PIK3CA canonical hotspot mutations, with the E545K variant being the most common  × 0.52 [Mice were maintained and handled in accordance with the Western University AUP 2015–045 protocol. We used the cell lines LAM1Immunohistochemistry (IHC) staining was carried out in the Molecular Pathology Core Facility (Western University) where 4-µM sections were cut and tested for expression of thyroid-related markers. Primary antibodies were used according to the manufacturer’s instructions: TTF-1 , PAX8 , Beta Catenin , Ki67  and Thyroglobulin . Slides were scanned using an Aperio ScanScope slide scanner.t-test was performed using Prism® 8 Graphpad Software Macintosh Version . P values < 0.05 were considered statistically significant. P values are defined as not significant p > 0.05, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001 and **** represents p < 0.0001.A Student’s unpaired, two-tail BRAFV600E mutation found in a significant fraction of ATCs [PIK3CAE545K, which is also commonly found in ATC [PIK3CA protein in LAM1:PIK3CAE545, but not LAM1EV  (p < 0.0001)    Fig. A. We als04) Fig. B. TogethEV and LAM1:PIK3CAE545K cells to determine whether the addition of PIK3CAE545K to the BRAFV600E background resulted in more resistance to clinically used therapy regimes. We first treated cells with etoposide, which is used clinically to treat ATC. We found no significant difference between the responses of the LAM1EV and LAM1:PIK3CAE545K cell lines , a specificity control (JHU029 – PIK3CA H1047L mutant), and the parental (LAM1) were compared to the LAM1:PIK3CA E545K for levels of PIK3CA E545K transcripts.Additional file 2: Table S1. Short tandem repeat profiling of LAM1 cell line. The STR profile for both the parental and edited LAM1 cell lines were identical to those reported by Copland et al. [Table S2. A hind flank model of disease progression. LAM1EV and LAM1:PIK3CAE545K cell lines were used for the generation of cell line xenograft hind flank models of disease (n = 5 per group). A total of 1 × 106 cells were injected into the hind flank of each athymic nude mouse. Mice were weighed twice per week and tumour dimensions (length and width) were measured once weekly when tumours were palpable using digital calipers. Tumour weights (mg) were measured and collected after dissection.d et al. . Table S


Proper identification of cell lines is a requirement of rigorous research involving cultured cells. Here we develop an innovative strategy to identify cell lines using transposable element profiles and apply this novel strategy one of the most important model species, Drosophila melanogaster. We clarify the provenance of several Drosophila cell lines and show that loss of heterozygosity affects genome evolution in Drosophila cell culture. Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines  relative to published reports, and also provide evidence that insertions from only a subset of long-terminal repeat retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data , which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabeling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here, we leverage the classical observation that transposable elements (TEs) proliferate in cultured  Cultured cell lines play essential roles in biological research, providing model systems to support discovery of basic molecular mechanisms and tools to produce biomolecules with medical and industrial relevance. Despite their widespread use, experiments in cultured cells often show non-reproducible outcomes, and increasing the rigor of cell line-based research is a priority of both funders and journals alike . The development of cell line identification protocols and standards for common model organisms like Drosophila is an important goal for increasing rigor and reproducibility in bioscience. Achieving this goal for a new species requires an understanding of the genome biology and cell line diversity of that organism, and should ideally take advantage of powerful, cost-effective modern genomic technologies.For most species beside humans, cell line authentication standards and protocols remain to be established  insertions are highly polymorphic among individual flies  and possibly also from the same donor genotype, including divergent sublines of the same cell line   data from the modENCODE project  and integrate our new data with legacy data  were obtained from the Drosophila Genomics Resource Center (DGRC), the Gorski lab  and the Strand lab (University of Georgia). DNA extractions were performed using Qiagen Blood and Tissue kit (Cat# 69504) for the mbn2 sample from the Strand lab and using the Zymo-Quick kit (Cat# D4068) for all other samples. Purified DNA was analyzed by Qubit and Fragment Analyzer to determine the concentration and size distribution, respectively. Samples were normalized to the same concentration before preparing libraries with the KAPA Hyper Prep Kit (Cat# KK8504). During library prep, DNA was fragmented by acoustic shearing with Covaris E220 Evolution before end repair and A-tailing. Single indices were ligated to DNA fragments. Libraries were purified and cleaned with Solid Phase Reversible Immobilization (SPRI) beads before PCR amplification. Final libraries underwent an additional round of bead cleanup before being assessed by Qubit, qPCR (KAPA Library Quantification Kit Cat# KK4854), and Fragment Analyzer. Libraries were then sequenced in paired-end 150-bp mode on an Illumina NextSeq500 high output flowcell and demultiplexed using bcl2fastq. Metadata, sequencing statistics, and SRA accession numbers for all cell line DNA-seq samples used in this study can be found in Supplementary Table S1.Public genome sequencing data for 26 samples of 18 Drosophila . The reference genome for these analyses was comprised of the major chromosome arms from the D. melanogaster dm6 assembly  and the TE library was the Berkeley Drosophila Genome Project canonical TE dataset v10.1 .Paired-end sequencing data from the modENCODE project  and depth of coverage . We chose not to normalize input datasets by downsampling to the lowest read length and coverage to avoid reducing the sensitivity of non-reference TE detection methods for higher-quality samples. Using complete samples allowed us to observe that the number of non-reference TE predictions per sample (Supplementary Table S2) showed a strong dependence on read length  or coverage  for all methods besides TEMP (https://github.com/bergmanlab/ngs_te_mapper2) in McClintock  and A4  profiles for nonoverlapping 10-kb windows of the dm6 genome using Control-FREEC  using tximport v1.18.0 . As reported previously for human cancer cell lines (Drosophila cell line sample had a unique profile of TE insertions (Supplementary File S1). The most parsimonious clustering of Drosophila cell lines using TE profiles revealed several expected patterns that indicate TE insertions reliably mark the identity of Drosophila cell lines  cluster closely together . More strikingly, we also observed that the mbn2 cell line originally reported by Overall, clustering patterns based on TE profiles suggest that misidentification is rare among the panel of cell lines sequenced by modENCODE. However, we observed two cases where the similarity of cell lines based on genome-wide TE profiles conflicted with expectations based on reported provenance. First, we unexpectedly found that the Sg4 cell line  clusters most closely with S3 cells, although the DGRC and FlyBase currently consider Sg4 to be a variant of S2 cells  that were not originally sequenced in the modENCODE cell line genome project mbn on a 2nd chromosome marked with three visible mutations . UnfortuDrosophila cell lines . Together, these data support the conclusions that multiple independent sublines of mbn2 cells all share a common origin and are likely to descend from a single divergent lineage of S2 cells. Based on these observations, we speculate that currently circulating mbn2 cells derive from a mislabeling or cross-contamination event with S2 cells in the Gateff lab that occurred prior to distribution to the Hultmark or Dorn labs , although the quantitative abundance of these TE families varies across cell lines. Other TE families appear to be represented in only one cell line or a subset of cell lines from the same lab origin . These results provide systematic genome-wide evidence for the classical observation that proliferation of different TE families in cultured Drosophila cells is cell-line dependent (Ivk proliferation), and that mbn2 cell lines are a divergent lineage of S2 cells .Based on these results, we next evaluated whether a small, experimentally tractable subset of TE families is sufficient to cluster and identify families . FinallyDrosophila cell lines raises the question of whether a single TE family could diagnostically mark the identity of a Drosophila cell line or subline. One such candidate for this possibility is the retroviral-like LTR retrotransposon ZAM in the closely related OSS and OSC ovarian somatic cell lines , but not in a putatively early passage subline of OSS cells .The observation that different TE families are amplified in distinct ZAM proliferation is restricted to a subset of OSS sublines or is in fact a specific marker for all OSS sublines, we performed an integrated analysis of TE predictions in WGS data from six OSS and OSC samples from our and two previous studies . Additionally, our analysis of OSS and OSC samples ultimately required tracking intra-sample TE allele frequencies, which is not available in other TE predictors that use single-end data  that improves speed and sensitivity relative to the original version and has been extended to estimate intra-sample TE allele frequencies .Because WGS data from ZAM has proliferated massively in the OSS_DGRC and OSS_C sublines , but is present in only one or two copies in OSS_E and all OSC sublines . These results revealed that sublines . The abuZAM abundance can only be explained by unlikely scenarios such as a massive loss of ZAM insertions on the branch leading to OSS_E, or independent parallel amplifications of ZAM on the OSS_C and OSS_DGRC sublines. An alternative hypothesis to explain the pattern of ZAM abundance is motivated by another observation made by bona fide OSS sublines would be OSS_C and OSS_DGRC, and ZAM proliferation could truly be a diagnostic marker of OSS cell line identity.Under the “reported provenance” hypothesis that OSS_E and OSS_C share a more recent common ancestor than they do with OSS_DGRC .To test these alternative hypotheses, we used ngs_te_mapper2 predictions as input to cluster OSS and OSC sublines using Dollo parsimony. We found two highly supported clusters, one containing only the OSS_C plus OSS_DGRC sublines and the other containing OSS_E plus all OSC sublines (ZAM amplification truly marks bona fide OSS cell lines (include the OSS line distributed by the DGRC), and that ngs_te_mapper2 TE predictions based on single-end WGS data can be effectively used to cluster Drosophila cell lines and reveal aspects of cell line history.Further support for the hypothesis that OSS_E is an OSC-like lineage can be found in patterns of SNP and CNV variation in these cell line genomes . OSS_C and OSS_DGRC have essentially identical BAF profiles across the entire genome . In contrast, OSS_E and OSC sublines share a BAF profile everywhere but the distal regions on chromosome arms 2L, 3L, and 3R  that are heterozygous in OSS_E are predicted to be homozygous in OSC sublines in distal LOH regions, but should maintain heterozygosity elsewhere in the genome. To test these predictions, we used intra-sample allele frequency estimates from ngs_te_mapper2 to classify the zygosity of TE insertions in OSS_E and OSC sublines. Evaluation of our classifier on simulated diploid genomes revealed it had >91% precision and crucially never falsely classified heterozygous insertions as homozygous (Supplementary Table S6), and is thus conservative with respect to detection of LOH using TE insertions. As predicted under our model, we observed that there are many shared TE insertions in distal LOH regions that are heterozygous in OSS_E but virtually all TE insertions in these regions are homozygous in OSC sublines . Consistent with the prediction of whole-arm LOH in the ancestor of the OSS_E/OSC lineage followed by ongoing transposition in cell culture, we observe that most shared TE insertions on chromosome X and arm 2R are homozygous , both of which are reported to have a diploid baseline autosomal copy number . Supplementary File S2 contains clustered TE profiles in the format of binary presence/absence data matrix including 34 Drosophila cell line samples . Supplementary File S3 includes data matrix of the number of non-reference TE insertion gain events per family on each branch of the most parsimonious tree used for the heatmap in Raw sequencing data generated in our study are available in the SRA under BioProject PRJNA689777. Supplementary material is available at figshare:


Loss-of-function events in tumor suppressor genes (TSGs) contribute to the development and progression of cutaneous malignant melanoma (CMM). Epigenetic alterations are the major mechanisms of TSG inactivation, in particular, silencing by promoter CpG-island hypermethylation. TSGs are valuable tools in diagnosis and prognosis and, possibly, in future targeted therapy. The aim of this narrative review is to outline bona fide TSGs affected by promoter CpG-island hypermethylation and their functional role in the progression of CMM. We conducted a systematic literature review to identify studies providing evidence of bona fide TSGs by cell line or animal experiments. We performed a broad first search and a gene-specific second search, supplemented by reference checking. We included studies describing bona fide TSGs in CMM with promoter CpG-island hypermethylation in which inactivating mechanisms were reported. We extracted data about protein role, pathway, experiments conducted to meet the bona fide criteria and hallmarks of cancer acquired by TSG inactivation. A total of 24 studies were included, describing 24 bona fide TSGs silenced by promoter CpG-island hypermethylation in CMM. Their effect on cell proliferation, apoptosis, growth, senescence, angiogenesis, migration, invasion or metastasis is also described. These data give further insight into the role of TSGs in the progression of CMM.  Cutaneous malignant melanoma (CMM) is the most aggressive form of skin cancer. If untreated, melanomas have a serious metastatic potential and cause a significant mortality . The worGenes important in the development and progression of cancer can be divided into oncogenes and tumor suppressor genes (TSGs). Whereas the former become active through gain-of-function events in proto-oncogenes, TSGs, on the other hand, normally encode proteins that prevent cancer development. They function mainly through the negative regulation of cell cycle progression, stimulation of DNA damage repair, induction of apoptosis, inhibition of metastasis  or gene transcription regulation . Their cIn recent years, the importance of the most common form of epigenetics namely DNA methylation in the biology of CM has been increasingly acknowledged. Moreover, DNA methylations are more easily reversible than genetic variation, and therefore offer a significant opportunity for cancer therapy. As DNA methylation is extremely interesting for therapeutic purposes, an overview of the bona fide TSGs methylated in melanomas is lacking in the literature. DNA methylation in mammals generally means the addition of a methyl group to the 5-carbon position of a cytosine in CpG sites by DNA-methyltransferases ,22,23. NOverall, we included 23 studies in this narrative review using systematic search . We founWFDC1 encodes a protease inhibitor involved in growth regulation and apoptosis. Liu et al. found WFDC1 expression to have reduced or lost 80% of the examined melanoma cell lines and tissues. Promoter hypermethylation contributes to WFCD1 gene silencing .Re-expression of the gene in cell lines lacking the gene caused no significant changes in proliferation, colony formation or invasion. A significant increase in migration was seen in one cell line, but not in the other (highly migratory) cell line. By contrast, in vivo assays showed significant tumor growth inhibition when transfecting the above cell lines in mice. The effect is possibly related to the upregulation of DKK-1, which is a Wnt-signaling pathway inhibitor .RASSF1A, RASSF8 and RASSF6 encodes a protein of the Ras-association domain family. RASSF1A encodes a microtubule-associated protein involved in the regulation of cell proliferation, migration and apoptosis. Immunohistochemistry showed decreased RASSF1A expression in melanoma cells compared with normal melanocytes or benign lesions. A reverse correlation between RASSF1A staining intensity and lymph node metastasis was observed . The genRASSF6 is involved in pro-apoptotic signaling pathways. Mezzanotte et al. found RASSF6 promoter methylation in the majority of the examined primary melanomas and brain metastases .Vector-induced expression of the gene in a cell line with methylated RASSF6 showed decreased cell growth and invasion. It is thought that RASSF6 works through MAPK-pathway and AKT inactivation .SOCS1 encodes a protein that is important in ubiquitination and negative regulation of pro-inflammatory cytokine signal transduction. Previous studies showed the frequent silencing of SOCS1 via promoter hypermethylation in human cancer, including melanoma ,29. TranGallagher et al. studied 1 parental and 3 derivative cell lines with variable tumorigenicity, cytokine resistance and metastatic potential. A total of 44 genes were downregulated in the derivative cell lines with respect to the parental cell line, of which 13 were reactivated after 2’ -deoxy-5-azacytidine (DAC) treatment (DAC functions as a DNA methylation inhibitor). Six of these  have 5’ CpG islands at their promotor site and are therefore likely to be regulated by DNA methylation . This waSYK encodes a protein that is not only involved in hematopoietic cell signaling, but also in regulating cellular senescence . SYK wasCHD13 encodes the adhesion molecule H-cadherin. H-cadherin is strongly reduced or lost in 70% of the melanoma cell lines examined by Kuphal et al. . On immuTRIM16 encodes a protein of the TRIM family, which is involved in the regulation of cell cycle, proliferation, differentiation, ubiquitination, apoptosis, tumor suppressor functions and oncogenesis . Sutton RUNX3 encodes a transcription and cell shape-regulating protein. Previous studies showed a significant decrease in RUNX3 expression in melanoma cell lines compared with melanocytes . In melaAPC encodes a protein that regulates cellular β-catenin, a key effector in the Wnt-signaling pathway. Additionally, it regulates cell cycle, adhesion, migration, microtubule assembly and chromosome segregation. Inactivating mutations and hypermethylation of APC have been found responsible for reduced expression in melanoma cell lines and tissue .KO of APC in melanoma cell lines by Worm et al. resulted in increased proliferation . However, a large (>70%) reduction in APC levels caused a decrease in invasiveness . Worm etMAPK13 encodes a protein involved in the regulation of proliferation, differentiation, survival and migration. Gao et al. found the gene to be expressed in normal melanocytes and fibroblasts, but not in melanoma cells due to promoter methylation. This process occurred in most of the examined primary melanomas, but not in benign nevi, and even higher percentages of methylation were found in metastases . MAPK13 RARβ encodes a retinoic acid receptor involved in vitamin A-mediated growth inhibition and melanocytic differentiation . The genAGTR1 encodes an Angiotensin II receptor and is thus involved in the renin-angiotensin-aldosterone system. The protein is expressed in normal human melanocytes as well as in radial growth phase melanoma cell lines ; however, AGTR1 is severely downregulated or undetectable in the vertical growth phase and in metastatic melanoma cell lines. The same trend was found in melanoma tissues in different stages of tumor progression. There was a strong correlation between methylation and transcriptional silencing in cell lines . It has SERPINB5 encodes maspin, a protein involved in cellular adhesion. The gene is expressed only in normal human melanocytes, but not in melanoma cell lines. All melanoma cell lines examined showed promoter methylation of the gene . Denk et14-3-3gression . 5-Aza-Cnescence .TCF21 encodes a transcription factor involved in mesenchymal-to-epithelial transition (MET) and in the repression of epithelial-to-mesenchymal transition (EMT is therefore important in cancer cell dissemination). All metastatic melanoma tissues examined by Arab et al. showed variable promoter methylation and corresponding TCF21 gene expression silencing. There was an inverse but not significant correlation between methylation status of TCF21 and overall survival . This stSPINT2 encodes a protein serving as proteolytic inhibitor of hepatocyte growth factor activator and thereby suppressing the HGF-MET-pathway involved in cell proliferation, dissociation, migration, angiogenesis and survival. Hwang et al. found a significantly decreased SPINT2 expression and increased methylation in metastatic melanomas compared with primary melanomas. Similar data were found in melanoma cell lines, compared with human primary melanocytes . Hwang eWe reviewed the literature on TSGs silenced by CpG island promoter hypermethylation in CMM. Overall, 23 studies were included describing 24 bona fide TSGs affected by promoter hypermethylation in CMM. We also found bona fide TSGs that were methylated and deleted in melanoma, but since DNA hypermethylation is reversible in nature and therefore offers a significant opportunity for cancer therapy, we decided to focus on the methylated bona fide TSG in melanoma, in this review. We excluded the following genes that were methylated and deleted in melanoma: PTEN, BRIMS1, FERMT3 (KIND3), AKAP12 (SSeCKS), CDKN2A, APAF-1, MTAP and MEN1.TSGs normally encode proteins that prevent cancer development by regulation of cell cycle progression, DNA damage repair, induction of apoptosis, inhibition of metastasis or gene transcription regulation . Loss-ofGallagher et al. performed transcriptomic studies and observed the downregulation of some genes in high-tumorigenic melanoma cell lines compared with low-tumorigenic melanoma cell lines, with six of them showing promoter CpG-islands. They treated cell lines and mice with a demethylating agent and observed attenuated tumor growth . They diSutton et al. demonstrated that TRIM16 is a marker of cell migration and metastasis and is a novel treatment target in melanoma . TRIM16 We provided a clear overview of bona fide TSGs methylated in melanomas, and we described their functional role in melanoma development. In the absence of a broad-based definition of bona fide TSGs, we used the following criteria: (a) overexpression of the gene in cell lines or introducing the gene in KO animals results in a decrease in cancer cell-specific traits; or (b) blocking the gene in cell lines or in animals results in an increase in cancer cell-specific traits. We included 24 bona fide TSGs methylated in melanomas, and we listed their chromosomal location, physiological role, inactivation mechanism, pathway, experiments conducted to meet the bona fide criteria and hallmark of cancer acquired by TSG inactivation. Future studies can focus on animal or cell line experiments with candidate TSGs, inactivation mechanisms of bona fide TSGs and reviewing TSGs affected by other (epi)genetic inactivation mechanisms. TSGs are valuable in diagnosis, prognosis and treatment strategies.We conducted a narrative review to overview literature on bona fide tumor suppressor genes that are methylated in melanoma. We used the electronic databases PubMed and Embase to search for eligible studies. The search strategy consisted of five concepts combined with AND. Concept 1 was tumor suppressor genes, concept 2 was melanoma, concept 3 was metastasis, concept 4 was NOT uveal or mucosal melanoma and concept 5 was methylation. These concepts were expressed in MeSH-terms (PubMed), Emtree-terms (Embase) or free text . The complete search strategy can be consulted in A manual inspection of the results was carried out and is presented in This review addresses only DNA hypermethylation in CMM because, compared to irreversible genetic variation, e.g., deletion, DNA hypermethylation is more easily reversible and offers options for cancer therapy. We subsequently split the remaining results in studies in those giving evidence of bona fide TSGs, on the one hand, and those focusing on observation of expression or methylation of the genes, on the other. We used the modified DaCosta criteria for bona fide TSGs: (a) overexpression of the gene in cell lines or introducing the gene in KO animals results in a decrease in cancer cell-specific traits; or (b) blocking the gene in cell lines or in animals results in an increase in cancer cell-specific traits. To ensure that we did not overlook important studies on bona fide TSGs, we conducted a second search. For those genes for which we could not find any evidence to be bona fide TSGs in the selected studies, we searched PubMed with the following strategy: name of the gene AND tumor suppressor gene AND melanoma. We included studies on bona fide TSGs methylated in melanoma. In addition, we included some studies by checking the references of the included articles. We extracted data of all studies in the ‘bona fide TSG’ group and summarized them in


Cancer diseases are currently one of the greatest health challenges in clinical medicine worldwide. Classic methods of treatment often lead to numerous side effects, including the development of multidrug resistance. For this reason, increasing hope is being placed on compounds of natural origin, mainly due to their pleiotropic effect on different types of cells, protective effect on normal cells and toxic effect on cancerous ones. The most studied group are the polyphenolic compounds, which include resveratrol. The effectiveness of polyphenols in the treatment and prevention of many diseases, including cancer of various origins, has become the basis of many scientific studies. The anticancer effect of resveratrol has been demonstrated at all stages of the carcinogenesis process. Additionally, whether administered by itself or in combination with cytostatics, it may play a significant role in the process of reversing multidrug resistance. A review of the effects of resveratrol in in vitro conditions proves that it has a stronger or weaker antiproliferative and proapoptotic effect on the cells of certain neoplasms of the gastrointestinal tract. Despite the differences in the effect of this compound on different types of cancer, a similar tendency can be observed especially regarding the correlation between the concentration of the compound and the incubation time on the one hand and the antitumour effect on the other hand. The information included in this review may prove helpful in planning in vivo and clinical studies in the future. The progress in medicine in terms of prevention programs, diagnostic tests and therapeutic strategies, as well as the awareness of industrialized societies in relation to the importance of physical activity and proper nutrition, have contributed to a significant improvement in the quality of life and the average life expectancy. Unfortunately, this is connected with an increasing incidence of aging-associated diseases, including cancer.Despite the implementation of cancer prevention, the incidence and mortality rates are constantly increasing everywhere in the world ,2. As a ABCB1 gene, encoding the P-glycoprotein (P-gp) protein, through which the drug is transported outside the cell, leading to a reduction in the effective concentration of the drug in the cell . Re. ReVeratoperties ,24,25. T extract . The cispe skins  27,28].,28.VeratSince then, this compound has been extracted from many other naturally occurring plants included in a varied diet, including bilberries, blueberries and peanuts ,30. HoweResveratrol is a phytoalexin, a compound produced by plants in response to the attack of pathogens and as a result of UV radiation and the action of heavy metals ,34. For The antitumour activity of resveratrol was first described in 1997 by Jang et al. ,41,42. DThe main goal of this review is to present the results of in vitro studies on the antiproliferative and proapoptotic effects of resveratrol on human gastrointestinal cancers.Pancreatic cancer is the ninth most common cancer in women and the tenth most common cancer in men ,50. The In the case of pancreatic cancers, the results of numerous in vitro studies indicate the potential antiproliferative effect of resveratrol, depending on the dose and the time of incubation with the compound.In a study on two pancreatic cancer cell lines, PANC-1 and AsPC-1, Xian-Zhong Ding’s team showed that after 48 and 72 h of incubation with a resveratrol solution at various concentrations, there was a statistically significant change in cell viability. These changes were dependent on the time and concentration of the compound tested. The greatest effects were observed at a concentration of 100 µM . HoweverThe studies of Jing Cui’s team also showed an inhibition of cancer cell survival dependent on the concentration and time of the effect of resveratrol. The tests were performed on the PANC-1, BxPC-3 and AsPC-1 pancreatic cancer lines, which were treated with resveratrol in a concentration range from 0 to 200 µM/L at 24, 48 and 72 h . StudiesAnother neoplastic model of pancreatic cancer—the Capan-2 line—was also used in order to study the effect of resveratrol on cell survival. After 24 h of exposure of the cells to resveratrol , a concentration-dependent decrease in cell viability was observed . The stuThe effect of resveratrol on cell survival was also studied by P. Liu’s team. Three pancreatic tumour lines were used in the study: PANC-1, CFPAC-1 and MIA PaCa-2, as well as normal pancreatic cells derived from tissue collected from a patient during surgery. The cells of the lines tested were exposed for 72 h to various concentrations of resveratrol. An increased cell growth inhibition was observed to take place with an increasing compound concentration. Compared with neoplastic cells, normal pancreatic cells showed greater resistance to the cytotoxic effect of resveratrol . The celMost studies confirm that the higher the dose of resveratrol, the stronger its antiproliferative effect on cells of various pancreatic cancer lines. At the same time, such a strong effect is not observed in normal pancreatic cells, which may translate into resveratrol being an effective and safe treatment option in the future.Apoptosis is a complex physiological process that follows a specific pattern that significantly influences the proper functioning of the organism. This process leads to the removal of unnecessary and damaged cells that could potentially pose a threat to the organism  . Due to For pancreatic cancer, resveratrol has been shown to be proapoptotic in many cases. In the studies of Xian-Zhong Ding’s team, the ability of resveratrol to induce apoptosis was examined by using flow cytometry. The pancreatic tumour cell lines PANC-1 and AsPC-1 were subjected to 72 h of incubation with 100 µM/L resveratrol. After this time, the induction of the apoptotic process was 27% and 34% for PANC-1 and AsPC-1, respectively .Similar tests were performed by Jing Cui et al. The cells of three pancreatic cancer lines  were incubated in resveratrol for 48 h at concentrations ranging from 0 to 200 µM/L. The analysis of the results showed that the induction of the apoptotic process was 26.20% for the PANC-1 cell line. A similar value was found for the BxPC-3 line: 22.08%. As for the AsPC-1 line, the percentage increase in apoptotic cells was only observed at a concentration above 150 µM/L . The teaStudies on resveratrol’s ability to induce the apoptotic process performed on the Capan-2 cancer cell line showed that the number of apoptotic cells after 24 h of incubation with 100 µM/L resveratrol solution increased significantly (from 12.92% to 21.31%) compared with cells not treated with the compound .The effect of resveratrol on apoptosis was also demonstrated by P. Liu’s team. Research shows that microRNAs can act as oncogenes or tumour suppressors; hence, they play an important role in the process of cancer initiation and progression . One of The effect of resveratrol on the activation of the executioner caspase-3, which is normally involved in typical apoptosis, was also studied. The tests were carried out on four pancreatic cell lines: PANC-1, MIA PaCa-2, Hs 766T and AsPC-1. The cells were incubated for 48 h with resveratrol (0–40 µM). Caspase-3 activity, which was measured with the help of a fluorometer, was the lowest for the Hs 766T line, while the highest activity was observed for the PANC-1 and the MIA PaCa-2 lines. For the AsPC-1 line, caspase-3 activity was intermediate in relation to the remaining cell lines. These studies did not include a reference to a normal pancreatic cell line .In conclusion, resveratrol is a proapoptotic compound in human pancreatic cancer, and it acts through many different pathways involved in this process, especially at high doses.Despite the observed reduction in the incidence of stomach cancer, it remains one of the most common neoplasms, especially among men . SimilarSeparate reports from recent years indicate that resveratrol is a compound that is also used in the treatment of gastric cancer .Xiaoping et al. analysed in vitro the effect of resveratrol on the survival of human gastric cancer cells as well as its potential mechanism of action. For this purpose, MGC-803 cells were exposed to different concentrations of resveratrol for 24 and 48 h. In this experiment, resveratrol was shown to inhibit cell growth depending on the dose and the duration of the effect. Accordingly, further experiments were performed to evaluate the effect of the compound on the cell cycle. The cells were incubated with resveratrol . The analysis showed that the treatment with resveratrol led to the inhibition of the cycle in the G0/G1 phase . Other sStudies on the effect of resveratrol on the inhibition of cell growth were also carried out on the gastric cancer model SGC-7901 cell line. A 48 h cell incubation with resveratrol (0–200 µM) resulted in an inhibition of cell proliferation proportional to an increasing concentration of the compound . SimilarFurther studies on the effect of resveratrol on neoplastic cells in gastric cancer were carried out on two cancer cell lines: the aforementioned SGC-7901 line and the BGC-823 line, as well as the benign epithelial cell line GES-1. The cell lines were exposed to various resveratrol concentrations ranging from 0 to 400 µM for 24, 48 and 72 h. A significant reduction in viability was observed in all cell lines tested. These changes were dose- and time-dependent .The proliferative activity of gastric cancer cells is significantly inhibited by resveratrol. The effect of the compound’s action is stronger when the concentration is higher and the exposure time is longer.Studies on the effect of resveratrol on the induction of the apoptotic process were carried out on a gastric cancer cell model: the SGC-7901 cell line. In this experiment, cells were incubated for 48 h with the compound in a concentration ranging from 0 to 200 µM. It was shown that the effect of resveratrol results in an increase in the production of ROS (reactive oxygen species), which according to the literature are involved in the apoptotic process . AdditioAs in pancreatic cancer, resveratrol also has a proapoptotic effect in gastric cancer, and the effect observed is stronger when the concentration of the compound is higher. However, there is less documented research on this tumour model.Liver cancer is also one of the most common cancers worldwide. It is diagnosed more often in men than in women . DespiteThe effect of resveratrol on cell viability was also studied in a liver cell model: the HepG2 line. In the experiment, cells were exposed to resveratrol (0–100 µM) for 24 and 48 h. The results of an MTT test showed a concentration- and time-dependent reduction of the viability in the cell line tested . P.-L. KIn conclusion, the tests performed on liver cancer cell models indicate the antiproliferative properties of resveratrol, primarily the inhibition of the cell cycle. The results are dependent on the concentration and time of the incubation with the compound, as well as the tissue origin of the cells tested.The mechanism of apoptotic induction by resveratrol in liver cancer cells in vitro has not been fully understood yet. However, it is known that the effect depends on the type of cancer lines studied, among which there are differences in the response to the effect of the compound.In the case of HepG2 cells, P.-L. Kuo’s team showed that resveratrol inhibits cell growth and induces the apoptotic process. The level of p53 protein in the HepG2 and Hep3B cell lines was analysed after incubation with resveratrol. In the HepG2 cell line, the expression level of this protein increased along with the increase in resveratrol concentration, while in the Hep3B cell line, no significant changes in p53 expression level were observed. These results provide evidence that, in this case, the proapoptotic effect of resveratrol is mediated by the p53 protein . ProteinIn other studies performed on the HepG2 cell line, the most pronounced effect on the induction of apoptosis was observed (by flow cytometry) at a concentration of 50 µM during a 48 h incubation with the compound .The TUNEL method was used to evaluate the effect of resveratrol on the induction of apoptosis in the liver cancer cell lines HepG2, Bel-7402 and SMMC-7721. After 24 h of incubation with 100 µM resveratrol solution, it was observed that resveratrol increased apoptosis in relation to untreated cells. Moreover, it was shown that resveratrol also influences the apoptotic proteins Bcl-2 (antiapoptotic) and Bax (proapoptotic), which leads to a decrease in the Bcl-2/Bax ratio .The results of all the studies described above suggest that resveratrol may play a significant role in the apoptosis of liver cancer cells by participating in the regulation of the expression of cycle-suppressor and proapoptotic proteins.Intestinal cancer is a common cancer characterised by a high mortality rate all over the world . It mostStudies carried out on the intestinal cancer lines HT-29 and WiDr have shown that the effect of resveratrol leads to an increasingly stronger inhibition of cell proliferation as the concentration of the compound increases .In another experiment, the intestinal adenocarcinoma cell lines HCA-17, SW480 and HT-29 were used. The cell lines were exposed to various concentrations of resveratrol. A concentration- and time-dependent inhibition of the number of cells was demonstrated in all the lines tested .Another team’s studies on the HT-29 cell line showed varying resveratrol effects depending on the concentration after 48 h of incubation with the compound. The MTT test showed that lower concentrations of resveratrol (1 and 10 µM) stimulated cell growth, while higher concentrations (25 and 50 µM) inhibited cell proliferation . By exteThe anticancer properties of resveratrol have also been shown in in vitro studies in which the research model was the HCT-116 and Caco-2 human intestinal cancer cell lines. A proliferation inhibition dependent on resveratrol concentration was observed in both cell lines .In another study performed on the intestinal cancer cell lines HCT-116, CO-115 and SW480, after a 24 h incubation with the compound at concentrations ranging from 0 to 50 µM, it was observed that cell viability decreased as the concentration of the compound increased .The effect of resveratrol on the proliferation of neoplastic intestinal cells was also observed in the DLD1 and HCT15 cell lines. Treatment with resveratrol at concentrations ranging from 0 to 40 µM for 24, 48 and 72 h resulted in a dose- and time-dependent cell growth inhibition .In intestinal cancers, resveratrol may act bi-directionally, depending on the concentration. Higher concentrations of the compound result in a significant inhibition of proliferation in particular cell systems, while lower concentrations can stimulate cell growth.Cyclooxygenase 2 (COX-2) overexpression is associated with physiological and pathological processes in the body, including the formation and progression of neoplasms ,99. One In intestinal cancer, resveratrol also has proapoptotic properties. This effect is most pronounced when higher concentrations of the compound are used.Cancer is a complex disease of various aetiologies, and the use of conventional treatments in most cases is associated with numerous side effects. Hence, the current scientific research focuses largely on the search for new therapeutic strategies, especially ones based on compounds of natural origin. High hopes are placed on the possibility of using these compounds in clinical conditions. The most often studied compounds are the polyphenolic compounds, including resveratrol. Due to the diverse range of biological effects, it is considered a promising anticancer agent.Based on the available scientific literature, in this study we have documented the antitumour effect of resveratrol, especially its role in inhibiting cell proliferation and inducing the apoptotic process.In vitro studies conducted over the last few decades show that resveratrol can be antiproliferative  and proapoptotic in various types of cancer.In this work, we focused on the effects of resveratrol in cancers of the gastrointestinal tract .When analysing the scientific reports presented in our study, the effect of resveratrol is strongly dependent on the type of cancer, the concentration of the compound and the duration of its action . In the The effect of resveratrol in stomach cancer (inhibition of cell proliferation) is also specific to certain cells. Changes in cell survival are observed in a wide range of concentrations (0–400 µM). As happens in pancreatic cancer, resveratrol exerts an inhibitory effect on stomach cancer cells in a time-dependent manner.In liver cancer, the effect of resveratrol on cell proliferation also shows a relationship between the concentration and the duration of the effect. However, in this neoplasm, changes are observed at lower concentrations of the compound (0–100 µM).A similar relationship between the concentration and the duration of the action of the compound and cell survival can be observed in intestinal cancer. Studies evaluating the antiproliferative properties of resveratrol showed a notable inhibition of cell proliferation in intestinal cancer at significantly lower concentrations of the compound (0–50 µM).Many of the in vitro studies presented indicate that the antitumor activity of resveratrol may be largely due to the induction of the apoptotic process. Translating the promising results of these in vitro studies into clinical studies is challenging due to the poor pharmacokinetic parameters of the compound—low bioavailability, poor water solubility and fast metabolism —hence, tIn in vitro studies, resveratrol elicits a weaker or stronger response depending on the type of cancer. However, in each case there is a correlation between the concentration and time of the action and the effectiveness of the compound. The promising results of the in vitro studies presented in this paper, as well as in many others, emphasize the need for further experiments, especially in vivo, on the effect of resveratrol to determine the therapeutic doses of the compound and the exact mechanisms of its action in relation to different types of cancer.