RhoA/ROCKI信号通路在紫杉醇诱导宫颈癌细胞周期阻滞中的作用

Effects of RhoA/ROCKI signal pathway on the taxol-induced cell-cycle arrest of cervical cancer cells

目的探讨RhoA/ROCKI信号通路在紫杉醇诱导宫颈癌细胞周期阻滞中的作用及其分子机制。方法 MTT法确定能引起细胞生长显著性抑制最小浓度(1μmol/L)的紫杉醇。流式细胞仪检测细胞周期和RhoA蛋白表达,Western blotting检测ROCKI、细胞周期蛋白(cyclins)及周期依赖性激酶(CDKs)的表达。结果紫杉醇处理C33A细胞后,G2/M细胞数和RhoA/ROCKI蛋白表达显著性增加,并呈时间依赖性;而cyclin B1、A、D1和CDK1蛋白表达显著性降低。上述现象被RhoA抑制剂C3转移酶全部反转;却被ROCKI蛋白抑制剂Y-27632部分反转,但与对照相比差异仍显著。紫杉醇或RhoA/ROCKI抑制剂对CDK2的表达无显著性影响。结论紫杉醇诱导了宫颈鳞癌C33A细胞的G2/M期阻滞,其机制与RhoA活性增强后抑制特异的cyclins和CDKs有关,而下游通路中ROCKI活性仅起到部分作用。

Objective To explore the effects of RhoA/ROCKI signal pathway in taxol-induced cell cycle arrest of cervical cancer cells. Methods The MTT method was used to determine the minimal inhibition concentration of taxol in C33A cells. Cell cycle and expression of RhoA protein were assayed by flow cytometry. Expressions of ROCKI, cyelins and eyelin-dependent kinases （CDKs） were determined by Western blotting. Results After cells were treated with 1μmol/L of taxol, the cell number of G2/M phase and the expression of RhoA/ROCKI protein increased dramatically in a time-dependent manner. However, the expressions of CDK1, cyclin B1, A and D1 proteins were decreased markedly. Moreover, these effects were totally reversed when cells pretreated with the inhibitor of RhoA （ C3 transferase） , and partly reversed by the treatment of ROCKI inhibitor （ Y-27632）, although the significance still existed as compared with the control. No marked effects were observed on the expression of CDK2 in cells treated with taxol or RhoA/ROCKI inhibitors. Conclusion Taxol-induced G2/M arrest in C33A cells was related to the decreases of specific cyelins and CDK, which is tightly regulated by the increased RhoA activity, but ROCKI may only play the role partly in this process.