GSDME Reverses The Chemoresistance Of MCF-7/Taxol Cells Via Pyroptosis

Breast cancer is one of the most common malignancies in women,with global morbidity and mortality accounting for 23%and 14%of cancer cases,respectively.At present,surgery combined with radiotherapy and chemotherapy is the main strategy for breast cancer treatment.However,some patients often have tumor treatment failure after a period of chemotherapy,and multidrug resistance?MDR?is an important cause of tumor metastasis and death.Currently,apoptosis and autophagy are two important forms of cell death,and they are also one of the important mechanisms of action of anti-tumor drugs.With the further exploration of the mechanism of resistance to cancer,studies have shown that chemotherapy drugs can also play a role through pyroptosis.Pyroptosis is a programmed death that depends on the formation of plasma membrane pores in members of the gasdermin protein family,also known as cellular inflammatory necrosis.The role of GSDME in regulating tumor cell death in tumor cells has become a hot topic.Shao Feng laboratory research found that if GSDME is highly expressed in cells,activated caspase-3 will induce pyroptosis rather than apoptosis by cleaving GSDME to generate an N-terminal domain.And studies have shown that the reduction of GSDME expression would cause resistance to etoposide to etoposide in certain melanoma cells like Mewo.Paclitaxel?Taxol?is a broad-spectrum chemotherapy drugs,which is commonly used in the treatment of solid tumors such as breast cancer.It is also one of the drugs that can easily induce the resistance of various tumor cells including breast cancer during the treatment.In this study,human breast cancer cell line MCF-7 was used as a parental cell,and a breast cancer anti-MCF-7/Taxol resistant cell model was established by low concentration gradient induction method.The chemoresistance biological characteristics were identified and the expression of the pyroptosis gene GSDME was compared.To study whether GSDME affects the sensitivity of MCF-7/Taxol cells to Taxol through pyroptosis,and provides new therapeutic ideas and experimental basis for clinical reversal of breast cancer resistance.ObjectiveTo investigate whether GSDME can affect the resistance of breast cancer cells to Taxol by regulating the occurrence of pyroptosis.Methods1.Establishment of MCF-7/Taxol resistant strains by Taxol low concentration gradient induction.2.Experimental group:MCF-7 group and MCF-7/Taxol group;si-NC group,si-GSDME group,si-NC+Taxol group and si-GSDME+Taxol group;Ad-NC group,Ad-GSDME group,Ad-NC+Taxol group and Ad-GSDME+Taxol group.3.The proliferation of MCF-7 cells was determined by CCK-8 assay,and the IC₅₀ concentration of Taxol was calculated.MCF-7/Taxol resistant cells were established by low concentration gradient induction method,and the establishment of chemoresistant cells was identified by CCK-8,cell counting,transmission electron microscopy,flow cytometry,real-time fluorescence quantitative PCR?qRT-PCR?and Western blot.To determine whether it is a multidrug resistance cell model and the expression of pyroptosis gene GSDME.4.Transfection of small interfering RNA negative control?si-NC?and two GSDEM small interfering RNA?si-GSDME?into MCF-7 cells by Lipofectamine3000 transfection method,and then treated with Taxol for 24h.The results of transfection were detected by qRT-PCR,and 2si-?GSDME1+GSDME2?was finally selected to be the experimental group.Same as described above,Ad-NC and Ad-GSDME were also transfected into MCF-7/Taxol cells by Lipofectamine 3000transfection,then treated with Taxol for 24 h,and the results of transfection were detected by qRT-PCR and Western Blot.The sensitivity of Taxol was detected by CCK-8,the change of pyroptosis was detected by flow cytometry,the release of LDH was detected by LDH release test,and the GSDME-F,GSDME-N and cleaved-caspase-3 proteins were detected by Western Blot.Results1 The successful establishment of anti-paclitaxel MCF-7/Taxol resistant cells in breast cancerThe drug sensitivity of Taxol in MCF-7/Taxol cells was significantly higher than that in MCF-7 cells at 24 h,48 h and 72 h?P?0.01?.Compared with MCF-7 cells,the G1 phase was prolonged,while the S phase was shortened in the cell cycle of MCF-7/Taxol cells?P?0.01?.The surface microvilli decreased,the nuclear-to-plasma ratio increased,the nucleolar enlargement increased in the transmission electron microscope,and the relative expression of the classical drug-resistant MDR1mRNA and P-gP protein both increased?P?0.01?.2 GSDME is low expressed in MCF-7/Taxol cellsFurther studies by qRT-PCR and Western Blot showed that compared with MCF-7 cells,the expression of GSDME mRNA and protein in MCF-7/Taxol cells was significantly decreased?P?0.01?.3 Taxol induces pyroptosis in breast cancer MCF-7 cellsAfter treatment with Taxol for 24 h,the incidence of pyroptosis,LDH release,GSDME-N and cleaved-caspase-3 protein were significantly increased in MCF-7cells compared with MCF-7/Taxol cells?P?0.01?.Under the inverted phase contrast microscope,the cell membrane is swollen and ruptured,and characteristic large bubbles appear from the cytoplasm.These results indicate that Taxol induces pyroptosis in breast cancer cells,and the occurrence is associated with GSDME expression levels.4 Knockdown of MCF-7 cells by GSDME on pyroptosis and drug sensitivityAfter transfection of si-GSDME into MCF-7/Taxol cells by RNAi technology,the expression of GSDME mRNA and protein in si-GSDME group was significantly lower than that in si-NC group?P?0.01?,and the sensitivity of cells to Taxol was significantly reduced.?P?0.01?.Compared with si-NC+Taxol group,the pyroptosis rate,LDH release level,GSDME-N and cleaved-caspase-3 protein were significantly decreased in the si-GSDME+Taxol group?P?0.01?.5 Effects of GSDME overexpressing MCF-7/Taxol cells on pyroptosis and drug sensitivityAfter transfection of Ad-GSDME into MCF-7/Taxol cells by liposome technique,the expression of GSDME mRNA and protein in Ad-GSDME group was significantly higher than that in Ad-NC group?P?0.01?,and cells were more sensitive to Taxol?P?0.01?.Compared with Ad-NC+Taxol group,the pyroptosis rate,LDH release level,GSDME-N and cleaved-caspase-3 protein in Ad-GSDME+Taxol group were significantly increased?P?0.01?.ConclusionIn conclusion,Taxol induced pyroptosis in breast cancer MCF-7 cells.GSDME can reverse the chemoresistance of paclitaxel-resistant MCF-7/Taxol cells in breast cancer by increasing pyroptosis.