effect of 20(S)-Rg3 in MCF-7 cells unexpectedly showed no significant difference. These results were consistent when Rg3 was treated in MDA-MB-453 cells (Figs. 4A, 4B). The results from flow cytometric analysis [i.e., fluorescence-activated cell sorting (FACS)] indicated that Rg5 significantly induced cell cycle arrest (Figs. 5A, 5B). This was further confirmed by the cell cycle assay with the data representing suppressed cell proliferation in MCF-7 cells after Rg5 treatment. Rg5 increased the number of cells in the G0/G1 phase and decreased the number of cells in the S phase. Based on these results, Rg5 may induce cell cycle arrest at the G0/G1 phase. Protein expression of cyclin D1, cyclin E2 and CDK4 was decreased, whereas the expression of p15INK4B, PFI-2 research buy p53 and p21WAF1/CIP1 was increased (Figs. 6A, 6B). As Fig. 7A shows, treatment with GW-572016 purchase Rg5 induced caspase-8 and caspase-9, caspase-7, caspase-6. The full-length Bid consequently disappeared in a dose-dependent manner. Poly (ADP-ribose) polymerase
(PARP) cleavage was detected in Rg5-treated MCF-7 cells, which indicated that Rg5 reduced cell viability by inducing apoptosis. Promotion of mitochondria-mediated intrinsic apoptotic pathway by Rg5 was evidenced by Bax/Bcl-2 dysregulation, activation of caspase-9, and release of cytochrome C (Fig. 7A). Apoptosis was evaluated by annexin V/FITC/PI dual staining. After 48 h, Rg5 significantly increased apoptosis at 25μM and 50μM and reduced apoptotic cells at 100μM, whereas necrotic cells were increased (Fig. 7B). The increased expression
of DR4 and DR5 on the cell surface was obvious when cells were treated at the 100μM concentration of Rg5 (Fig. 8A). Activation of p38 mitogen-activated protein kinases (MAPKs) is necessary for apoptosis induced by exposure to ultraviolet radiation, cytokines, chemotherapy, ceramide, and serum deprivation . When SPTLC1 cells were treated with Rg5 (50μM and 100μM), p38 MAPKs were activated with the generation of reactive oxygen species (data not shown) (Fig. 8C). Survivin, an inhibitor of apoptotic proteins, is highly expressed in most types of cancer and is a regulator of mitosis; survivin-targeting cancer treatment is validated with great efficacy and no serious toxicity . The expression of survivin was suppressed at high concentrations of Rg5 (Fig. 8D). Apoptotic cells were visualized with DAPI as fluorescent probes. When cells were incubated for 48 h with Rg5 at indicated concentrations (i.e., 0μM, 50μM, and 100μM), the cells displayed the typical apoptosis morphology such as fragmented and condensed nuclei with cellular shrinkage (Fig. 9B). Cells treated with Rg5 at the 100μM concentration showed a necrosis-like morphology (Fig. 9C). Red ginseng is fresh ginseng that is dry-steamed once using water vapor. Black ginseng refers to ginseng that is steamed nine times. Fine Black ginseng refers to the fine roots (i.e., hairy roots) of BG steamed nine times. As Fig.