Anticancer Effect of Rh2, a Histone Deacetylase Inhibitor, in HepG2 Cells and HepG2 Cell-Derived Xenograft Tumors Occurs via the Inhibition of HDACs and Activation of the MAPK Signaling Pathway

PURPOSE: To investigate the effect of 20(S)-ginsenoside Rh2 (Rh2) on anti HepG2 liver cancer cells and HepG2 cell-derived xenograft tumors, and explore the underlying mechanisms. MATERIALS AND METHODS: The activity of total HDACs and HAT were assessed with a HDACs colorimetric kit. Expression of HDAC1, HDAC2, HDAC6, p-ERK, ERK, p-P38, P38, p-JNK and JNK proteins was tested by Western blotting.H3K9 and H3K14 proteins were also checked by immunofluorescence, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining. Activity of Renilla luciferase (HIF) was detected using the Luciferase Reporter Assay system reagent. Gene expression for CyclinD1, Bcl-2, Bax, HIF, IL-1, IL-6, IL-10 and TNF-α was tested by q-PCR. Expression levels of CD31 and Ki-67 was tested by immunohistochemical staining. RESULTS: Total HDAC activity was decreased and total histone acetyltransferase (HAT)activity was increased in a time-dependent manner. Expression of HDAC1 and p-JNK proteins was significantly increased, expression levels of p-ERK was decreased. H3K9 and H3K14 fluorescence protein were increased. Flow cytometric analysis of the cell cycle revealed that the percentage of cells in the G0/G1 phase in the treatment group(64.35±1.36%) was significantly increased compared with the untreated group(61.61±1.23%).The apoptotic rate of the HepG2 group was 10.03±1.92%, which increased to 17.87±1.67% in the treatment group. Expression levels of the transcription factor HIF were also increased in HepG2 cells following induction by Rh2. Expression of CyclinD1 and Bcl-2 at the genetic level was significantly decreased, while expression levels of Bax, HIF, IL-1, IL-6, IL-10 and TNF-α was increased. In vivo, the expression levels of both CD31 and Ki-67 proteins were significantly down-regulated in the treatment group compared with the control group. CONCLUSIONS: The effects of Rh2 were suggested to occur through the inhibition of total HDAC activity, which subsequently induced MAPK signaling and down-regulated the expression of HIF.