Inhibiting Autophagy Enhances PA-MSHA Lethality For Human Breast Cancer Cells

ObjectiveTo investigate the effects of ER stress and autophagy on PA-MSHA (Pseudomonas aeruginosa-mannose sensitive hemagglutinin) inhibiting proliferation and inducing apoptosis of breast cancer cell lines and the mechanism, and to explore the detailed mechanisms of action in human breast cancer cells both in vitro and in vivo.MethodsPart1:MDA-MB-231HM cells were treated with PA-MSHA at different concentrations and different times. Changes of cell super-microstructure were observed by transmission electron microscopy. Western blot was used to evaluate the expression level of ER stress-related molecules and autophagy-related molecules.Part2:MDA-MB-231,MDA-MB-231HM and MDA-MB-468were treated with PA-MSHA,3-MA and changes of cells were observed by microscope. Apoptosis was measured by flow cytometry (FCM) with PI staining and ANNEXIN V-FITC staining. shRNA was used to downregulate IRE1expression to determine whether IRE1expression is critical for the inducing of autophagy by PA-MSHA on breast cancer cells. Apoptosis induced by PA-MSHA,3-MA was measured by flow cytometry (FCM) with PI staining, ANNEXTN V-FITC staining and Hoechst33258staining under fluorescence microscopy. Western blot was used to evaluate the expression level of autophagy-related molecules.Part3:2×106MDA-MB-231HM-shCON cells and MDA-MB-231HM-shIRE1cells suspended in0.1ml PBS were implanted into the mammary fat pad of mice. These mice were given0.1ml PA-MSHA(2.2×1010cells/ml) or PBS s.c treatment daily. Tumor volume and apoptosis was measured. ResultsPart1:A time-dependent and concentration-dependent ER stress and autophagy of PA-MSHA was observed in MDA-MB-231HM cells.Part2:Microscope, FCM with PI staining and ANNEXIN V-FITC showed that PA-MSHA together with3-MA could induce apoptosis in breast cancer cells stronger than PA-MSHA or3-MA alone. More apoptosis can be induced by PA-MSHA in MDA-MB-231and MDA-MB-231HM cells transfected with shIRE1.Part3:In vivo, PA-MSHA treatment significantly suppressed mammary tumorigenesis in xenograft in MDA-MB-231H-shIRE1cell-transplanted mice. Tumor sample analyses by TUNEL confirmed induction of stronger apoptosis in MDA-MB-231HM-shIRE1cell transplanted mice than control group.ConclusionsPA-MSHA may induce ER stress and autophagy while treating breast cancer cell lines. The involvement of the IRE1pathway plays a critical role in the induction of autophagy via ER stress. Inhibition of IRE1or combination usage of chemical autophagy inhibitors can enhance the cytotoxicity and apoptosis induced by PA-MSHA.