Inhibition Of Autophagy Enhances Apoptosis Induced By The PI3K/mTOR Inhibitor NVP-BEZ235in Glioblastoma U251Cells

Background: Glioblastoma is the most aggressive and the most frequent common tumor,comprising approximately50%of the cerebral gliomas. Surgical removal of the tumor is thefirst-line therapy. Unfortunately, these tumor cells are highly mobile and usually infiltrate thesurrounding tissues. Therefore, surgery has to be followed by chemotherapy and radiationtherapy to further reduce the number of remaining tumor cells. Despite recent advances in allthese therapies, the response is still very poor, and the median survival time for patients withglioblastoma remains about12months. Therefore, novel therapeutic strategies are urgentlyneeded.The PI3K/AKT/mTOR signaling pathway has been focused in recent years due to itspotential role in cancer. The PI3K/AKT/mTOR signaling pathway acts as a convergence fornumerous upstream signals and stimulates the activity of a multitude of downstream effectorsin turn, thereby mediating cellular survival, growth, and so on. Therefore, it is not surprisingthat aberrant and deregulation activation of this signaling is a common molecular event inmany malignancy types, include glioblastoma, making them an attractive target foranti-cancer therapy.Whereas, apart from participation in cell proliferation, PI3K/AKT/mTOR pathway alsoinhibits cell autophagy, which would protect cell against apoptosis caused bychemotherapeutic drugs. Autophagy is a cellular lysosomal degradation pathway that isessential for regulation of cells survival and death to maintain cellular homeostasis. One ofthe key regulators of autophagy is mTOR. Inhibition of mTOR signaling (e.g., by the mTORinhibitor rapamycin) induces autophagy. Autophagy can be either a pro-survival or deathmechanism depending on the circumstances, thus generating variable impact on the outcomeof cancer therapy.NVP-BEZ235, a novel duel PI3K/mTOR inhibitor, showed great antitumor benefit andprovided a treatment strategy in malignant.Objective: We take the hypothesis that whether combination use of PI3K/mTORinhibitor NVP-BEZ235and autophagy nhibitors would enhance the apoptosis of glioblastomaU251cells induced by NVP-BEZ235.Method: The effect of NVP-BEZ235with different densities on U251cells viability wasmeasured by MTT assay. The effect of NVP-BEZ235with different densities on cell nucleus of U251was detected by Hoechst staining. The expression of apoptosis-related proteinscaspase-3and cytochrome C were determined by Western blot analysis. The expression ofautophagy apoptosis-related proteins LC3, Beclin1and p62were also determined by Westernblot analysis. Furthermore, the combinated effect of NVP-BEZ235and autophagy inhibitor3-MA or lysosome inhibitor CQ on the viability and apoptosis of U251cells were detected.Result:1. NVP-BEZ235inhibited the growth of U251cells.2. The inhibit effectinduced by NVP-BEZ235is dose dependent and time dependent.3. NVP-BEZ235enhancedthe activation of apoptosis related protein caspase-3and the expression of cytochrome C inU251cells.4. NVP-BEZ235regulated the expressions of autophage related proteins LC3,Beclin1and p62in U251cells.5. Combination of NVP-BEZ235and autophagy inhibitor3-MA or lysosome inhibitor CQ enhanced the apoptosis in U251cells induced byNVP-BEZ235.Conclusion: Autophagy inhibition enhanced the apoptosis of glioblastoma U251cellinduced by PI3K/mTOR dual inhibitor NVP-BEZ235’s anticancer efficacy.