摘要
目的:研究大黄素衍生物A对卵巢癌淋巴结定向高转移细胞SKOV3-pm4的增殖、迁移作用及对Rac1、CDC42基因及蛋白表达的影响。方法实验分为空白对照组、不同浓度药物组(大黄素衍生物A)、Rac1抑制剂组、Rac1激活剂组。以四甲基偶氮唑蓝(MTT)法测定大黄素衍生物A对SKOV3-pm4细胞增殖的抑制作用。免疫荧光法观察SKOV3-pm4细胞中Rac1和CDC42蛋白的分布情况。用细胞划痕实验观察SKOV3-pm4细胞迁移情况,分别采用实时荧光定量PCR (RT-PCR)技术和蛋白免疫印迹法(Western blot)检测细胞中Rac1、CDC42基因和蛋白的表达水平。结果 MTT法检测显示大黄素衍生物A(5~50 mg/L)能抑制SKOV3-pm4细胞的增殖,24 h的半数抑制浓度(IC50)为34.46 mg/L。细胞划痕实验显示4 mg/L的大黄素衍生物A能抑制SKOV3-pm4细胞迁移,且呈时间±赖性。免疫荧光观察到细胞中Rac1和CDC42蛋白均广泛分布于细胞质中,Rac1蛋白在细胞核周围较为密集。RT-PCR和Western blot法检测结果显示随着大黄素衍生物A药物浓度的梯度增加,Rac1基因和蛋白的表达水平均降低,与空白对照组比较,差异均有统计学意义(P均<0.05),而CDC42基因和蛋白的表达变化不明显。结论大黄素衍生物A能抑制SKOV3-pm4细胞增殖和迁移能力,其作用途径可能是通过调控Rac1信号传导通路而实现;Rac1可能是大黄素衍生物A潜在的作用靶点。
Objective To study the effects of Emodin Derivative A on SKOV3-pm4 ovarian cancer cells with lymph node-oriented metastasis,particularly on expression of Rac1 and CDC42 mRNA and protein. Methods SKOV3-pm4 cells were treated for 24 h with different concentrations of Emodin Derivative A in the presence or absence of Rac1 inhibitor or activator. Cell proliferation was assessed by MTT assay,cell migration,by scratch assay;Rac1 and CDC42 expression,by RT-PCR and Western blotting;and distribution of these two proteins,by immunofluorescence. Results Emodin Derivative A at doses of 5-50 mg/L inhibits the proliferation of SKOV3-pm4 cells (IC50 34.46 mg/L). Rac1 and CDC42 protein were present throughout the cytoplasm,though Rac1 staining was denser around the nucleus than in other parts of the cell. Expression of Rac1 mRNA and protein decreased as the dose of Emodin Derivative A increased. CDC42 expression,however,did not change significantly with drug dose. Emodin Derivative A at a dose of 4 mg/L inhibited cell migration. Conclusions Emodin Derivative A can inhibit migration and proliferation of SKOV3-pm4 cells,and the drug may act by targeting Rac1.
出处
《中国癌症防治杂志》
CAS
2015年第3期166-172,共7页
CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT
基金
国家自然科学基金资助项目(81360502)
广西自然科学基金资助项目(2014GXNSFAA118161)