S-adenosylmethionine inhibits the growth of cancer cells by reversing the hypomethylation status of c-myc and H-ras in human gastric cancer and colon cancer

Abstract

A global DNA hypomethylation might activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-Adenosylmethionine (SAM) serves as a major methyl donor in biological transmethylation events. The object of this study is to explore the influence of SAM on the status of methylation at the promoter of the oncogenes c-myc, H-ras and tumor-suppressor gene p16 (INK4a), as well as its inhibitory effect on cancer cells. The results indicated that SAM treatment inhibited cell growth in gastric cancer cells and colon cancer cells, and the inhibition efficiency was significantly higher than that in the normal cells. Under standard growth conditions, C-myc and H-ras promoters were hypomethylated in gastric cancer cells and colon cancer cells. SAM treatment resulted in a heavy methylation of these promoters, which consequently downregulated mRNA and protein levels. In contrast, there was no significant difference in mRNA and protein levels of p16 (INK4a) with and without SAM treatment. SAM can effectively inhibit the tumor cells growth by reversing the DNA hypomethylation on promoters of oncogenes, thus down-regulating their expression. With no influence on the expression of the tumor suppressor genes, such as P16, SAM could be used as a potential drug for cancer therapy.

Keywords: S-Adenosylmethionine; cancer therapy; epigenetics; gastric cancer; hypomethylation.

Fig 1

MTT assay showing cell growth viability of cancer cells and normal cells in response to SAM treatment. (A) Gastric cancer cells (MGC-803). (B) Colon cancer cells (HT-29). (C) Normal cells (normal chang liver cells). * p <0.05.

Fig 2

The colony formation assay showing cell growth inhibition of cancer cells and normal cells in response to SAM treatment. Representative colony images are shown. (A), (B) with or without SAM treatment in gastric cancer cells (MGC-803). (C), (D) with or without SAM treatment in colon cancer cells (HT-29). (E), (F) with or without SAM treatment in normal cells (normal chang liver cells). (G), (H), (I) data analysis of number of colony. * p <0.05.

Fig 3

The analysis of methylation status of c-myc, H-ras and P16(INK4a) promoter by MSP assay in cancer cells MGC-803 and HT-29 and normal cells with or without SAM treatment. (A) c-myc promoter. (B) H-ras promter. (C) p16 promoter. M, DNA ladder markers; ME, amplified by methylated primers; UM, amplified by non-methylated primers.

Fig 4

Quantitative RT-PCR analysis of c-myc, H-ras and p16 expression level in cancer cells and normal cells with or without SAM treatment. (A) c-myc mRNA expression. (B) H-ras mRNA expression. (C) p16 mRNA expression. The ratio of c-myc, H-ras and p16 mRNA expression and GAPDH mRNA expression was shown. * p <0.05.

Fig 5

Western blot analysis of C-MYC, H-RAS and P16 expression level in cancer cells and normal cells with or without SAM treatment. (A) C-MYC, H-RAS, P16 and Actin protein expression. (B) Data analysis. The ratio of C-MYC, H-RAS and P16 protein expression and Actin expression was shown. * p <0.05.

Fig 6

Expression of C-MYC, H-RAS and P16 protein assayed by cell immunofluorescence in cancer cells and normal cells with or without SAM treatment. The cells with expressed C-MYC (A), H-RAS (B) and P16 (C) were stained as a green fluorescence or red fluorescence, respectively. The statistical analysis of expression of C-MYC,H-RAS and P16 protein assayed by cell immunofluorescence was shown in (D), (E), and (F) respectively. * p <0.05. a) The cells scored by a bright field microscopy (× 200); b) C-MYC or H-RAS positive cells scored by counting the number of cells with green fluorescence (× 200).