The Mechanism Of NVP-BEZ235 Combined With Chloroquine Inhibit The Proliferation And Migration In Triple Negative Breast Cancer Cells

Objective:To explore the inhibitory effect of NVP-BEZ235 combined with CQ on the proliferation and metastasis of triple negative breast cancer cellsMethods:The consequence of NVP-BEZ235 and CQ alone or combination on the proliferation of triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 were detected by MTT assay.The Chou-Talalay method was used to calculate Combination index(CI).Cloning experiments were used to detect cell proliferation ability.Furthermore,immunofluorescence staining experiments were used to observe the formation of autophagosomes in triple negative breast cancer cells after combined therapy.Hoechst33342 staining was used to detect the number of bright blue apoptotic bodies in cancer cells.The scratch healing experiment and the Transwell cell migration experiment were used to detect the effect of combined medication on the horizontal and vertical migration ability of MDA-MB-231 and MDA-MB-468 cells.The expression level of autophagosome marker protein,EMT markers,apoptosis associated proteins,key members of Akt/mTOR signaling pathway-associated proteins were detected by Western bloting assay.Results:1.The consequence of MTT assay showed that compared with the single agent,the combination of NVP-BEZ235 and CQ were more obvious inhibited the ability of proliferation in triple-negative breast cancer cells,also the 48h inhibition rates of MDA-MB-231 and MDA-MB-468 cells were 49%and 47%,respectively(P<0.01);2.The CI values of the two drugs were all less than 1,which had a synergistic effect;3.The results of clonal colony formation experiments showed that the combined application of NVP-BEZ235 and CQ had a more obvious inhibitory effect on the proliferation of triple-negative breast cancer cells compared with the drug alone group(P<0.01);4.The results of immunofluorescence staining experiments showed that the fluorescence particles in the cytoplasm of the combined drug group was significantly increased compared with the single drug group;5.Compared with the single agent group,the number of apoptotic bodies increased gradually by Hoechst33342 staining;6.The Wound healing and Transwell migration assay demonstrated that the horizontal and longitudinal migration ability were supressed in the combination group compared with the single agent group(P<0.05);7.Western bloting experiments showed that the expressions of autophagy marker proteins LC3Ⅱand p-62 in the combination group were significantly up-regulated;the ratio of apoptosis marker proteins Cleared caspase 9,Cleared PARP,Cleared caspase 3 and Bax/Bcl-2 was significantly increased;The expression levels of epithelial marker E-cadherin were significantly up-regulated,and the expression levels of mesenchymal markers Vimentin,Slug,and Snail were down-regulated;the levels of Akt/mTOR signaling pathway-related proteins p-AktSer473,p-4EBP1,and p-S6 were significantly reduced.Conclusions:1.The combination of NVP-BEZ235 and CQ can promote the apoptosis of triple-negative breast cancer cells and inhibit the proliferation and migration of tumor cells by blocking the process of autophagy.2.The mechanism of NVP-BEZ235 combined with CQ against triple-negative breast cancer cells is related to the regulation of Akt/mTOR signaling pathway and EMT process.