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, 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.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Institutes of Health National Institute of Cancer [Grants R01-CA093638, R01-CA133470].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- PI3K
- phosphatidylinositol 3-kinase
- NVP-BEZ235
- 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
- ATG5
- autophagy related 5
- ATM
- ataxia telangiectasia mutated
- DER
- dose enhancement ratio
- DNA-PKcs
- DNA-dependent protein kinase catalytic subunit
- 4E-BP1
- 4E binding protein 1
- E64d
- 2S,3S-trans-(ethoxycarbonyloxirane-2-carbonyl)-l-leucine-(3-methylbutyl) amide
- EGFR
- epidermal growth factor receptor
- GFP
- green fluorescent protein
- Gy
- gray
- HR
- homologous recombination
- H2AX
- H2A histone family member X
- γ-H2AX
- phosphorylated H2AX
- LY294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
- 3-MA
- 3-methyladenosine
- MEF
- mouse embryonic fibroblast
- mTOR
- mammalian target of rapamycin
- NHEJR
- nonhomologous end joining repair
- P-
- phosphorylated-
- PAGE
- polyacrylamide gel electrophoresis
- PARP
- poly(ADP-ribose) polymerase
- PI-103
- 3–4-(4-morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl-phenol
- PIK3CA
- phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α
- PTEN
- phosphatase and tensin homolog
- RAD001
- dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone
- siRNA
- small inhibitory RNA.
- Received June 7, 2012.
- Accepted September 18, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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