DNA甲基转移酶抑制剂对结肠癌细胞增殖、凋亡及细胞周期影响的体外研究

In vitro effect of DNA methyltransferase inhibitor on proliferation,apoptosis and cell cycle of colon cancer cells

目的探讨DNA甲基转移酶抑制剂SGI-1027对结肠癌LoVo细胞增殖、凋亡及细胞周期的影响。方法取对数生长期LoVo细胞,实验组加入终浓度分别为0.1、1、5、10μmol/L SGI-1027的培养液,对照组用含10%胎牛血清的RPMI1640培养基同步培养。MTT法检测SGI-1027作用于LoVo细胞24、48、72 h的增殖抑制率;倒置相差显微镜观察细胞形态学变化;SGI-1027处理后每个浓度收集细胞1×10-6个,采用膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）/碘化丙啶（PI）双染或PI单染流式细胞术分别检测SGI-1027处理后的细胞凋亡率和细胞周期时相分布情况;Western blotting检测SGI-1027对PI3K/Akt通路的影响。结果倒置相差显微镜观察显示,随SGI-1027浓度和作用时间的增加,LoVo细胞的凋亡形态学改变更为显著。除0.1μmol/L SGI-1027处理24 h后的LoVo细胞增殖抑制率与对照组无差异外,随着药物浓度的增加和作用时间的延长,SGI-1027对LoVo细胞的增殖抑制作用不断增强。与对照组比较,SGI-1027（0.1-10μmol/L）处理LoVo细胞24、48h的凋亡率均升高,且各浓度间的差异均有统计学意义（P〈0.05）。与对照组比较,1-10μmol/L SGI-1027处理后G0/G1期细胞比例及PTEN水平均高于对照组,S期和G2/M期细胞比例及Akt水平均低于对照组,差异有统计学意义（P〈0.05）。结论SGI-1027可抑制LoVo细胞的增殖并诱导细胞凋亡及G0/G1期阻滞,可能与其抑制PI3K/Akt通路激活有关,且SGI-1027作用的时间和浓度均对LoVo细胞恶性行为有影响。

Objective To investigate the in vitro effect of DNA methyltransferase inhibitor SGI-1027 on proliferation,apoptosis and cell cycle of colon cancer LoVo cells,as well as the possible mechanism. Methods The LoVo cells in the logarithmic growth phase were used in this study,and the LoVo cells exposured to SGI-1027 with final concentrations of 0. 1,1,5 and 10 μmol / L were chosen as experimental group. Inverted phase contrast microscope was used to observe morphology of LoVo cells. The cells in control group were synchronously cultured in RPMI 1640 with 10% fetal bovine serum. The proliferation inhibition rates of SGI-1027 on LoVo cells were detected by adding MTT solution after 24,48 and 72 h. After SGI-1027 treatment,1 × 10-6 cells per concentration were harvested for the detection of cell apoptosis rate and cell cycle phase distribution by flow cytometry with Annexin-V fluorescein isothiocyanate（ FITC） / propidium iodide（ PI） double staining and PI staining,respectively. The effect of SGI-1027 on PI3 K / Akt pathway was evaluated by Western blotting. Results Inverted phase contrast microscope showed that the morphological chages of LoVo cells were more obvious treated by SGI-1027 in dose and time dependent manner. In addition to the treatment with 0. 1 μmol / L SGI-1027 for 24 h,the proliferation inhibitory rates were increased in time and dose dependent manner. Compared with control group,the apoptotic rates of LoVo cells treated by SGI-1027（ 0. 1-10 μmol / L） at 24,48 h were all increased,and the differences between different concentrations were statistically significant（ P〈 0. 05）. Compared with control group,the proportion of G0/ G1 phase and the level of PTEN increased after SGI-1027 treatment（ 1-10 μmol / L）,but the proportions of both S phase and G2/ M phase cells as well as the level of Akt decreased（ P〈 0. 05）. Conclusion SGI-1027 could inhibit the proliferation of LoVo cells and induce apoptosis and G0/ G1ar-rest,and the time and concentration of SGI-1027 treatment have effect on the malignant behavior of LoVo cells.