Abstract
Autophagy is originally named as a process of protein recycling. It begins with sequestering cytoplasmic organelles in a membrane vacuole called autophagosome. Autophagosomes then fuse with lysosomes, where the materials inside are degraded and recycled. To date, however, little is known about the role of autophagy in cancer therapy. In this study, we present that temozolomide (TMZ), a new alkylating agent, inhibited the viability of malignant glioma cells in a dose-dependent manner and induced G2/M arrest. At a clinically achievable dose (100 μM), TMZ induced autophagy, but not apoptosis in malignant glioma cells. After the treatment with TMZ, microtubule-associated protein light-chain 3 (LC3), a mammalian homologue of Apg8p/Aut7p essential for amino-acid starvation-induced autophagy in yeast, was recruited on autophagosome membranes. When autophagy was prevented at an early stage by 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, not only the characteristic pattern of LC3 localization, but also the antitumor effect of TMZ was suppressed. On the other hand, bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase, that prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes, sensitized tumor cells to TMZ by inducing apoptosis through activation of caspase-3 with mitochondrial and lysosomal membrane permeabilization, while LC3 localization pattern stayed the same. These results indicate that TMZ induces autophagy in malignant glioma cells. Application of an autophagy inhibitor that works after the association of LC3 with autophagosome membrane, such as bafilomycin A1, is expected to enhance the cytotoxicity of TMZ for malignant gliomas.
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Abbreviations
- TMZ:
-
temozolomide
- LC3:
-
microtubule-associated protein light-chain 3
- 3-MA:
-
3-methyladenine
- GBM:
-
glioblastoma multiforme
- AA:
-
anaplastic astrocytoma
- AVO:
-
acidic vesicular organelle
- PI3K:
-
phosphatidylinositol-3 kinase
- GFP:
-
green fluorescent protein
- DMSO:
-
dimethylsulfoxide
- Br-dUTP:
-
bromolated deoxyuridine triphosphate nucleotides
- FITC:
-
fluorescent isothiocyanate
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Acknowledgements
We thank Dr. T Yoshimori and Dr. N Mizushima for GFP-LC3 expression vector and the Schering–Plough Research Institute for TMZ. We also thank Dr. A Yamamoto and Dr. N Mizushima for helpful suggestions. This study was supported in part by the USPHS Grant 1R01 CA88936 (SK) awarded by the National Cancer Institute, in part by a start-up fund from The University of Texas MD Anderson Cancer Center, and in part by a generous donation from the Anthony D Bullock III Foundation.
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Kanzawa, T., Germano, I., Komata, T. et al. Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11, 448–457 (2004). https://doi.org/10.1038/sj.cdd.4401359
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DOI: https://doi.org/10.1038/sj.cdd.4401359
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