Growth Inhibition of Human MDA-MB-231 Breast Cancer Cells by δ-Tocotrienol Is Associated with Loss of Cyclin D1/CDK4 Expression and Accompanying Changes in the State of Phosphorylation of the Retinoblastoma Tumor Suppressor Gene Product

Abstract

Tocotrienols, a subgroup within the vitamin E family of compounds, have shown antiproliferative and anticancer properties, however, the molecular basis of these effects remains to be elucidated. In this study, the effect of δ-tocotrienol on cell cycle arrest was assessed by studying the retinoblastoma protein (Rb) levels and phosphorylation status, levels of E2F (a transcription factor critically involved in the G1/S-phase transition of the mammalian cell cycle; originally identified as a DNA-binding protein essential for early region 1A-dependent activation of the adenovirus promoter designated E2), and other cell cycle controlling proteins in estrogen receptor-negative MDA-MB-231 breast cancer cells. The cell growth assay demonstrated that exposure of the MDA-MB-231 cells to δ-tocotrienol (1-20 μM) resulted in a dose- and time-dependent inhibition of cell growth as compared with vehicle treated cells and the magnitude of growth inhibition was higher at 10 and 20 μM treatment for 48 and 72 h. The phosphorylation status of Rb plays a central role in the control of the cell cycle at the G0/G1-phase. δ-Tocotrienol treatment reduced the total Rb and its phosphorylation at the Ser780, Ser795, Ser 807/811 and Thr826 positions in a dose- and time-dependent fashion. The site-specific inhibition of the phosphorylation of Rb by δ-tocotrienol was tightly associated with a marked reduction in the expression of cyclin D1 and its regulatory partner cyclin-dependant kinase 4 (CDK4), which is responsible for the phosphorylation of Rb at Ser780, Ser795, Ser 807/811 and Thr826. In addition, δ-tocotrienol also reduced the expression of E2F that occurred simultaneously with the loss of Rb phosphorylation and inhibition of cell cycle progression. Interestingly, δ-tocotrienol also caused a marked reduction in the expression of G2/M regulatory proteins including cyclin B1 and CDK1. To the best of our knowledge, this study was the first to reveal that the target of cell proliferative inhibitory action of δ-tocotrienol in a model estrogen receptor-negative human breast cancer cell line MDA-MB-231 is mediated by the loss of cyclin D1 and associated suppression of site-specific Rb phosphorylation, suggesting its future development and use as an anticancer agent.