[Ginsenoside 20(S)-Rg3 upregulates tumor suppressor VHL gene expression by suppressing DNMT3A-mediated promoter methylation in ovarian cancer cells]

Abstract

Objective: To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau (VHL) gene in ovarian cancer cells.

Methods: Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL, DNMT1, DNMT3A and DNMT3B by real-time PCR and Western blotting, respectively. The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, were detected using real-time PCR. VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells. VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice.

Results: Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels (P < 0.05) and upregulated DNMT3B expression only at the mRNA level, but did not cause significant changes in either the mRNA or protein level of DNMT1. Treatment of the cells with 2 and 5 μmol/L 5-Aza-CdR obviously increased VHL mRNA expression by by over 3 folds (P < 0.05). 20(S)-Rg3 significantly decreased the methylation level in the promoter region of VHL gene, and this effect was abrogated by DNMT3A overexpression in the cells (P < 0.05). Immunohistochemisty showed a significantly increased VHL expression but a lowered DNMT3A expression in subcutaneous SKOV3 cell xenografts in 20 (S)-Rg3-treated nude mice.

Conclusions: Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.

Keywords: ginsenoside; methylation; ovarian cancer; von Hippel-Lindau.

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20(S)-Rg3上调卵巢癌细胞SKOV3的VHL 20(S)-Rg3 upregulates VHL in ovarian cancer cell line SKOV3. A: Real-time PCR shows elevatated VHL mRNA level in 20(S)-Rg3-treated cells; B: Western blotting shows increased VHL protein level in 20(S)-Rg3-treated cells. ^*P < 0.05.

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20(S)-Rg3降低VHL基因启动子区的甲基化水平 20(S)-Rg3 reduces methylation level in VHL gene promoter.A: Real-time PCR shows increased VHL mRNAlevel in 5-Aza-CdRtreated SKOV3 cells; B: The promoter region of VHL gene is predicted to contain CpG islands; C: MSP results show significantly decreased methylation level in the promoter region of VHL gene by 20(S)-Rg3. U=Unmethylated. M=Methylated. ^*P < 0.05.

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20(S)-Rg3通过抑制DNMT3A介导的DNA甲基化而上调VHL 20(S)-Rg3 upregulates VHL via suppressing DNMT3A-mediated methylation in the promoter region of VHL gene. A: Real-time PCR results show that 20(S)-Rg3 treatment decreases DNMT3A mRNA level and increases DNMT3B mRNA level without affecting DNMT1 mRNA in the cells; B: Western blotting results show that 20(S)-Rg3 treatment decreases DNMT3A and increases DNMT3B protein expressions without affecting DNMT1 expression in the cells; C: Real-time PCR shows reduced VHL mRNA level in 20(S)-Rg3-treated cells overexpressing DNMT3A; D: Western blotting results show lowered VHL protein expression in 20(S)-Rg3-treated cells overexpressing DNMT3A; E: MSP results show increased methylation level in the promoter region of VHL gene in 20(S)-Rg3-treated cells overexpressing DNMT3A. U=Unmethylated. M= Methylated. Ns : non-significant; ^*P < 0.05.

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20(S)-Rg3治疗的SKOV3细胞裸鼠皮下移植瘤组织中VHL表达增强而DNMT3A表达减弱 Immunohistochemical staining showing increased VHL and lowered DNMT3A expressions in subcutaneous tumors derived SKOV3 cells in 20(S)-Rg3-treated nude mice (Original magnification, ×200; insets, ×400).