ABSTRACT
OBJECTIVE@#To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms.@*METHODS@#We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor).@*RESULTS@#In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis.@*CONCLUSIONS@#ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.
Subject(s)
Female , Humans , Acetates , Pharmacology , Apoptosis , Genetics , Autophagy , Autophagy-Related Proteins , Metabolism , Benzopyrans , Pharmacology , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Gene Expression Regulation , MCF-7 Cells , Morpholines , Pharmacology , Proto-Oncogene Proteins c-akt , Metabolism , Pyrans , Pharmacology , TOR Serine-Threonine Kinases , Metabolism , Triazines , Pharmacology , Ubiquitin-Conjugating Enzymes , MetabolismABSTRACT
Objective To investigate the effect of salinomycin on cancer stem cell formation of prostate cancer cell line DU145 and its possible mechanisms,providing theoretical basis for the clinical application of salino-mycin. Methods (1)DU145 cells were treated with salinomycin. The percentage of ALDH+cells,which was used as the marker of cancer stem cells,was detected by flow cytometry.(2)After treated with salmonin,DU145 cells were subjected to Western-Blot analysis for the expression of mTORsignal pathway-related proteins such as p-70s6k, p-p70s6,p-s6 and so on. 3)DU145 cells were treated with salinomycin combined with mTOR signal pathway inhibi-tor rapamycin,and the ALDH+cancer stem cells were detected using flow cytometer. Results (1)Salmonomycin significantly inhibited ALDH-positive cancer stem cells in DU145cell line(inhibition rate in 77.8%),which was twice as high as that of traditional anticancer drug paclitaxel(which has a inhibition rate of 38.64%). This results suggesting that salinomycin would have the effect of inhibiting cancer stem cells. (2)The expression ofm-TOR p-70s6k,p-p70s6 and p-s6 in mTOR signaling pathway was inhibited by salinomycin in a time-dependent and dose-dependent manner,suggesting that salinomycin would inhibite mTOR signaling pathway.(3)Salinomycin combined with rapamycin can decrease the proportion of ALDH-positive DU145 cancer stem cells(inhibition rate in 77.95%), suggesting that salinomycin may inhibit ALDH-positive DU145 stem cells through the mTOR signaling pathway. Conclusion Salinomycin may play an important role in inhibiting cancer stem cells by inhibiting mTOR pathway signaling.