Vitamin C inhibits benzo[a]pyrene-induced cell cycle changes partly via cyclin D1/E2F pathway in human embryo lung fibroblasts

Objective: To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELP) cells. Methods: The stable transfectants, HELP transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot Flow cytometric analysis was employed to detect the distributions of cell cycle. Results: B[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELP cells. Vitamin C decreased the expression levels of cyclin D1, E2F1 and E2F4 in B[a]P-stimulated HELP cells. Dose-dependent relationships were not found between the different concentrations vitamin C (10, 100, 500, 1000, and 5000 μmol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELP cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELP cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone Conclusions: B[a]P progressed HELP cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.