Simvastatin inhibits cancer cell growth by inducing apoptosis correlated to activation of Bax and down-regulation of BCL-2 gene expression

Abstract

The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been proven to be effective in lowering cholesterol and as anti-lipid agents against cardiovascular disease. Recent reports demonstrate an anticancer effect induced by the statins through inhibition of cell proliferation. Probably, these effects are due to suppression of the mevalonate pathway leading to the depletion of various downstream products that play an essential role in cell cycle progression, cell signaling and membrane integrity. To date, although many hypotheses have been proposed, the exact mechanism at the basis of cancer cell growth arrest induced by statins is not known. In this study, we have demonstrated that simvastatin, at a dose of 20 µM for 24-72 h, induced in cancer cells but not in normal cells precise features of apoptosis including increased DNA fragmentation while, at the molecular level simvastatin induced overexpression of the pro-apoptotic gene Bax together with an inhibition of BCL-2, the gene that has the well-known function of protecting cells from apoptosis. The simvastatin-mediated induction of apoptosis in similar cancer cells but not in normal cells is very interesting and may be at the basis of cancer therapy using statins, usually in combination with chemotherapy or to be used as a cancer protective drug. Simvastatin may, thus, play a dual prophylactic role as a lipid-lowering drug for the prevention of heart disease and as an anticancer agent to prevent certain types of cancers.

Figure 1

Internucleosomal DNA fragmentation. Treatment of MCF7 cells with 20 μM simvastatin for 24 h induces internucleosomal DNA fragmentation. DNA was extracted after treatment with simvastatin for 1 day (lane 2) or 3 days (lane 3). Lane 1, untreated MCF7 cells; lane 4, SAEC human normal small airway epithelial cells treated with simvastatin 20 μM for 3 days. Lane M, 100-bp ladder (Boehringer, Mannheim, Germany) used as a size marker.

Figure 2

TUNEL apoptosis analysis. Positive control of TUNEL reaction is obtained by inducing apoptosis with hydrogen peroxide in breast cancer MCF7 cells and in SAEC human normal small airway epithelial cells where a clear fluorescein green coloration appears inside the nuclei. No sign of apoptosis is induced by simvastatin in non-transformed human fibroblast. In breast cancer MCF7, hepatocellular HepG2, lung carcinoma NCH lung, and human gastric cancer cells NCI gastric showed strong apoptosis, as indicated by the clear green fluorescence in the cell nuclei.

Figure 3

PCR analysis of Bax and BCL-2 expression. The PCR expression profile of Bax and BCL-2 genes demonstrate that, in all cancer cells, there is an increase in Bax expression and a diminution in the expression of BCL-2 respect to the untreated control cells. On the contrary, there are no differences in the expression of these genes in the SAEC human normal small airway epithelial cells. Actin is reported as the internal control.

Figure 4

Real-time PCR analysis of Bax and BCL-2 genes. The real-time PCR analysis of Bax and BCL-2 genes demonstrate that, in all simvastatin-treated cancer cells (S), there is an increase in Bax expression and a diminution in the expression of BCL-2 with respect to controlling untreated cells (C). On the contrary, there are no differences in the expression of these genes in the SAEC normal small airway epithelial cells. The values have been normalized with respect to actin. The graph represents three independent experiments with each bar as mean ± SD. ^*P<0.05 compared to control group.

Figure 5

Apoptosis protein analysis. BCL-2 and Bax protein was determined by Western blot analysis after 24 and 72 h of treatment with 20 μM simvastatin on MCF7 breast cancer cells and SAEC human normal small airway epithelial cells. Actin was reported as internal control. C, the cells are maintained for 72 h without drug addition. It is evident that the incubation with simvastatin caused time-dependent increase in Bax expression and decrease in BCL-2 protein expression only in MCF7 cancer cells.