P5-06-07: The Mechanisms of Autophagy Involved in the Effects of Everolimus (RAD001) on Overcoming Multidrug Resistance of Breast Cancer Cell Line MCF-7/ADR.
Authors: Liu Z-B , Hou YF , Xu W-H , Ling H , Shao Z-M 
Published: June 2012

To investigate the effects of everolimus on overcoming multidrug resistance of breast cancer cell line MCF-7/ADR and to explore its mechanisms of action. MCF-7 and MCF-7/ADR cells were treated with ADM, everolimus alone or by combination at different concentrations and different times. Changes of cell morphology were observed by microscopy. Apoptosis induced by ADM or everolimus were measured by flow cytometry with PI staining and ANNEXIN V-FITC staining. Western blot was used to evaluate the expression level of apoptosis and autophagy-related molecules. To investigate specifically the role of autophagy in everolimus treated breast cancer cell lines, we used a siRNA approach, targeting autophagy gene, ATG5. The CI for each experimental combination was calculated. Dose-dependent inhibition of cell proliferation was observed with ADM and everolimus for both 24 and 48h. MCF-7/ADR was resistant to ADM. The addition of everolimusdid increase the sensitivity of MCF-7/ADM cells to ADM, as indicated by the ID50 by 2.6 fold for 24h; but for 48h, everolimus is even able to reduce the ID50 of ADM much higher to 5–10 times. Thus, inhibition of mTOR does appear to restore ADM sensitivity in MCF-7/ADR cells, especially for a longer synergistic time (48h). Everolimus alone induced a decrease in the phosphorylation of two downstream molecules of mTOR, including p70S6K and 4E-BP1, confirming the role of everolimus in mTOR signaling regulation, whereas everolimus in combination with ADM caused an almost complete suppression of p70S6K and 4E-BP1 phosphorylation. When cells were exposed to both everolimus and ADM for 24h, there was no loss of procaspase-3 nor was there a concentration-dependent increase in cleaved caspase protein. On the contrary, ADM administration induced the proteolytic cleavage of caspase 3, and when ADM was combined with everolimus, a further increase in the expression of cleaved caspase 3 was detected in 48h. Cells treated with CQ markedly enhanced ADM-induced cell death in MCF7/ADR cells in early stage (24h). As observed with immunoblot analysis, cotreatment of everolimus and ADM increased expression level of ATG5 and LC3-II to -I ratios, also accelerated the rate of cell death. That's why cotreatment with CQ resulted in a rapid drop in cell viability with complete cell death observed by CCK-8 test. Immunoblot analysis also revealed non significant change of ATG5 and LC3-II within 48h of everolimus on the base of ADM treatment. Silencing ATG5 resulted in a marked downregulation of ATG5 but increased the level of proteolytic cleavage of caspase 3 induced by ADM alone and cotreatment of everolimus and ADM when cells were exposed to both everolimus and ADM for 24h. This result was consistent with our finding using the Annexin-V apoptotic assay. For 48h, the results of proteolytic cleavage of caspase 3 were similar as when cells weren't silenced by ATG5 siRNA. In conclusion, autophagy plays bidirectional roles in the mechanisms of tumor cells suppression of everolimus combining MCF-7/ADR. Especially in late stage, inhibiting autophagy helps to overcoming resistance of everolimus and sensitizes cancer cells to ADM-induced apoptosis.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-07.