Liraglutide Induces Pyroptosis Of Human Breast Cancer MCF-7 Cells Through Regulation Of Autophagy-CTSB-inflammasome Axis

Objective: Diabetes and breast cancer have become important causes of death in China and around the world.In recent years,an increasing number of epidemiological data indicate that diabetes is positively correlated with the risk of breast cancer.Liraglutide,a glucagon-like peptide-1(GLP-1)analogue,has been widely used as a new antidiabetic agent in the treatment of diabetic patients.It has been reported that in addition to its hypoglycemic effect,liraglutide can reduce the risk of various malignancies,such as breast cancer.Studies have found that liraglutide could damage breast cancer cells by regulating the expression of inflammatory cytokines and inflammation-related signaling pathways,but the underlying mechanism has not been fully elucidated.This study intends to investigate the effect of liraglutide on proliferation and migration of breast cancer line MCF-7 cells and its possible molecular mechanism from the perspective of pyroptosis.Methods: In this study,MCF-7 cells were selected as the research object.The effect of liraglutide on the proliferation rate of MCF-7 cells was detected by MTT assay.Transwell assay was used to detect the effect of liraglutide on cell migration ability.Hoechst 33342/PI staining assay was performed to observe the integrity of the cell membrane.Western blot was used to detect the expression of NLRP3,caspase-1,IL-1? and autophagy-related proteins LC3 and P62 in MCF-7 cells.Pyroptosis inhibitor glycine was used to detect the occurrence of pyroptosis in MCF-7 cells.The q RT-PCR assay was used to analyze the relative expression levels of caspase-1 and IL-1? m RNA.The autophagosomes of MCF-7 cells were observed by transmission electron microscopy(TEM).Acridine orange(AO)staining was used to label acidic vesicular organelles(AVOs)structures.Moreover,the roles of NLRP3 inflammasome,CTSB and autophagy in liraglutide-induced MCF-7 cells pyroptosis were investigated using NLRP3 inhibitor MCC950,CTSB inhibitor CA-074 Me,autophagy inhibitor 3MA and autophagy inducer Rapa.Results: Liraglutide inhibited the proliferation and migration of MCF-7 cells in a dose-dependent manner.The effective condition of liraglutide detected by MTT assay was 400 nmol/L for 48 hours.Liraglutide induces MCF-7 cells pyroptosis: the expression levels of NLRP3,caspase-1 p20,IL-1? protein and m RNA,the ratio of PI-positive MCF-7 cells were significantly increased relative to control,and pyroptosis inhibitor glycine could attenuate liraglutide-induced pyroptosis.Further studies showed that liraglutide-induced pyroptosis and migration suppress depends on the activation of NLRP3 inflammasome in MCF-7 cells: liraglutide treatment resulted in a significant increase in the expression of caspase-1 p20 and IL-1? protein and m RNA,however,protein and m RNA expression of caspase-1 p20 and IL-1? and the above indicators of pyroptosis were significantly decreased after treatment with NLRP3 inhibitor MCC950,and the ability of cells to migrate increased significantly.CTSB mediatesliraglutide-stimulated the activation of NLRP3 inflammasome and pyroptosis and inhibition of cell migration: western blot and immunofluorescent staining of CTSB results showed that CTSB,inflammation-related proteins and PI intake increased,and migration decreased;but the above indicators were significantly inhibited by CTSB inhibitors CA-074 Me,suggests that the liraglutide can induce MCF-7 cell release CTSB into the cytoplasm and activate the NLRP3 inflammasome and pyroptosis.Liraglutide-induced autophagy was involved in the release of CTSB,activation of NLRP3 inflammasome,pyroptosis and suppression of cell migration in MCF-7 cells: Western blot,TEM and AO staining results showed that liraglutide enhanced autophagy in MCF-7 cells,but the autophagy inhibitor 3-MA pretreatment results showed that the expression levels of cytoplasmic CTSB,NLRP3,caspase-1 p20 and IL-1? protein were significantly reduced,and Rapa reversed this trend after activating autophagy,suggesting that liraglutide-induced autophagy plays an important role in pyroptosis and cell migration via regulating the release of CTSB in MCF-7 cells.Conclusion: Liraglutide upregulated the level of autophagy,degraded autophagic lysosome and released CTSB into cytoplasm,and further activate the NLRP3 inflammasome and pyroptosis.This study elaborated that autophagy-CTSB-NLRP3 inflammasome signaling pathway plays an important role in liraglutide-induced pyroptosis in MCF-7 cells,providing theoretical basis for further study on the internal mechanism of liraglutide's anti-breast cancer effect.