Oridonin Enhances The Anticancer Activity Of NVP-BE235 Against Neuroblastoma Through Enhanced Autophagy

Background and Objective The aberrant activation of PI3K/Akt/m TOR signaling pathway plays an important role in the oncogenesis,prognosis and chemotherapy resistance of neuroblastoma.However,NVP-BEZ235,a potent dual PI3 K and m TOR inhibitor have not shown beneficial effects on neuroblastoma especially in terms of apoptosis induction as a single agent.We therefore attempted to explore an effective combination regimen to enhance the anticancer activity of NVP-BEZ235.Oridonin is a ent ? kaurane diterpenoid extracted from the plant Rabdosia rubescens.Previous studies have been demonstrated that oridonin as a supplement may potentiate the therapeutic effects of anticancer drugs gemcitabine and imatinib.In this research,we used oridonin to combine with NVP-BEZ235,and investigated the effects and molecular mechanism of the co-treatment on neuroblastoma.Methods 1.The effect of NVP-BEZ235 on the proliferation and apoptosis of NB cells was measured by CCK-8 assay and western blot analysis.We next analyzed the effect of NVP-BEZ235 on cell cycle progression in NB cells by flow cytometry using PI staining of DNA content and western blot analysis.2.The effect of oridonin and NVPBEZ235 co-treatment on the proliferation and apoptosis of NB cells was measured by CCK-8 assay,AO/EB double fluorescence stain and western blot analysis.Then,the proliferation and apoptosis index of NB xenograft tumor tissues were also assessed by Ki-67 immunohistochemistry and TUNEL assay after the combination treatment of oridonin and NVPBEZ235.3.The autophagy activity of NB cells was examined after the treatment of VP-BEZ235,oridonin or their combination through western blot.The recombinant p LKO.1 lentivirus expressing sh RNA against Beclin-1 gene were constructed and packaged in vitro with 293 T cells;SHSY-5Y cells were infected with collected virus and the expressions of Beclin-1 were assayed by Western blotting.We then performed flow cytometric analysis of Beclin-1 deficient cells stained with FITC-labeled Annexin V and PI to examine the population level of apoptotic response to the combination treatment.The expression of autophagy and apoptosis-associated proteins in Beclin-1 deficient cells were evaluated through western blot.Results 1.NVP-BEZ235 caused a time-and dose-dependent inhibition of cell proliferation in NB cells,but did not induce NB cell apoptosis.Flow cytometry revealed that the percentage of NB cells in the G0/G1 phase increased significantly and the rate of S and G2/M phase reduced after NVP-BEZ235 treatment;the expression of G1-phase cyclins cyclin D1 and cyclin E1 also reduced remarkably.2.Co-treatment with oridonin and NVPBEZ235 induced enhanced anti-proliferation and cell apoptosis in NB cells.Moreover,the combination of oridonin and NVP-BEZ235 had a significant effect on suppression of NB xenograft tumor growth;the proliferation of tumor tissues measured by Ki-67 immunostaining was markedly reduced and the rate of TUNEL-stained apoptotic cells increased significantly in combination treated mice as compared to vehicle control.3.Apoptotic pathway was activated in response to the combination of oridonin and NVPBEZ235,coming together with its apoptosis-induced effect.Sh RNA-mediated knockdown of Beclin-1 decreased the fraction of apoptotic cells in response to the co-treatment,and the activity of autophagy and the expression of apoptosis-associated proteins reduced.Conclusions 1.NVP-BEZ235 as a single agent can effectively block NB cell proliferation through inhibition of cell cycle progression but not activation of apoptosis.2.The combined treatment of oridonin and NVPBEZ235 led to programmed death of NB cells in a synergistic manner,dramatically enhancing the antitumor efficacy on human NB cells in vitro and in vivo.3.Enhanced autophagy played a key role in the generation of antineoplastic effects of NVP-BEZ235 co-treatment with oridonin in neuroblastoma cells.