雪胆素乙通过破坏微丝结构和促进p21Cip1表达抑制前列腺癌PC-3细胞的增殖

摘要

OBJECTIVE To explore effects of cucurbitacin Ⅱb (Cu Ⅱb) on human prostate cancer PC-3 cells and its underlying mechanism. METHODS The proliferation of cells was detected by MTS assay after PC-3 cells were treated with CuⅡb 0. 064 -200 μmol·L-1 for 48 h. After treated with Cu Ⅱb 2 and 20 μmol·L-1 for 24 h, cell morphologic changes were observed under phase contrast microscopy. After exposed with Cu Ⅱb 2 and 20 μmol·L-1 for 48 h, cell cycle distribution was measured by flow cytometry using propidium iodide ( PI) staining. When cultured with Cu Ⅱb 20 μmol·L -1 for 1, 4 and 24 h dividedly, the changes of microfilament and microtubule structures were assessed by immunofluorescence staining. After separately treated with CuⅡb 20μmol·L-1 for 1, 4 and 24 h, the protein expression levels of F-actin, G-actin, p21Cip1, cyclin A, cyclin B1, cyclin D1 and cyclin E were detected by western blotting ( P < 0. 05 ). RESULTS CuⅡb inhibited the proliferation of PC-3 cells in a concentration-dependent manner. CuⅡb 20 μmol · L-1l increased the cell rates of G2/M phase (tetraploid )to(45.3±1.8)% from ( 27.7 ± 1.5)% in control group (P < 0. 01 ) , indicating an arrest of cell cycle progression. The cells became shrunk after Cu H b treatment. Meanwhile, CuⅡb 20 μmol · L-1 decreased G-actin levels and induced severe F-actin aggregation(P <0.05) , but had minimal effect on the microtubules. In addition, CuEb 20 μmol · L-1 also elevated p21Cip1 expression and downregulated cyclin A level in PC-3 cells, whereas the other cyclins were slightly upregulated(P <0. 05). CONCLUSION CuⅡb can significantly inhibit the proliferation of human prostate cancer PC-3 cells probably through induction of actin aggregation, disruption of microfilaments, upregulation of tumor suppressor p21Cip1 expression and arrest of cell cycle progression.%目的 分析雪胆素乙(CuⅡb)对人前列腺癌PC-3细胞增殖及细胞周期的影响,探讨其作用机制.方法 MTS法检测CuⅡb0.064～200μmol· L-1作用48 h后PC-3细胞增殖;CuⅡb2和20 μmol·L-1 作用24 h,相差显微镜观察细胞形态;CuⅡb2和20 μmol·L-1作用48 h,流式细胞术分析细胞周期分布;免疫荧光染色分析CuⅡb 20 μmol·L-1分别作用1,4和24h后微丝和微管细胞骨架变化;免疫印迹法测定CuⅡb20 μmol·L-1分别作用1,4和24 h后G肌动蛋白、F肌动蛋白、p21 Cip1及细胞周期蛋白A,B1,E和D1的表达.结果 CuⅡb以浓度依赖方式抑制PC-3细胞的增殖(r=0.9817,P＜0.05).CuⅡb 20 μmol·L-1作用48 h,使细胞周期阻滞于G2/M期,从溶剂对照组的(27.7±1.5)％上升为(45.3±1.8)％(P＜0.01),相差显微镜见细胞发生明显收缩.CuⅡb 20 μmol· L-1作用24 h,使G肌动蛋白水平显著下降,F肌动蛋白发生严重聚集(P＜0.05),而对微管只有轻微影响.与溶剂对照组相比,CuⅡb 20 μmol· L-1作用24 h后,细胞p21Cip1表达明显升高,细胞周期蛋白A表达显著下调,其他细胞周期蛋白表达上调(P＜0.05).结论 CuⅡb能明显抑制人前列腺癌PC-3细胞的增殖,其机制可能是通过诱导肌动蛋白聚集,破坏微丝骨架,促进抑癌因子p21Cip1表达,阻滞细胞周期的进程.