NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, enhances the radiosensitivity of human glioma stem cells in vitro
Abstract
Wen-juan WANG1, Lin-mei LONG1, Neng YANG1, Qing-qing ZHANG1, Wen-jun JI1, Jiang-hu ZHAO1, Zheng-hong QIN1, Zhong WANG2, Gang CHEN2, Zhong-qin LIANG1, *
1Department of Pharmacology, Medical School of Soochow University, Suzhou 215123, China; 2Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215123, China
Aim: NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor and shows dramatic effects on gliomas. The aim of this study was to investigate the effects of NVP-BEZ235 on the radiosensitivity and autophagy of glioma stem cells (GSCs) in vitro.
Methods: Human GSCs (SU-2) were tested. The cell viability and survival from ionizing radiation (IR) were evaluated using MTT and clonogenic survival assay, respectively. Immunofluorescence assays were used to identify the formation of autophagosomes. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and fluorescence microscope. Western blot analysis was used to analyze the expression levels of proteins. Cell cycle status was determined by measuring DNA content after staining with PI. DNA repair in the cells was assessed using a comet assay.
Results: Treatment of SU-2 cells with NVP-BEZ235 (10–320 nmol/L) alone suppressed the cell growth in a concentration-dependent manner. A low concentration of NVP-BEZ235 (10 nmol/L) significantly increased the radiation sensitivity of SU-2 cells, which could be blocked by co-treatment with 3-MA (50 µmol/L). In NVP-BEZ235-treated SU-2 cells, more punctate patterns of microtubule-associated protein LC3 immunoreactivity was observed, and the level of membrane-bound LC3-II was significantly increased. A combination of IR with NVP-BEZ235 significantly increased the apoptosis of SU-2 cells, as shown in the increased levels of BID, Bax, and active caspase-3, and decreased level of Bcl-2. Furthermore, the combination of IR with NVP-BEZ235 led to G1 cell cycle arrest. Moreover, NVP-BEZ235 significantly attenuated the repair of IR-induced DNA damage as reflected by the tail length of the comet.
Conclusion: NVP-BEZ235 increases the radiosensitivity of GSCs in vitro by activating autophagy that is associated with synergistic increase of apoptosis and cell-cycle arrest and decrease of DNA repair capacity.
Keywords: human glioblastoma; glioma stem cells; irradiation; radiosensitivity; apoptosis; autophagy; LC3; PI3K/mTOR pathway; NVP-BEZ235; 3-MA; cell cycle arrest; DNA repair
This work was supported by grants from the National Natural Science Foundation of China (Grant Nos 30873052, 81072656, 81102466), Jiangsu Province’s outstanding Medical Academic Leader program (No LJ201139) and Priority Academic Program Development of Jiangsu Higher Education Institutions, PAPD, Jiangsu Province’s Key Medical Department in 2011.
* To whom correspondence should be addressed.
E-mail liangzhongqin@suda.edu.cn
Received 2012-11-19 Accepted 2013-02-26
Keywords:
1Department of Pharmacology, Medical School of Soochow University, Suzhou 215123, China; 2Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215123, China
Aim: NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor and shows dramatic effects on gliomas. The aim of this study was to investigate the effects of NVP-BEZ235 on the radiosensitivity and autophagy of glioma stem cells (GSCs) in vitro.
Methods: Human GSCs (SU-2) were tested. The cell viability and survival from ionizing radiation (IR) were evaluated using MTT and clonogenic survival assay, respectively. Immunofluorescence assays were used to identify the formation of autophagosomes. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and fluorescence microscope. Western blot analysis was used to analyze the expression levels of proteins. Cell cycle status was determined by measuring DNA content after staining with PI. DNA repair in the cells was assessed using a comet assay.
Results: Treatment of SU-2 cells with NVP-BEZ235 (10–320 nmol/L) alone suppressed the cell growth in a concentration-dependent manner. A low concentration of NVP-BEZ235 (10 nmol/L) significantly increased the radiation sensitivity of SU-2 cells, which could be blocked by co-treatment with 3-MA (50 µmol/L). In NVP-BEZ235-treated SU-2 cells, more punctate patterns of microtubule-associated protein LC3 immunoreactivity was observed, and the level of membrane-bound LC3-II was significantly increased. A combination of IR with NVP-BEZ235 significantly increased the apoptosis of SU-2 cells, as shown in the increased levels of BID, Bax, and active caspase-3, and decreased level of Bcl-2. Furthermore, the combination of IR with NVP-BEZ235 led to G1 cell cycle arrest. Moreover, NVP-BEZ235 significantly attenuated the repair of IR-induced DNA damage as reflected by the tail length of the comet.
Conclusion: NVP-BEZ235 increases the radiosensitivity of GSCs in vitro by activating autophagy that is associated with synergistic increase of apoptosis and cell-cycle arrest and decrease of DNA repair capacity.
Keywords: human glioblastoma; glioma stem cells; irradiation; radiosensitivity; apoptosis; autophagy; LC3; PI3K/mTOR pathway; NVP-BEZ235; 3-MA; cell cycle arrest; DNA repair
This work was supported by grants from the National Natural Science Foundation of China (Grant Nos 30873052, 81072656, 81102466), Jiangsu Province’s outstanding Medical Academic Leader program (No LJ201139) and Priority Academic Program Development of Jiangsu Higher Education Institutions, PAPD, Jiangsu Province’s Key Medical Department in 2011.
* To whom correspondence should be addressed.
E-mail liangzhongqin@suda.edu.cn
Received 2012-11-19 Accepted 2013-02-26