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Inhibition of autophagy as a strategy to augment radiosensitization by the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.

ABSTRACT: 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, p110?, 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 p110? or mTOR failed to decrease the phosphorylation of DNA-PKcs, suggesting that the effect of the drug was direct rather than mediated via p110? 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.

SUBMITTER: Cerniglia GJ

PROVIDER: S-EPMC3502620 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Inhibition of autophagy as a strategy to augment radiosensitization by the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.

Cerniglia George J GJ Karar Jayashree J Tyagi Sonia S Christofidou-Solomidou Melpo M Rengan Ramesh R Koumenis Constantinos C Maity Amit A

Molecular pharmacology 20120918 6

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 al ...[more]

PMID: 22989521

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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.

Dataset Information

Inhibition of autophagy as a strategy to augment radiosensitization by the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.

Publications

Inhibition of autophagy as a strategy to augment radiosensitization by the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.

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