The Crosstalk Mechanisms Of Cell Autophagy Initiated By SAHA-CTSB In Breast Cancer Cells

ObjectiveBreast cancer(BC)is one of the most common malignant tumors in women,and the age of onset is mainly in 40?60 years-old women in perimenopausal period.In Europe,the United States and other developed countries,the incidence of breast cancer is ranked the first position in women with malignant tumors.In recent years,with the acceleration of city construction and increase of the life pressure,the incidence rate of breast cancers in China is increasing year by year,and the illness age is more and more lowered.It affects women's health.Acetylation and deacetylation of histone proteins play an important role in the regulation of gene expression in eukaryotes.The acetylation state of chromatin can lead to the expressions of tumor suppressor genes and pro-apoptotic genes.As a Histone deacetylase inhibitors(Histone Deacetylase Inhibitor,HDACi),SAHA(Suberoylanilide Hydroxamic Acid,SAHA)can increase the acetylation status in specific chromatin regions,thereby affecting gene expressions.It has become a new class of anticancer drugs.A large number of studies have found that SAHA can result in growth arrest and death in a variety of tumors,such as MCF-7,MDA-MB-231,MDA-MB-435 and SKBr-3 breast cancer cell lines.Cathepsin B(CTSB)is a lysosomal cysteine protease.Recent studies showed that CTSB expression is involved in the cell proliferation,invasion,metastasis and angiogenesis in tumor cells(breast cancer,ovarian cancer,bladder cancer,lung cancer,colon cancer,melanoma,gastric cancer).Therefore,the overexpression of CTSB in tumor cells is thought to be associated with aggressive and poor prognosis.However,some studies have shown that the expression of CTSB may play an important role in apoptosis and autophagy in some tumor cells.Autophagy is a biological effect that can eliminate the abnormal proteins and aging organelles in the body.It is beneficial to maintain the homeostasis for the cell.In hypoxia,energy deficiency,inflammation and other stress conditions,autophagy can inhibit the functions of toxic or carcinogenic proteins,repress the organelle aggregation,protect cells and depress the carcinogenesis.Furthermore,autophagy can supply unlimited nutrients for tumor growth and promote the proliferation of cancer cells.In recent years,with the deepening research on autophagy,scholars have found that autophagy play an important role in the treatment and prevention of breast cancer,and more and more attention has been paid to it.The purpose of this study is to investigate the role of SAHA in the proliferation of two breast cancer cells MCF-7 and MDA-MB-231,and to assess the expressions of CTSB in breast cancer cells.Belong to the members of lysosomal cysteine protease,CTSB can mediate breast carcinogenesis by autophagy.The study will construct a good experimental basis on breast cancer proliferation regulated by the crosstalk between SAHA-CTSB and other signal pathway,but also provide a theoretical basis on the novel target of gene therapy in breast cancer treatment.Methods1.Culture MCF-7 and MDA-MB-231 breast cancer cells.2.Utilize RTCA to monitor the proliferation changes by SAHA treatment in MCF-7 and MDA-MB-231 cells.3.Employ BioStationIM time-lase live cell imaging acquisition to measure the cell proliferation changes by SAHA treatment in MCF-7 and MDA-MB-231 cells.4.Use CTSB inhibitor Cystatin C to knock down the function of CTSB.5.Employ Western Blot,ELISA and In Cell Western to measure the expression of CTSB and screen the optimal concentrations of Cystatin C.6.Employ MuseTM to analyze the cell viability in MCF-7 and MDA-MB-231 cells with SAHA and Cystatin C treatment.7.Employ immunocytochemistry to measure the expression of LC311 in MCF-7 and MDA-MB-231 cells with SAHA and Cystatin C treatment.8.Analyze the expression of autophagy-related factors in breast cancer with SAHA and Cystatin C treatment by RT-QPCR Array and Western Blot.9.Analyze the expression of MAPK signaling pathway in MCF-7 and MDA-MB-231 cells with SAHA and Cystatin C treatment by MAPK antibody Array.Results1.RTCA and BioStationIM results showed that SAHA could inhibit the proliferation of breast cancer cell line MCF-7 and MDA-MB-231.2.Western Blot,ELISA,In Cell Western and Muse analysis showed that the high expression of CTSB was accompanied by the SAHA inhibitory effect in breast cancer cells.Contrarily,blocking-up the expression of CTSB can reverse the effect of SAHA.3.Immunofluorescence,RT-QPCR Array,Western Blot and MAPK antibody Array showed that SAHA mediated the autophagy by activating CTSB in breast cancer cells,and it was associated with MAPK signaling pathway.Conclusion1.SAHA can inhibit the proliferation of MCF-7 and MDA-MB-231 breast cells with the high expression of CTSB.2.The blocking-up the expression of CTSB can inhibit the anti-tumor effect of SAHA.3.SAHA mediates the autophagy by activating CTSB in breast cancer cells,and it is associated with MAPK signaling pathway.