Study On The Effect Of Autophagy On Osteosarcoma Cell Toxicity Induced By PI3K/mTOR Inhibitor NVP-BEZ235

Phosphatidylinositol-3- kinase/protein kinase B/the mammalian target of Rapamycin(PI3K/Akt/m TOR) is an important signaling pathway for tumor cell survival and apoptosis. Dysregulation of the mammalian target of PI3K/AKT/m TOR signaling has been found in many human cancers, including osteosarcoma. NVP-BEZ235 is a novel,dual PI3K/m TOR inhibitor that has exhibited promising activity against cancer cell. Autophagy is a cellular lysosomal degradation pathway essential for regulation of cell survival and death to maintain homeostasis.This process is negatively regulated by mammalian target of m TOR signaling and often counteracts efficacy of certain cancer therapeutic agents. Inhibition of autophagy may enhance NVP-BEZ235 induced apoptosis. Therefore, we investigated the role of autophagy in NVP-BEZ235 induced apoptosis of osteosarcoma cells, and the relevant molecular mechanisms.Methods(1) The effect of NVP-BEZ235 and(or) autophagy inhibitor 3-MA on the survival rate of U2 OS osteosarcoma cell line was detected by MTT.(2) The expressions of PI3K/Akt m TOR signaling pathway associated protein(Akt and P70S6K), apoptosis associated protein(cytochrome c, caspase-3 and PARP) and autophagy related protein(LC3 and Beclin 1 and p62) were detected by immunoblot method(Western blot)(3) Cell apoptosis was detected by flow cytometry following Annexin VFITC/ PI double staining.(4) Mitochondrial membrane potential was detected by flow cytometry following JC-1 staining.(5) The effects of autophagy inhibition on NVP-BEZ235 induced cytotoxicity in U2 OS cells were examined by transfection of si RNA Atg5 and autophagy inhibitor 3-MA.Results(1) Compared with adjacent normal tissues, the expression levels of p-AKT and p-p70S6 K were significantly increased in osteosarcoma tissues.(2) NVP-BEZ235 decreased the survival rate of U2 OS cells in a dose-dependent and time-dependent manner(3) NVP-BEZ235 could significantly increase the expression of cell apoptosis pathway relevant protein(cytochrome C cleaved caspase-3and cleaved PARP) and increase the apoptosis rate in U2 OS cells.(4) NVP-BEZ235 increased the expression of LC3 and Beclin 1, and down regulated the expression of p62.(5) Atg5 si RNA transfection and autophagy inhibitor 3-MA significantly enhanced the decrease of the cell survival rate and apoptosis induced by NVP-BEZ235.Conclusion:NVP-BEZ235 induced apoptosis of osteosarcoma cells and induced autophagy in osteosarcoma cells. And inhibition of autophagy can significantly enhance the cytotoxic action of NVP-BEZ235 in osteosarcoma cells. Therefore, induction of autophagy by NVP-BEZ235 might appear to be a survival mechanism that may counteract its anticancer effects, and blockade of autophagy could enhance NVP-BEZ235’s anticancer efficacy. Thus, our studies provide rationale for clinical development of combinations of NVP-BEZ235 and autophagy inhibitors for the treatment of HCC and other malignancy.