| Objective: To detect the effect of different concentrations of S-adenosyl-L-methionine(SAMe)on proliferation and migration of HepG2 cells.Then exploratorily research the mechanism of SAMe on regulating the transcription,expression and subcellular distribution of oncogene C-myc at the molecula level,which should be a basis for the further study of SAMe in the treatment of the liver cancer drugs. And the study also provides a more feasible direction for the therapeutic pharmaceuticals of the liver cancer.Methods: HepG2 cells were cultured in vitro, 10 groups of different concentrations(2.5,5.0,7.5,10.0,12.5,15.0,17.5,20.0,22.5,25.0μmol/L) of SAMe were used to stimulate HepG2 cells. MTT was used to detect the proliferative capability of the HepG2 cells per day until 8 days treatment.Then screening out the best half inhibitory concentration (IC50) of the SAMe applying to the HepG2 cells line in the shortest time.And at this concentration of the SAMe, Wound Healing Assay was used to test the alteration of the HepG2 cells' migration ability.HepG2 cells in logarithmic growth phase were singled out,and then cultured the cells in vitro by using the SAMe of drug half inhibitory concentration (IC50) for 5 days. After that, RT-PCR,Western Blotting and Immunocytochemistry were used to measure the alteration of the oncogene C-myc's transcription, expression and the variation of its protein's subcellular localization.Results: 10 different concentrations of SAMe were used to treat HepG2 cells for 8 days and cell proliferation were detected every 24h.The half inhibitory concentration (IC50) of SAMe were 24.1μmol / L, 22.5μmol / L, 19.3μmol / L, 17.1μmol / L, 15.2μmol / L, 14.2μmol / L, 12.8μmol / L, 11.2μmol / L respectively ,with time and the double dose dependent manner.The best IC50 of SAMe treated to the HepG2 cells within the shortest time was the concentration of 15.2μmol / L for 5 days,which was coincidant with that in relevant references,Under the treatment of the SAMe(15.2μmol / L,5d),the migration of HepG2 cells was also influenced, the scratch healing rate of the SAMe treated cells' group was only 76.78% compared with the untreated group, this difference had a statistically significant (P = 0.04 *: P <0.05); In addition, with half inhibition concentration (IC50) of SAMe(15.2μmol / L) 5-days treatment,the transcription level of the C-myc oncogene's mRNA of HepG2 cells decreased significantly, the SAMe treated group was only 58.3%(P = 0.012 *: P <0.05) compared with the untreated group; the result of the Western Blotting detection also showed that the C-myc protein expression of SAMe treatment group was decreased to 52.6% (P = 0.0017 *: P <0.05) of that of the untreated group. However, from immunocytochemistry testing result,we found that the C-myc protein located in the cytoplasm of the HepaG2 cells, positioning of the C-myc protein has not changed between the SAMe treatment group and the untreated group.Conclusions: 1. The SAMe can inhibit the proliferation and migration of the HepG2 cells with dose and time dependent manner. 2. As one chemical compound, SAMe can significantly regulate the transcription and the protein expression of the oncogene C-myc;however,no subcellular relocalization of the oncogene C-myc protein in the liver cells after treated with SAMe.In general,we demonstrate that proliferation and migration of HepG2 cells ae change after treayted by SAMe,as well as the transcription and expression of oncogene C-myc are up-regulated,indicating that the SAMe may has a broad prospect in the research of the treatment of the liver cancer drugs. |
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