重楼皂苷Ⅶ对人结肠癌细胞株的抑制增殖及诱导凋亡作用研究

Polyphyllin Ⅶ inhibits proliferation of colon cancer cells and induces apoptosis

目的研究重楼皂苷Ⅶ(PP7)对人结肠癌细胞的增殖抑制作用及诱导凋亡机制。方法采用不同浓度的PP7作用于人结肠癌细胞株HCT116,以噻唑蓝法检测细胞增殖活力,倒置显微镜观察细胞形态变化;软琼脂克隆形成实验检测细胞集落形成能力;流式细胞法检测细胞凋亡;蛋白质印迹法检测凋亡相关蛋白半胱氨酸蛋白酶3(Caspase-3)和多聚二磷酸腺苷核糖聚合酶(PRAP)的表达。结果噻唑蓝法检测结果显示PP7可抑制HCT116细胞增殖,且抑制作用具有浓度剂量依赖性,二甲基亚砜以及PP7 1、2μmol/L处理后细胞增殖活力依次为(95. 8±3. 5)%、(61. 7±5. 8)%、(38. 6±4. 5)%,两两比较差异均有统计学意义(均P <0. 05)。PP7能明显抑制HCT116细胞的克隆形成能力,尤其PP7 2μmol/L浓度处理后不能形成任何菌落。PP7处理24 h后细胞凋亡明显增加,且Caspase-3和PRAP相对表达水平随PP7浓度(0、1、2μmol/L)增加而增加[Caspase-3:1. 00比(1. 86±0. 23)、(2. 57±0. 37),PRAP:1. 00比(2. 18±0. 34)、(2. 96±0. 45)],两两比较差异均有统计学意义(均P <0. 05)。结论 PP7能抑制人结肠癌HCT116细胞生长及诱导细胞发生凋亡,提示PP7可能是一种新的结肠癌治疗药物。

Objective To investigate the mechanism of polyphyllin Ⅶ( PP7) inhibiting proliferation of human colon cancer cells and promoting cell apoptosis. Methods Human colon cancer cell line HCT116 was treated with different concentrations of PP7. Cell proliferation was tested by methyl thiazolyl tetrazolium( MTT)assay. Morphology changes of cells were observed through inverted microscope. Cell colony formation assay was performed using soft agar medium. Cell apoptosis was detected by flow cytometry,and apoptosis-related proteins such as Caspase-3 and poly adenosine diphosphate-ribose polymerase( PRAP) were detected by western blotting.Results MTT assay showed that PP7 inhibited proliferation of HCT116 cells in a dose-dependent manner;cell proliferation activity was( 95. 8 ± 3. 5) % for dimethyl sulfoxide,( 61. 7 ± 5. 8) % for 1 μmol/L PP7,and( 38. 6 ± 4. 5) % for 2 μmol/L PP7,with significant differences among them( P < 0. 05). PP7 could significantly inhibit cell colony formation and no colony was observed after 2 μmol/L PP7 treatment. Cell apoptosis was significantly promoted after 24 hours of PP7 treatment. Caspase-3 and PRAP expressions increased with the concentration( 0,1,2 μmol/L) of PP7[Caspase-3: 1. 00 vs( 1. 86 ± 0. 23) vs( 2. 57 ± 0. 37);PRAP: 1. 00 vs( 2. 18 ±0. 34) vs( 2. 96 ± 0. 45) ]. Conclusion PP7 can inhibit growth and promote apoptosis of human colon cancer cell line HCT116,suggesting that PP7 may be a new choice in treating colon cancer.