Kwang Youl Lee

GINSENOSIDE-RG1, ONE OF THE MAJOR ACTIVE MOLECULES FROM PANAX GINSENG, IS A FUNCTIONAL LIGAND OF GLUCOCORTICOID RECEPTOR

We have examined the possibility that a component of Panax ginseng, ginsenoside-Rg1 (G-Rg1), acts by binding to the glucocorticoid receptor (GR). G-Rg1 competed for [3H]dexamethasone (Dex) binding to GR with a specific affinity of 1-10 microM and activated a glucocorticoid responsive element-containing luciferase reporter gene. The dose-dependence patterns of G-Rg1 and Dex for these two effects were nearly identical, although two to three orders of magnitude higher concentration of G-Rg1 than that of Dex was required for the same magnitude of response. At the cellular level, the growth of FT02B cells was suppressed by G-Rg1 as well as by Dex, each of whose effects were abolished by RU486. These results demonstrate that G-Rg1 is a functional ligand of GR.

ACTIVATION OF CASPASE- 3 PROTEASE VIA A BCL-2-INSENSITIVE PATHWAY DURING THE PROCESS OF GINSENOSIDE RH2-INDUCED APOPTOSIS

We have demonstrated that ginsenoside Rh2 (G-Rh2), a ginseng saponin with a dammarane skeleton, induces apoptosis of human hepatoma SK-HEP-1 cells as evidenced by analyses of DNA fragmentation, flow cytometry and changes in cell morphology. Ac-YVAD-CMK or Ac-DEVD-CHO effectively prevented G-Rh2-induced DNA fragmentation, indicating the involvement of caspase-like proteases in the process of apoptosis. In addition, G-Rh2 induced the processing of caspase-3 to an active form, p17. In stable Bcl-2 transfectants, G-Rh2 also induced DNA fragmentation, while staurosporine-induced DNA fragmentation was totally blocked. As it did in wild-type cells, G-Rh2 induced the proteolytic activation of caspase-3 protease and subsequent cleavage of PARP in the bcl-2 transfectants. In summary, G-Rh2 contains an apoptotic inducing activity in SK-HEP-1 cells which functions via Bcl-2-insensitive activation of caspase-3, followed by proteolytic cleavage of PARP.

Ginsenoside-Rh2 blocks the cell cycle of SK-HEP-1 cells at the G1/S boundary by selectively inducing the protein expression of p27kip1

The mechanism of action by which ginsenoside-Rh2 (G-Rh2) suppresses the proliferation of SK-HEP-1 cells is reported. The results from flow cytometric analyses show that G-Rh2 arrested the cell cycle at the G1/S transition phase. The cyclin E-dependent kinase activity which had been immunoprecipitated with cyclin E-specific antibody was down-regulated in the cells in response to G-Rh2. The IC50 value required to down-regulate the kinase activity by 50% was approximately 0.75 microM. Immunoblotting analyses show that G-Rh2 selectively induced the expression of p27kip1 in a dose-dependent manner whereas it had no effect on the levels of cyclin E, cdk2, and p21WAF1. In addition, our data show that G-Rh2 reduced the protein levels of cdc25A at doses higher than 10 microM. Collectively, these data suggest that ginsenoside-Rh2 arrests the cell cycle at the G1/S transition phase by selectively inducing protein expression of p27Kip1 and, as a consequence, down-regulating cyclin E-dependent kinase activity.

Ginsenoside-Rg1 down-regulates glucocorticoid receptor and displays synergistic effects with cAMP

Ginsenoside-Rg1 (G-Rg1) from the roots of Panax ginseng C. A. Meyer has been shown to bind to the glucocorticoid receptor (GR). To further explore the effect of G-Rg1 binding to GR, a luciferase reporter gene containing two copies of a glucocorticoid response element was constructed and transiently transfected into FTO2B rat hepatoma cells. A dose-dependent induction of the reporter gene was observed in response to G-Rg1, and the inductive effect was blocked by treatment with the antiglucocorticoid RU486. In addition, both G-Rg1- and dexamethasone (Dex)-induced transcription was synergistically enhanced by the treatment of dibutyryl cAMP (Bt2-cAMP). G-Rg1 treatment also led to the down-regulation of intracellular GR content, which was similar to the effect of Dex. By showing that G-Rg1 down-regulates GR and induces GR-mediated transcription synergistically with cAMP, we conclude that G-Rg1 is a functional GR ligand in FTO2B cells.

ALOESIN UP-REGULATES CYCLIN E/CDK2 KINASE ACTIVITY VIA INDUCING THE PROTEIN LEVELS OF CYCLIN E, CDK2, AND CDC25A IN SK-HEP-1 CELLS

In the present study, we show that aloesin, which is a low molecular weight ingredients present in Aloe vera, stimulates the proliferation of cultured human hepatoma SK‐HEP‐1 cells. The incorporation of [3H] thymidine into DNA in the cell cultures was significantly increased at a dose of 10 μM aloesin. The aloesin‐induced DNA synthesis appears to require newly synthesized proteins because cycloheximide treatment blocked the DNA synthesis evoked by this compound. We then examined whether this compound increases the intracellular levels of cell cycle regulators by immunoblotting. The data showed that aloesin increased the levels of cyclin E, CDK2, and CDC25A in SK‐HEP‐1 cells. In addition, immuno‐complex kinase assays showed that aloesin up‐regulated the enzyme activity of cyclin E/CDK2 kinase in a dose‐dependent manner. Collectively, these results suggest that aloesin stimulates the proliferation of SK‐HEP‐1 cells by inducing the intracellular levels of cyclin E/CDK2 kinase complex and CDC25A, which, together, result in the up‐regulation of cyclin E‐dependent kinase activity.

Ginsenoside‐Rg1 positively regulates cyclin E‐dependent kinase activity in human hepatoma SK‐HEP‐1 cells

In the present study, we describe that ginsenoside‐Rg1 (G‐Rg1) stimulates the proliferation of cultured human hepatoma SK‐HEP‐1 cells. The incorporation of [3H] thymidine into DNA was increased in the cells in response to G‐Rg1. The stimulatory effect of G‐Rg1 on DNA synthesis in SK‐HEP‐1 cells require newly synthesized proteins, since cycloheximide blocked the DNA synthetic activity stimulated with G‐Rg1. Thus, we examined whether G‐Rg1 induces the intracellular protein levels of regulatory proteins for cell cycle progression using immunoblottings. The results from immunoblottings showed that G‐Rg1 induced the levels of cyclin E and cdk2 proteins in the cells. Furthermore, the results from immuno‐complex kinase assays for cyclin E‐dependent kinase showed that G‐Rg1 up‐regulates the kinase activity in a dose‐dependent manner. These results suggest that G‐Rg1 stimulates cell‐growth of SK‐HEP‐1 cells by inducing the intracellular levels of cyclin E/cdk2 complex, which in turn up‐regulates cyclin E‐dependent kinase activity.