Resveratrol and quercetin cooperate to induce senescence-like growth arrest in C6 rat glioma cells

Abstract

Glioma is the most frequent and malignant primary human brain tumor with dismal prognosis despite multimodal therapy. Resveratrol and quercetin, two structurally related and naturally occurring polyphenols, are proposed to have anticancer effects. We report here that resveratrol and quercetin decreased the cell number in four glioma cell lines but not in rat astrocytes. Low doses of resveratrol (10 microM) or quercetin (25 microM) separately had no effect on apoptosis induction, but had a strong effect on caspase 3/7 activation when administered together. Western blot analyses showed that resveratrol (10 microM) and quercetin (25 microM) caused a reduction in phosphorylation of Akt, but this reduction was not sufficient by itself to mediate the effects of these polyphenols. Most important, resveratrol and quercetin chronically administered presented a strong synergism in inducing senescence-like growth arrest. These results suggest that the combination of polyphenols can potentialize their antitumoral activity, thereby reducing the therapeutic concentration needed for glioma treatment.

Figure 1

Resveratrol (Rsv) and quercetin (Quer) reduced the number of human, mouse, and rat gliomas. Glioma cell lines from rat (C6), mouse (GL261), and humans (U87‐MG and U138‐MG) were treated with 10 or 50 µM Rsv, 25 or 100 µM Quer, and 10 µM Rsv plus 25 µM Quer for 24, 48, and 72 h; cells were then counted in a hemocytometer. Each time point represents the mean ± SEM of three independent experiments. anova was followed by SNK post test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Resveratrol (Rsv) increased propidium iodide (PI) incorporation in C6 cells but not in astrocyte cell cultures. C6 cells were treated with 10 or 50 µM Rsv, 25 or 100 µM quercetin (Quer), and 10 µM Rsv plus 25 µM Quer. Astrocyte cultures were treated with 1, 10, 25, or 50 µM Rsv. At the end of the treatments (72 h), the images were obtained using a Carl Zeiss inverted microscope. Representative light micrographs of C6 (a), astrocyte cell cultures (c), fluorescence micrographs of C6 (b), and astrocyte cell cultures (d) stained with PI (×100) are shown. The numbers below the pictures depict values of PI staining (ratio of PI labeled cells/total number cells, normalized to control) as mean ± SEM of three independent experiments. anova was followed by SNK post test ***P < 0.001.

Figure 3

Resveratrol (R) induces caspase 3/7 activation, annexin V staining, and redudes mitotic index. C6 cells were treated with Rsv (10, 25, or 50 µM) and quercetin (Q) (25 or 100 µM) and 10 µM Rsv plus 25 µM Quer for 48 or 72 h. Caspase 3/7 activity was estimated by fluorogenic assay for the effector caspase 3/7 (a). Cells were stained with annexin V‐FLUOS and propidium iodide (PI) and positive cells were observed using an inverted fluorescence microscope (×200). The same fields are shown. Control cells (left panels), 50 µM Rsv (middle panels), and 10 µM Rsv plus 25 µM Quer (right panels) observed under light microscopic image (upper panel), with annexin V staining (middle panel) and PI staining (lower panel) after 72 h of treatments (b). Cells were treated as indicated above for 24, 48, and 72 h and cells in mitosis were visualized by PI staining. The results represent mean ± SEM from three independent experiments. anova was followed by SNK post test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Resveratrol (Rsv) and quercetin (Quer) decreased the phosphorylation of Akt. C6 cells were treated with 10 µM Rsv, 25 µM Quer, or 10 µM Rsv plus 25 µM Quer, for 30 min, 4 h, and 24 h, and with 10 µM LY294002 for 30 min. Lysates were subjected to western blot analysis for pS473Akt and total Akt antibodies (a) and PTEN antibody (b). Representative blots of three independent experiments are shown and the numbers below the blots depict values of densitometric evaluation as mean ± SEM. Number of C6 cells treated as indicated for 72 h (c). The results represent mean ± SEM from three independent experiments. anova was followed by SNK post test. *P < 0.05, **P < 0.01.

Figure 5

Resveratrol (Rsv) and quercetin (Quer) induce senescence in C6 cells. C6 cells were cultivated in the continuous presence of 10 or 25 µM Rsv, 25 µM Quer, 10 µM Rsv plus 25 µM Quer, or 10 µM LY294002 for 12 days and cumulative population doublings were plotted against time (a). After this time cells were stained for senescence‐associated‐β‐galactosidade (SA‐β‐gal) and photographed (b). The bar graph represents the ratio of SA‐β‐gal‐positive cells to total cells of at least three fields of three independent experiments (c). Colony formation efficiency of C6 cells 7 days after 12 days Rsv or Quer treatment or 6 days of cotreatment (d), and SA‐β‐gal staining at the end of the colony formation assay (e). anova was followed by SNK post test. *P < 0.05, **P < 0.01, ***P < 0.001.