Effects of R type and S type ginsenoside Rg3 on DNA methylation in human hepatocarcinoma cells

Abstract

Ginsenoside Rg3, a bioactive constituent isolated from Panax ginseng, exhibits antitumorigenic, antioxidative, antiangiogenic, neuroprotective and other biological activities are associated with the regulation of multiple genes. DNA methylation patterns, particularly those in the promoter region, affect gene expression, and DNA methylation is catalyzed by DNA methylases. However, whether ginsenoside Rg3 affects DNA methylation is unknown. High performance liquid chromatography assay, MspI/HpaII polymerase chain reaction (PCR) and reverse transcription‑quantitative PCR were performed to assess DNA methylation. It was demonstrated that 20(S)‑ginsenoside Rg3 treatment resulted in increased inhibition of cell growth, compared with treatment with 20(R)‑ginsenoside Rg3 in the human HepG2 hepatocarcinoma cell line. It was additionally revealed that treatment with 20(S)‑ginsenoside Rg3 reduced global genomic DNA methylation, altered cystosine methylation of the promoter regions of P53, B cell lymphoma 2 and vascular endothelial growth factor, and downregulated the expression of DNA methyltransferase (DNMT) 3a and DNMT3b more than treatment with 20(R)‑ginsenoside Rg3 in HepG2 cells. These results revealed that the modulation of DNA methylation may be important in the pharmaceutical activities of ginsenoside Rg3.

Figure 1.

Chemical structure and HPLC chromatogram of 20(R)-ginsenoside Rg3 and 20(S)-ginsenoside Rg3. The X-axis shows the retention time and the Y-axis shows the voltage. The position of the asymmetric carbon atom in ginsenoside Rg3 is marked with a red circle. From the HPLC chromatograms of standards, the 20(R)-ginsenoside Rg3 retention time was ~11.5 min, compared with the 20(S)-ginsenoside Rg3 retention time of 10.5 min. (A) R type; (B) S type. HPLC, high performance liquid chromatography.

Figure 2.

Inhibitory effect of ginsenoside Rg3 on HepG2 cell proliferation. (A) Growth inhibition curve for HepG2 cells treated with ginsenoside Rg3. HepG2 cells were treated with various concentrations (0, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 µg/ml) of ginsenoside Rg3 using a doubling dilution every 48 h, and cell viability was measured using a Cell Counting Kit-8 assay. (B) Linear regression equation of logarithm (base 10) concentration and inhibition ratio. The X-axis shows the logarithm (base 10) concentration of ginsenoside Rg3, and the Y-axis shows the mean value of the inhibition ratio.

Figure 3.

Chromatogram and curve for standard dC and 5-MedC. (A) Chromatogram of standard nucleotide mixture. The same concentrations of a mixture of 5-MedC, dG, dT, dC and dA were diluted into various concentrations and detected using high performance liquid chromatography. dC and MedC are marked on the chart. The X-axis shows retention time and the Y-axis shows the voltage. (B) Linear regression curves for the dC and MedC. The X-axis shows the concentration of dC and MedC, and the Y-axis shows the peak area value. 5-MedC, 5-methyl-2′-deoxycytidine; dC, 2′-deoxycytidine.

Figure 4.

Alterations in DNA methylation in HepG2 cells treated with ginsenoside Rg3. HepG2 cells were left untreated, or were treated with 326.84 µg/ml 20(R)-ginsenoside Rg3 or 178.03 µg/ml 20(S)-ginsenoside Rg3, and the DNA was purified and hydrolyzed prior to detection using high performance liquid chromatography. The percentage of cytosine methylation was calculated, and the values in the treatment groups were compared with those in the untreated group. *P<0.05, vs. control.

Figure 5.

Effect of ginsenoside Rg3 on the gene expression and promoter region DNA methylation of P53, BCL2 and VEGF. (A) Effect of ginsenoside Rg3 on the mRNA expression of Bcl2, p53 and VEGF. The values in the treated groups were compared with those in the untreated control group (*P<0.05 and **P<0.01). (B) Example of the effect of ginsenoside Rg3 on DNA methylation at the promoter region of Bcl2. (C) Example of the effect of ginsenoside Rg3 on DNA methylation at the promoter region of VEGF. (D) Example of the effect of ginsenoside Rg3 on the DNA methylation at the promoter region of P53. BCL2, B cell lymphoma 2; VEGF, vascular endothelial growth factor; L, DNA ladder; U, undigested; M, digested with MspI; H, digested with HpaII.

Figure 6.

Effect of ginsenoside Rg3 on the expression of DNMT1 and DNMT3. *P<0.05 and **P<0.01, vs. untreated control. DNMT, DNA methyltransferase.