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Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma.

ABSTRACT: Recent studies have identified that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is an important feature of osteosarcoma, where it promotes cell proliferation, survival, and chemo-resistance. Here, we studied the therapeutic potential of NVP-BEZ235, a novel dual PI3K/mTOR dual inhibitor, on osteosarcoma cells in vivo and in vitro. NVP-BEZ235 was cytotoxic and cytostatic to a panel of osteosarcoma lines (MG-63, U2OS and SaOs-2), where it induce apoptosis and cell-cycle arrest. At the molecular level, NVP-BEZ235 inhibited PI3K-AKT-mTORC1 activation and downregulated cyclin D1/cyclin B1 expressions, while increasing MEK/Erk phosphorylation in osteosarcoma cells. MEK/Erk inhibitors PD98059 and MEK-162 increased NVP-BEZ235 activity on osteosarcoma cells. In vivo, oral NVP-BEZ235 inhibited U2OS xenograft in SCID mice, and its anti-tumor efficiency was further enhanced by MEK-162 co-administration. Taken together, our findings indicate that dual inhibition of PI3K and mTOR with NVP-BEZ235, either alone or in combination with MEK/Erk inhibitors, may be an efficient treatment for osteosarcoma.

SUBMITTER: Zhu YR

PROVIDER: S-EPMC4622008 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Project description:BackgroundThe phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition.Materials and methodsWe investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual ?H2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well.ResultsBoth compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of ?H2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor.ConclusionsThe results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.

Project description:We investigated the effect of 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl} propanenitrile (NVP-BEZ235) (Novartis, Basel Switzerland), a dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor currently being tested in phase I clinical trials, in radiosensitization. NVP-BEZ235 radiosensitized a variety of cancer cell lines, including SQ20B head and neck carcinoma cells and U251 glioblastoma cells. NVP-BEZ235 also increased in vivo radiation response in SQ20B xenografts. Knockdown of Akt1, p110α, or mTOR resulted in radiosensitization, but not to the same degree as with NVP-BEZ235. NVP-BEZ235 interfered with DNA damage repair after radiation as measured by the CometAssay and resolution of phosphorylated H2A histone family member X foci. NVP-BEZ235 abrogated the radiation-induced phosphorylation of both DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated. Knockdown of either p110α or mTOR failed to decrease the phosphorylation of DNA-PKcs, suggesting that the effect of the drug was direct rather than mediated via p110α or mTOR. The treatment of cells with NVP-BEZ235 also promoted autophagy. To assess the importance of this process in radiosensitization, we used the autophagy inhibitors 3-methyladenine and chloroquine and found that either drug increased cell killing after NVP-BEZ235 treatment and radiation. Knocking down the essential autophagy proteins autophagy related 5 (ATG5) and beclin1 increased NVP-BEZ235-mediated radiosensitization. Furthermore, NVP-BEZ235 radiosensitized autophagy-deficient ATG5(-/-) fibroblasts to a greater extent than ATG5(+/+) cells. We conclude that NVP-BEZ235 radiosensitizes cells and induces autophagy by apparently distinct mechanisms. Inhibiting autophagy via pharmacologic or genetic means increases radiation killing after NVP-BEZ235 treatment; hence, autophagy seems to be cytoprotective in this situation. Our data offer a rationale for combining NVP-BEZ235 along with an autophagy inhibitor (i.e., chloroquine) and radiation in future clinical trials.

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Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma.

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