Original Contribution
Resveratrol enhances the therapeutic effect of temozolomide against malignant glioma in vitro and in vivo by inhibiting autophagy

https://doi.org/10.1016/j.freeradbiomed.2011.10.487Get rights and content

Abstract

The alkylating agent temozolomide (TMZ) is the major chemotherapeutic drug used clinically in the treatment of malignant gliomas. This study investigated the mechanism behind TMZ-induced cell death and the possibility that resveratrol might increase TMZ efficacy. TMZ induced both apoptotic cell death and cytoprotective autophagy through a reactive oxygen species (ROS) burst and extracellular signal-regulated kinase (ERK) activation, which was suppressed by resveratrol, resulting in a decrease in autophagy and an increase in apoptosis, suggesting that the ROS/ERK pathway plays a crucial role in the fate of cells after TMZ treatment. Isobolographic analysis indicated that the combination of TMZ and resveratrol has a synergistic effect. Moreover, an in vivo mouse xenograft study also showed that coadministration of resveratrol and TMZ reduced tumor volumes by suppressing ROS/ERK-mediated autophagy and subsequently inducing apoptosis. Taken together, our data indicate that TMZ-induced ROS/ERK-mediated autophagy protected glioma cells from apoptosis, and the combination of resveratrol with TMZ could improve the efficacy of chemotherapy for brain tumors.

Graphical abstract

Highlights

► Temozolomide (TMZ) induces both apoptosis and autophagy in the glioma cells. ► ROS/ERK-mediated autophagy induced by TMZ plays a pro-survival role. ► Resveratrol enhances TMZ-induced apoptosis by inhibiting ROS and autophagy. ► Resveratrol exhibits a synergistic effect on TMZ-mediated cytotoxicity. ► Co-administration of resveratrol and TMZ reduces tumor volumes in vivo.

Section snippets

Reagents and antibodies

Media such as RPMI 1640, MEM, and DMEM were purchased from Hyclone (Logan, UT, USA). Vitamin C and 3-(4,5-dimethyl-2-thiazolyl)-2,5-dimethyl-2H-tetrazolium bromide (MTT) were purchased from Merck (Darmstadt, Germany). TMZ, resveratrol, acridine orange, 3-methyladenine (3-MA), bafilomycin A1 (BAF), chloroquine (CQ), ammonium chloride (AC), and tiron were purchased from Sigma (St. Louis, MO, USA). 2,7-Dihydrodichlorofluorescein diacetate (DCFH-DA), dihydroethidium (HEt), and dihydrorhodamine 123

TMZ induces apoptosis in glioma cells

To examine the cytotoxicity of TMZ in various glioma cells, the U87 MG (obtained from a Caucasian strain), GBM8401, and GBM cell lines (both from Asian strains) were subjected to MTT assay to determine cell viability. Referring to the concentrations (0–1000 μM) used in the study of Aoki et al. [24], the cells were treated with 0–500 μM TMZ for 72 h. As shown in Fig. 1A, TMZ inhibited cell growth in a dose-dependent manner in three glioma cell lines. The IC50 of TMZ was calculated as 397.2, 391.4,

Discussion

Recent studies have demonstrated the median overall survival rate for glioblastoma patients to be 10.2–14.6 months [29]. However, progress remains poor, even with treatment that combines surgery, radiation, and/or chemotherapy [30]. Although TMZ is a major chemotherapeutic agent used clinically to mediate GBM cell death, the increase in median survival time of 2 months is modest [31]. In this study, we demonstrated both in vitro and in vivo that resveratrol, a natural polyphenol, potentiated the

Acknowledgments

This study was sponsored by grants from the National Science Council (NSC99-2320-B-038-008-MY3) and Shin Kong Wu Ho-Su Memorial Hospital, Taiwan (SKH-TMU-96-11) to C.M.S.

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