Specific mTOR inhibitor rapamycin enhances cytotoxicity induced by alkylating agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) in human U251 malignant glioma cells
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Loss of the PTEN tumor suppressor gene and amplification of the epidermal growth factor receptor (EGFR), which is common in malignant gliomas, result in activation of the mammalian target of rapamycin (mTOR). Rapamycin is a highly specific inhibitor of mTOR and induces a cytostatic effect in various glioma cell lines. DNA-damaging agents such as nitrosourea are widely used in malignant glioma treatment; therefore, we investigated the effect of rapamycin on cell growth and death in combination with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU, nimustine hydrochloride) in human glioma cells. In U251 malignant glioma (U251MG) cells, we confirmed that rapamycin enhanced ACNU-induced apoptosis. We found that rapamysin inhibited ACNU-induced p21 induction, and knocking down of p21 protein by siRNA enhanced ACNU-induced apoptosis in U251MG cells. Furthermore, adenovirus-mediated over-expression of p21 protein rescued U251MG cells from apoptosis induced by ACNU and rapamycin. Finally, treatment of intracerebral U251MG xenografts with a combination of rapamycin and ACNU in vivo resulted in statistically prolonged median survival (P < 0.05). These results suggest that rapamycin in combination with DNA-damaging agents may be efficacious in the treatment of malignant gliomas.
KeywordsApoptosis Malignant glioma p21 The mammalian target of rapamycin (mTOR) 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)
This work was supported in part by a Grant-in-Aid for Scientific Research to Eiji Kohmura (15659337), Atsufumi Kawamura (16591440), and Takashi Sasayama (17790968) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
We are grateful to Hideyuki Saya (Kumamoto University, Kumamoto, Japan), for valuable suggestions on various analyses. We would like to thank Ayumi Katoh and Nami Takase for technical assistance in the Western blot assays and in vivo experiments.
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