DNA Methylation Regulates GSDME Expression In MCF-7/Taxol Cells And Its Role In Chemotherapy Resistance

Breast cancer（BC）is the highest incidence of cancer in women worldwide and one of the leading causes of cancer-related death worldwide.The main strategy for clinical treatment of BC includes surgery combined with radiotherapy and chemotherapy.Most patients have some improvement after treatment.However,with the widespread use of chemotherapy drugs,patients have developed drug-resistance.Paclitaxel（Taxol）is one of the most widely used chemotherapeutics for breast cancer patients.However,the development of drug-resistance greatly reduces the effectiveness of treatment.Therefore,it is urgent to explore the molecular mechanism of potential drug-resistance and enhance the sensitivity of BC patients to chemotherapy drugs.BC is a heterogeneous disease with extensive genetic mutations and chromosomal abnormalities.The occurrence and development of BC involve heredity and epigenetics.The latter includes DNA methylation,chromosome remodeling,non-coding RNA regulation,and histone modification,among which DNA methylation is the most important epigenetic modification.Studies have found that DNA methylation causes most tumor cells to silence tumor suppressor genes or/and activate oncogenes,which interferes with tumor cell death.Moreover,abnormal DNA methylation is not only involved in the occurrence of human cancers but also related to chemotherapeutic drug-resistance.Chemotherapeutic drugs can improve the therapeutic effect of tumor cells with high GSDME expression by inducing pyroptosis.Therefore,targeting GSDME-mediated pyroptosis may improve the chemotherapy sensitivity of cancer cells.5-Aza-2-deoxycytidine（decitabine）is an inhibitor of DNA methyltransferases（DNMTs）,which enters DNA during the S phase of the cell cycle and affects gene expression by changing epigenetic patterns.Studies have found that decitabine can improve the sensitivity of tumor cells to conventional chemotherapy drugs.It has been clinically used to treat myelodysplastic syndrome（MDS）and hematological malignancies.Therefore,epigenetic therapy with decitabine may be a promising strategy for cancer treatment.Pyroptosis is a programmed cell death mediated by the Gasdermin family.In recent years,GSDME,one of the family members,has become the focus of oncology research.Stimulation of chemotherapeutic drugs causes tumor cells with a high expression of GSDME to undergo pyroptosis and causes tumor cells with a low expression of GSDME to undergo apoptosis.In terms of killing cancer cells,pyroptosis is more destructive than apoptosis.Therefore,loss of GSDME expression leads to cancer cells resistant to chemotherapy agents.In this study,the human breast cancer sensitive cell MCF-7 was used as the research object,and the breast cancer Taxol-resistant cell line MCF-7/Taxol was established by the low-concentration gradient induction method.By comparing the relationship between the expression of GSDME and epigenetic changes in the two cells,to explore whether DNA methylation affects the expression of GSDME,which in turn affects the chemotherapy sensitivity of MCF-7/Taxol cells to Taxol.ObjectiveTo investigate the mechanism of DNA methylation regulating the expression of GSDME in MCF-7/Taxol cells and its relationship with chemotherapy resistance.Methods1.Construction of MCF-7/Taxol resistant cell line by low concentration gradient induction method.2.Experimental group:MCF-7 group（Sensitive cells）and MCF-7/Taxol group（Drug-resistant cells）;MCF-7 group（Sensitive cells）,MCF-7/Taxol group（Drug-resistant cells）and MCF-7/Taxol+DAC group（Cells treated with decitabine）.3.The proliferation of MCF-7 cells and MCF-7/Taxol cells was determined by cell counting kit-8（CCK-8）assay,and the IC₅₀ concentration of Taxol was calculated.MCF-7/Taxol resistant cells were established by a low concentration gradient induction method,and the sensitivity of Taxol was identified by CCK-8,quantitative real-time PCR（q RT-PCR）,and Western blotting（WB）.The expression level of GSDME in breast cancer cells was observed by q PCR and WB analyses.Epigenetic changes were detected by Methylated DNA immunoprecipitation-sequencing（Me DIP-seq）and methylation-specific PCR（Me DIP-PCR）.4.Decitabine was used to stimulate the drug-resistant cells（MCF-7/Taxol+DAC）.Breast cancer cells were divided into sensitive group,drug-resistant group,and decitabine treatment group.CCK-8 assay was used to detect cell proliferation.pyroptosis was detected by LDH assay,flow cytometry,and WB analyses.The colony-forming assay was used to detect cell proliferation.Cell scratch healing test was used to detect cell migration.Transwell experiment was used to detect cell invasion.Results1 MCF-7/Taxol cells are highly resistant to Taxol.Compared with MCF-7 cells,the resistance index of Taxol in MCF-7/Taxol cells was significantly higher（P≤0.001）.Compared with MCF-7 cells,the expression levels of ABCB1 m RNA and PGP protein in MCF-7/Taxol cells were significantly increased（P≤0.001）.2 The expression of GSDME is regulated by epigenetic mechanisms in MCF-7/Taxol cellsCompared with MCF-7 cells,the expression of GSDME was significantly reduced in MCF-7/Taxol cells（P≤0.01）;Compared with MCF-7 cells,the methylation level of GSDME in the enhancer region was increased significantly in MCF-7/Taxol cells（P≤0.05）;Compared with MCF-7/Taxol cells,the expression level of GSDME was increased significantly in MCF-7/Taxol+DAC cells（P≤0.001）.3 Taxol induces pyroptosis in breast cancer MCF-7 cells and MCF-7/Taxol+DAC cellsAfter treatment with Taxol for 24 h,the LDH release level,the incidence of pyroptosis,and the expression of GSDME-N active fragments were significantly decreased in MCF-7/Taxol cells compared with MCF-7 cells（P≤0.001）.After treatment with Taxol for 24 h,the LDH release level,the incidence of pyroptosis,and the expression of GSDME-N active fragments were significantly increased in MCF-7/Taxol+DAC cells compared with MCF-7/Taxol cells（P≤0.001）.These results indicate that Taxol induces pyroptosis in breast cancer cells,and the occurrence is associated with the expression levels of GSDME.4 Combined treatment with decitabine and Taxol enhances the chemotherapy sensitivity of MCF-7/Taxol cellsCompared with MCF-7 cells,the cell growth inhibition rate of MCF-7/Taxol cells was significantly reduced（P≤0.001）.Compared with MCF-7/Taxol cells,the cell growth inhibition rate of MCF-7/Taxol+DAC cells was significantly increased（P≤0001）.These results suggest that combined treatment with decitabine and Taxol could enhance the chemosensitivity of MCF-7/Taxol cells.5 Decitabine treatment affects the biological function of MCF-7/Taxol cellsCompared with MCF-7 cells,the colony forming ability,the migration ability,and the invasion ability of MCF-7/Taxol cells were significantly increased（P≤0.001）.Compared with MCF-7/Taxol cells,the colony forming ability,the migration ability,and the invasion ability of MCF-7/Taxol+DAC cells were significantly reduced（P≤0.001）.The above results indicate that decitabine treatment can change the biological function of MCF-7/Taxol cells.ConclusionThe low-concentration gradient induction method was used to construct breast cancer Taxol-resistant cell line MCF-7/Taxol and identified it as a highly resistant model.The expression of GSDME was highly in MCF-7 sensitive cells,and cells die in the form of pyroptosis with the treatment of Taxol;The expression of GSDME was decreased in MCF-7/Taxol resistant cells due to DNA methylation,which leads to suppression of pyroptosis and shows resistance to Taxol.Demethylation agent decitabine treatment of drug-resistant cells increases the expression of GSDME,which can induce pyroptosis and increase the sensitivity of cells to Taxol.Combined treatment with Decitabine and Taxol may be a new method to overcome the resistance of MCF-7/Taxol cells to Taxol.