Ginsenoside Rh2-induced G1 phase cell cycle arrest in human breast cancer cell is mediated by cooperation P15^INK4B with p27^Kip inhibitors.

2603

Ginsenoside Rh2 (Rh2), a purified ginseng saponin, has been known to have anti-cancer effects in cancer cells but the mechanism of cell cycle arrest is not fully understood. We used MCF7 (wild type p15) and MDA-MB-231 (p15 deletion) human breast cancer cells to gain further insights into the mechanism of Rh2-induced cell cycle arrest. We observed that Rh2 (40 μM) treatment significantly inhibited cell growth in a concentration dependent manner and caused G1 phase cell cycle arrest in cell cycle progression in both MCF7 and MDA-MB-231 cells. The G1-S transition in mammalian cells requires the activity of cyclin D-dependent kinase Cdk4 and/or Cdk6 and the cyclin E-dependent kinase Cdk2. The Rh2-mediated G1 phase cell cycle arrest was accompanied by downregulation of the protein levels of Cdk2, 4, 6 and cyclin D1, cyclin E. In addition, Rh2 treatment markedly reduced the level of phosphorylated retinoblastoma protein (pRb), enhanced association of unphosphorylated Rb and the transcription factor E2F1, and decreased of E2F1 transcriptional activity in reporter luciferase assay. Unlike MDA-MB-231, MCF7 cells contain high levels of the universal Cdk inhibitor p27^Kip1. The treatment with Rh2 elevated expression of the Cdk4 specific inhibitor p15^ink4B and significantly induced the recruitment of p27 to Cdk4. The recruitment of p27 coincided with increased binding p27 with Cdk4. These results suggested that Rh2 inhibited the growth of MCF7 cells by inducing protein expression of p15 and reducing the protein level of Cyclin D which resulted in down regulation of cyclin/Cdk kinase activity, decreasing phosphorylation of pRb, and inhibiting E2F release. Furthermore Rh2 induced G1 arrest was also caused by inhibition of cyclin D-Cdk4 through the cooperative action of p15^INK4Band p27 ^Kip. ¹This investigation was supported by Kangwon Bio-Nuri grant, Korea Research Foundation. ²This investigation was supported by NCI grant CA129347.