Increased Reactive Oxygen Species and Distinct Oxidative Damage in Resveratrol-suppressed Glioblastoma Cells

Abstract

Background and Aim: Glioblastoma multiforme (GBM) is a highly aggressive brain malignancy that lacks reliable treatments. Resveratrol possesses anti-cancer effects, but its activity against glioblastoma cells is variable for unknown reasons. One mechanism through which anti-cancer drugs exert their effects is oxidative damage caused by increased reactive oxygen species (ROS) production. Thus, the present study examined the relationship between oxidative stress and sensitivity to resveratrol in glioblastoma cells. Methods: Two GBM cell lines (U251 and LN428) were exposed to 100 μM resveratrol for 48 h, and proliferation and apoptosis were assessed. ROS generation was evaluated using 2'-7'-dichlorodihydrofluorescein diacetate-based flow cytometry and fluorescent microscopy. Immunocytochemical staining and western blotting were conducted at regular intervals to profile the expression patterns of superoxide dismutase-2 (SOD2), catalase, caspase-9, caspase-3, and sulfotransferases (SULTs) in untreated and resveratrol-treated GBM cells. Results: Resveratrol-treated U251 cells, but not resveratrol-treated LN428 cells, exhibited remarkable growth arrest and extensive apoptosis accompanied by elevated intracellular ROS levels and attenuated SOD2 and catalase expression. Mitochondrial impairment and more distinct increases in the expression of activated caspase-9 and caspase-3 were detected in U251 cells following resveratrol treatment. The levels of resveratrol metabolic enzymes (SULT1A1 and SULT1C2) were lower in U251 cells than in LN428 cells. Conclusions: Resveratrol increased ROS generation and induced oxidation-related cellular lesions in U251 cells by activating an ROS-related mitochondrial signal pathway. The levels of SULTs and ROS may indicate the therapeutic outcomes of resveratrol treatment in GBM.

Keywords: glioblastoma multiforme; mitochondrial damage; oxidative activity; reactive oxygen species; resveratrol.

Figure 1

Evaluation of resveratrol sensitivities of U251 and LN428 cells. Resveratrol sensitivities of U251 and LN428 cells were evaluated by MTT assay (A), hematoxylin and eosin morphological staining (B) and fluorescent TUNEL labeling (C). N, without resveratrol treatment; R, treated by 100 µM resveratrol. *, P <0.05 in comparison with N group; **, P <0.01 in comparison with N group.

Figure 2

Mitochondrial spheroid formation and reactive oxygen species (ROS) accumulation in resveratrol-sensitive U251 cells. (A) Transmission electron microscopic examination (×40,000) of the double membrane-defined mitochondrial spheroids (white arrow) in resveratrol-treated U251 cells. N, without resveratrol treatment; R, treated with 100 μM resveratrol for 48 h. (B) The cells were treated with 100 µM resveratrol for 0, 6, 12, 24, 36, or 48 h and stained with 2′-7′-dichlorodihydrofluorescein diacetate. Variation of the intracellular fluorescence intensity in U251 and LN428 cells was determined using flow cytometry. (C) Fluorescence intensity analysis of U251 and LN428 cells. (D) ROS levels in U251 and LN428 cells were measured using fluorescence microscopy. All data are presented as the mean ± SD of three independent experiments. Compared with the 0 h sample, *, P < 0.05; **, P < 0.01.

Figure 3

Sequential analyses of superoxide dismutase-2 (SOD2) and catalase (CAT) levels in resveratrol-treated U251 and LN428 cells. (A) Immunocytochemical evaluation of SOD2 and CAT expression in U251 and LN428 cells treated with 100 µM resveratrol for 0, 6, 12, 24, 36, or 48 h. (B) Western blot and gray density analyses of SOD2 and CAT expression in U251 and LN428 cells. β-actin was used as the quantitative control.

Figure 4

Caspase-9 and caspase-3 levels and sulfotransferase 1A1 (SULT1A1) and SULT1C2 expression patterns in untreated and resveratrol-treated U251 and LN428 cells. (A) Western blot evaluation of pro-caspase-9, pro-caspase-3, activated caspase-9, and activated caspase-3 U251 and LN428 cells cultured normally (N) and in the presence of 100 µM resveratrol for 48 h (R). (B) Fractionation of pro-caspase-9, pro-caspase-3, activated caspase-9, and activated caspase-3 in normally cultured (N) and resveratrol-treated (R) U251 and LN428 cells according to the western blot results. (C-D) Western blot and immunocytochemical demonstration of SULT1A1 and SULT1C2 downregulation in U251 cells compared with that in LN428 cells before (N) and after treatment with 100 µM resveratrol for 48 h (R).