| DNA methylation is the most prevalent modification to the DNA of the eukaryotic cells and there are a lot of ways of the modification to the DNA base. Recently, DNA methylation is the hotspot to be pain close attention in the domain of the embryology and oncobiology. The 5mC in the molecular of the DNA is very important for the differentiation and development of the embryo. It is reported that the DNA methylase made a great role in the tumorigenesis and the tumor development, and DNA methylase inhibitors could deteriorate the DNA methylation, include the prevention of the mutation of the methylated CpG, the reactivation of the methylated genes and so on, to act the role of the anti-tumor activity. DNA methylase inhibitors include three kinds: cytosine analogue, SAM analogue, SAM metabolism inhibitors. Cytosine analogue was taken more interest in the study of the domain of the anti-tumor treatment as DNA methylase inhibitors. 5-Aza-5′-deoxycytidine as a new one of the cytosine analogue, the synthesis and pharmacologic actions and other concerning had not been reported. This paper is aimed to explore the way of 5-Aza-5′-deoxycytidine synthesis and to separate theα,βisomer and to apply HPLC into measuring the content of the single isomerαorβ. And then the MTT assay was used to study the growth suppression of the single isomerαorβto the hepatoma carcinoma cell BEL-7402 and the gastric carcinoma cell SGC-7901 and to detect growth curves of cells. Flow cytometry was adopted to discover the influence of the single isomerαorβto the cell cycle distribution of the above two cells.The main contents were divided into two sections as follows:1. Study on the synthesis, separation and assay of the 5-Aza-5′-deoxycytidine. To synthesis 5-Aza-cytosine with the cyanoguanidine as raw material and 1, 2, 3-O-triactyl-5-deoxy-D-ribofuranose was prepared from the reaction of the breakage of the glucosidic bond of 5'-Deoxy-2', 3'-diactyl-inosine, which was gained from inosine by halogenation, hydrogenization and acetylation in turn. 5-Aza-2′, 3′-O-biactyl -5′-deoxy cytidine was synthesized through the kondensation action with the above two intermediates and the single isomerαandβwere separated by the silica gel chronmatographic column. The title compound 5-Aza-5′-deoxy cytidine was prepared by the ammonolysis of the single isomerαandβ5-Aza-2′, 3′-O-biactyl -5′-deoxycytidine, and theλmax was detected by the ultraviolet scanning. HPLC method is adopted to determine the content of the isomerαandβ5-Aza-5′-deoxycytidine. Chromatography conditions are as follows: Hypersil ODS reversed-phase chromatographic column (4.6mm×200mm, 5μm); methanol: water (70:30) as the mobile phase; flow rate is 1ml·min-1; detection wavelength is set at 223nm (β), 210nm (α). At the result, the content of theα5-Aza-5′-deoxycytidine is 98.8% and theβisomer is 99.6%.2. Study on the antitomour activity of the 5-Aza-5'-deoxycytidineThe hepatoma carcinoma cell BEL-7402 and gastric carcinoma cell SGC-7901 lines were treated with different concentrations (0, 5, 10, 50, 100, 200μmol/L) of theαandβisomers of 5-aza-5'dC to explore the antitumor activity of 5-aza-5'dC. And then MTT assay was used to detect the growth curves of cells. Flow cytometry was adopted to discover cell cycle distribution. As result Proliferation of the hepatoma carcinoma cell BEL-7402 and gastric carcinoma cell SGC-7901 were both inhibited by theβisomer 5-aza-5'dC treatment, while the 50% inhibiting concen- tration to the hepatoma carcinoma cell BEL-7402 (IC50: 32.6μmol/L) were lower than to the gastric carcinoma cell SGC-7901 (IC50: 75.7μmol/L), and theαisomer was inferior, the 50% inhibiting concentration to the two cell lines both over 100μmol/L. The hepatoma carcinoma cell BEL-7402 blocked in G0/G1 phase and G2/M phase, while SGC-7901 was blocked in G2/M phase. In conclusion, to the hepatoma carcinoma cell BEL-7402 and the gastric carcinoma cell SGC-7901,βisomer 5-aza-5'dC can inhibit the proliferation and block cell cycle. |
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