Growth Inhibition And Demethylation By A Component Of Natural Drug, CDP, In Cancer Cell Lines

Background and Objective Epigenetic mechanism results in the heritable silencing of genes without any changes in their nucleic sequences and it involved DNA methylation, RNA associated silencing and histone modification.It has been wildly accepted that hypermethylation of CpG islands in promoter is a vital mechanism for the silencing of many tumor suppressor genes (TSGs) and cancer related genes, such as p16, GSTP1 and E-cadherin. As well as known, methyl group is transferred from S-adenosylmethionine (SAM) to cytosine by DNA methyltransferases (DNMTs) and 5-methylcytisine is formed. Some inhibitors of DNMTs, 5-azacytidine and 5-aza-2'-deoxycytidine, can block the hypermethylation of the newly synthesized DNA strand, reverse hypermethylation and re-express silenced genes through covalent bonds with DNMTs. However, their clinical utility was limited to some extent by the side effects, such as mutagenesis, cytotoxicity and myelosuppression. There is a great need to finding effective and nontoxic substitutes for this kind of analogs. Based on previous studies, we reported here that the component of natural drug, CDP,an analog of SAM, would inhibit growth of four tumor cell lines and demethylation in promoter CpG island oipl6 gene.Methods Human hepatocellular carcinoma cell line Huh-7, breast cancer cell line T47D , MCF-7 and MDA-MB-435 were treated in the presence of different concentrations CDP (25,50,75,lOOumol/L) and DNA methyltransferase inhibitor, 5-azazcytidine(l,2,4,8,lOumol/L). The growth inhibition was assayed by MTT; the methylation status of promoter in pi 6 gene was analyzed by MSP(methylation-specific PCR).Results ?The cancer cell lines of Huh-7, T47D, MCF-7 and MDA-MB-435 were identified by MSP. The methylation specific bands of CpG islands \npl6, GSTP1, E-cadherin genes' promoters appeared in Huh-7 and T47D, MCF-7, MDA-MB-435, respectively. The unmethylation specific bands of the four genes were not existed in any of the four cell lines;(DAfter treatment with CDP at the presence of concentration of 25 ^ 50 > 75, 1 OOumol/L, significant inhibition of cell growth was observed in Huh-7, T47D and MCF-7 at all of the concentrations from the 2nd to the 7th day(p<0.05), while in MDA-MB-435 at the concentration of 50> 75 > 100umol/L from 3rd to the 6th day (p<0.05);?Compared with control group, the cell growth was significantly inhibited in Huh-7 at the present of concentration of K 2, 4> 8> lOumol/L of 5-aza-C from the 3rd to the 7th day (jxQ.OS), in T47D from 4rd to the 7th day (p<0.05), in MDA-MB-435 from the lnd to the 7th day (p<0.05), while in MCF-7 at the present of concentration of 2^ 8, lOumol/L from the 2nd to the 7th day (p<0.Q5);?Treated with 50umol/L or 75umol/L of CDP, the survival rate of MDA-MB-435 was the highest among these four tumor cell lines from the 1st tothe 4th day, and that of the MCF-7 was the lowest;?Treated with 4umol/L or 8umol/L of 5-aza-C, the survival rate of Huh-7 was the highest among these four tumor cell lines from the 1st to the 4th day, and that of the MDA-MB-435 was the lowest;?After treating the cells with 25, 5CK 75, lOOumol/L of CDP for 6 days, the methylation specific bands of CpG island in pi 6 gene' promoter were still existed in Huh-7 and T47D, while unmethylation specific bands appeared at all of the concentrations of CDP in Huh-7, while at the concentration of 5CK 75, 100umol/LinT47D;?Treated with 50umol/L of CDP, the methylation specific bands of CpG island in pi6 gene' promoter were still existed in Huh-7 and T47D, unmethylation spedific bands appeared at 12h and lasted to 144h in Huh-7 while at 72h and lasted to 144h.Conclusion Like 5-aza-C, CDP inhibited the proliferation of Huh-7, T47D, MCF-7 and MDA-MB-435 at a dose, time-dependent mode, and has the ability to reverse the hypermethylated pi6 genes. These results suggest that CDP has great potential in the development of effective anticancer drugs.