The Effect Of Autophagy Inhibitor Chloroquine On Triple-negative Breast Cancer Cells And Its Mechanism

BackgroundDue to the lack of effective therapeutic targets for triple-negative breast cancer(TNBC),the therapy mainly relies on surgery,radiotherapy and chemotherapy.However,the therapeutic effect is not ideal,and the recurrence-free survival and overall survival in patients are low.Therefore,it is urgent to investigate the mechanism of tumorigenesis of triple-negative breast cancer and search for new therapeutic targets and more effective treatments.Autophagy,precisely regulated by a series of autophagy-related genes(ATG),is an evolutionarily conserved process for the turnover of intracellular substances in eukaryotes.Recent researches showed that autophagy plays an important role in tumorigenesis,proliferation,metastasis,energy metabolism,and so on.Exploring the relationship between autophagy and tumorigenesis and development has become an important direction of cancer research.Chloroquine(CQ),a commonly used autophagy inhibitor,has been demonstrated to effectively inhibit the proliferation of tumor cells including melanoma,lymphoma and bladder cancer et al.However,the role and mechanism of CQ in triple-negative breast cancer is not well defined.ObjectiveOn the basis of studying the relationship between autophagy-related genes and triple-negative breast cancer,the effect of chloroquine on the proliferation,cycle distribution,apoptosis,migration and invasion of triple-negative breast cancer cells and its underlying mechanism is investigated.Methods1.Using bioinformatics methodsthe GEO database to to analyze the expression of autophagy-related genes in triple-negative breast cancer tissues.2.Using CRISPR/Cas9 technology to knock out the Beclinl gene in triple-negative breast cancer cells and screening stable monoclonal knockout cells;using CCK-8 assay,clone formation assay and xenograft model to determine the effect of'Beclinl-knockout on the growth of triple-negative breast cancer cells in vitro and Jin vtvo.3.Using CCK-8 assay and clone formation assay to determine the effect of CQ on the proliferation of triple-negative breast cancer cells.4.Using flow cytometry to determine the effect of CQ on cell cycle and apoptosis;using wound healing assay and transwell assay to determine the effect of CQ on migratory and invasive ability.5.Using western blotting to determine the effect of'CQ on the expression of cell cycle,apoptosis,EMT related proteins and autophagy-related markers.Results1.By analyzing the GEO database,we observed a variety of autophagy-related genes were increased in TNBC samples.2.The Beclinl gene was successfully knocked out using CRISPR/Cas9 technology in MDA-MB-231 cells and two stable monoclonal Beclinl-knockout cell lines were screened;Beclinl-knockout significantly inhibited proliferation and colony formation and in vivo tumor growth of MDA-MB-231 cancer cells.3.CQ could effectively inhibit proliferation and colony formation of MDA-MB-231 and HCC1937 cells in vitro.4.Low concentration of CQ induced G0/G1 cell cycle arrest,but high concentration triggered G2/M cell cycle arrest after 24 h in both MDA-MB-231 and HCC1937 cells.CQ obviously induced apoptosis after 48 h.CQ could effectively inhibit the migratory and invasive ability of MDA-MB-231 and HCC1937 cells.5.Compared with the control group,the expression of cell cycle proteins CDK2,CDK4 and CyclinD3 were all decreased in MDA-MB-231 cells after CQ treatment;the expression of P21 in HCC1937 cells was gradually increased;the expression of the cell epithelial marker molecules were up-regulated,and the expression of the mesenchymal marker molecules were down-regulated after CQ treatment.The expression of apoptosis-related proteins and autophagy-related markers were elevated in TNBC cells.ConclusionAutophagy participates in the development and progression of triple-negative breast cancer.Chloroquine,suppressing autophagy,could effectively suppress the proliferation of TNBC cells by inducing cell cycle arrest and apoptosis and affeet the migration and invasion ability through EMT.