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

Abstract

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.<br />.

Keywords: 20(S)-ginsenoside Rh2; HepG2 cells; MAPK; histone deacetylase; hypoxia-inducible factor.

Figure 1

Rh2 Inhibits the Activity of HDACs and Increases the Activity of HATs. ELISAs were Used to DetermineHDAC and HAT Activity. (A) HDAC activity decreasedin a time-dependent manner in the treatment group. (B) HAT activity was increased in the treatment group. (C) Expression levels of HDAC1, HDAC2 andHDAC6 were analyzed using western blotting. β-actin was used asthe referenceprotein.(D) HDAC1 activity was analyzed using an ELISA. All experiments were repeated at least three times.Rh2, 20(S)-ginsenoside Rh2; HDAC, histone deacetylase; HAT, histone acetyltransferase

Figure 2

Rh2 Increases Histone Acetylation. (A-D)Cells were incubated with Rh2 for 48h,and fluorescence microscopy was used to analyze the localization of H3K9 andH3K14 in HepG2 cells (magnification, x400;scale bar, 200-μm). All experiments were repeated at least three times. Rh2, 20(S)-ginsenoside Rh2; H3K9, histone 3 lysine 9; H3K14, histone 3 lysine 14

Figure 3

R2 Up-Regulates the Expression Levels of MAPK Signaling Pathway Proteins and Down-Regulated the Expression Levels of HIF. (A and B) HepG2cells were treated with Rh2 for 12,24 or 48h, and western blotting was used to analyze the expression levels of p-ERK,ERK,p-P38,P38,p-JNK and JNK.β-actin was used as the referenceprotein. (C) Activity of Renilla luciferase (HIF) was detected using the Luciferase Reporter Assay system reagent. (D) Reverse transcription-quantitative PCR was used to determine the mRNA expression levels of HIF. All experiments were repeated at least three times.*P<0.05 vs. control. Rh2, 20(S)-ginsenoside Rh2; p-, phosphorylated; HIF, hypoxia-inducible factor

Figure 4

Rh2 Inhibits Proliferation, Promotes Apoptosis and Down-Regulates the Expression Levels of mRNAs Associated with Tumor Angiogenesis in HepG2 Cells. (A) HepG2 cell cycle distribution was analyzed following treatment with Rh2 for 48h. Flow cytometry was used to determine the cell cycle distribution. Blue areas represent the G0/G1 phase, red areas represent the G2/M phase and green areas represent the S phase. Percentage of cell cycle distribution is presented as the mean ± SD. (B) Annexin V-FITC/PI double staining was performed to analyze the levels of apoptosis in HepG2 cells induced byRh2 for 48 h. (C and D) Reverse transcription-quantitative PCR was performed to analyze the mRNA expression levels of Bax, Bcl-2, cyclinD1,IL-1, IL-6, IL-10 and TNF-α. All experiments were repeated at least three times.*P<0.05 vs. control. Rh2, 20(S)-ginsenoside Rh2; PI, propidium iodide; IL, interleukin

Figure 5

Rh2 Decreases Tumor Volume and Diameter, and Inhibits Tumor Angiogenesis. (A) Mean volume of the tumors from each group is shown. Data are presented as the mean ± SD. (B) Diameter of each tumor from the different groups is shown. (C-F)Immunohistochemical staining was used to analyze the expression levels of CD31 and Ki-67 in the tumor tissue (magnification, x400; scale bar, 200-μm). All experiments were repeated at least three times.Rh2, 20(S)-ginsenoside Rh2

Figure 6

Rh2 Inhibits Total Activity of HDAC and HAT, Activates the MAPK Signaling Pathway and Down-Regulates the Expression Levels of mRNAs Associated with Tumor Angiogenesis in Vivo (A and B) ELISAs were used to analyze the activity of histone deacetylases and histone acetyltransferases in the tumor tissue. (C and D)Western blotting was used to analyze the protein expression levels of p-ERK, p-P38 and p-JNK. (E and F) Reverse transcription-quantitative PCR was used to analyze the mRNA expression levels of Bax, Bcl-2, cyclin D1, IL-1, IL-6, IL-10 and TNF-α. All experiments were repeated at least three times.*P<0.05 vs. control. p-, phosphorylated; IL, interleukin