Resveratrol at high doses acts as an apoptotic inducer in endothelial cells

Abstract

Purposes: Resveratrol is a phenolic compound found in grapes and other food products. In order to assess the availability of resveratrol as an angio-inhibiting drug, we examined whether resveratrol plays an important role in bovine aortic endothelial cells (BAECs) for cell apoptosis and cell migration.

Methods and materials: Endothelial cell apoptosis was observed as detected by the Hoechst staining and the caspase-3 activity. Additionally, Western blotting was performed for monitoring the activities of various cell signaling molecules.

Results: Resveratrol was shown to act as a pro-apoptotic agent. The pro-apoptotic effect of resveratrol was as great as that of etoposide, a well-known anti-cancer drug. In addition, resveratrol had an inhibitory effect on endothelial cell migration. The demonstrated efficacy of resveratrol suggests that resveratrol may be utilized as an anti-angiogenic drug. To determine the underlying mechanisms, we further investigated which signaling molecules are activated by resveratrol. Extracellular signal-regulated kinase (ERK) was activated by the treatment with resveratrol in BAECs, whereas endothelial nitric oxide synthetase (eNOS), Akt, and Jun N-terminal kinase (JNK) were inhibited. The pretreatment with PD compound, an ERK inhibitor, had no effect on the pro-apoptosis induced by resveratrol.

Conclusion: Resveratrol plays an important role in endothelial cell apoptosis, indicating that resveratrol can be utilized as a potent anti-angiogenic drug.

Keywords: Apoptosis; Caspase-3; Cell signaling; Endothelial cells; Resveratrol.

Fig. 1

Resveratrol induces endothelial cell apoptosis. Confluent BAECs were starved for more than 4 h and then the cells were incubated for additional hours (0, 4, 8, 12, 24 or 72 h) with the indicated doses of resveratrol (RES). Consecutively, the cells were incubated for additional hours (0, 4, 8, 12, 24 or 72 h) We then counted the apoptotic cells (round shrunken cells) under the microscope. The line graphs represent the percentages of apoptotic cells (means±S.E.). The experiments were independently performed at least three times.

Fig. 2

The resveratrol-induced apoptosis is mediated by activation of caspase-3. Confluent cells were incubated with 0.5% FBS-DMEM containing none (CON), 100 µM etoposide (ETO) or 100 µM resveratrol (RES) for 36 h. The cell were then stained with Hoechst 33258 and we observed the nuclei under a fluorescence microscope (A). Arrows indicate the fragmented nuclei. In panel B, after the cells were lysed, proteins in the cell lysates were resolved by SDS-PAGE, electrotransferred to PVDF membranes and immunoblotted with caspase-3 antibodies.

Fig. 3

Resveratrol blocks the endothelial cell migration. Confluent endothelial cells were serum-starved and then scraped with a razor blade after 24 h starvation. The cells were subsequently incubated in media containing 20% fetal bovine serum (CON) or various concentration of resveratrol (RES). After 26 h, we then observed the migrated cells under the microscope. The line on each photograph indicates the boundary lines immediately after scraping. The photographs are representative of at least three different observations. We then counted the cells that migrated over the lines. Quantification was performed by counting the number of cells that migrated in the same field. The bar graphs show the mean number of migrated cells±S.E. (n=3).

Fig. 4

Resveratrol modulates a variety of cell signaling molecules. Serum-depleted cells (grown in 0.5% FBS-DMEM) were stimulated with 100 µM resveratrol treatment as a function of time. After the cells were lysed, the proteins in cell lysates were resolved by SDS-PAGE, electrotransferred to PVDF membranes and immunoblotted with the appropriate antibodies.

Fig. 5

ERK is not associated with the resveratrol-induced apoptosis. (A) Apoptosis was observed as described in Fig. 1. Interestingly, 10 µM of PD compound had no effect on apoptosis. The bar graphs represent means±S.E. (n=3). (B) Serum-depleted cells (grown in 0.5% FBS-DMEM) were stimulated with 100 µM resveratrol treatment for 24 h. After the cells were lysed, proteins in the cell lysates were resolved by SDS-PAGE, electrotransferred to PVDF membranes and immunoblotted with caspase-3 antibodies.