手霉素联合顺铂对人卵巢癌细胞3AO的增殖抑制作用

Inhibitory effects of manumycin combined with cisplatin on the proliferation of human ovarian cancer cell line 3AO in vitro and in vivo

目的:研究手霉素联合顺铂对人卵巢癌细胞3AO体外增殖及裸鼠移植瘤生长的抑制作用,并探讨其机制。方法:MTT法检测手霉素及手霉素联合顺铂对3AO细胞的体外增殖抑制作用;建立人卵巢癌裸鼠移植瘤模型,采用免疫组织化学SP法检测手霉素及手霉素联合顺铂对移植瘤组织survivin、NF-κB、血管内皮生长因子(vascular endothelial growth factor, VEGF)和caspase-3蛋白的表达,FCM检测移植瘤细胞周期和细胞凋亡率的变化。结果:手霉素对3AO细胞的增殖有明显抑制作用,且呈时间和剂量依赖效应(P<0.05)。手霉素可增强顺铂对3AO细胞的增殖抑制作用,随着手霉素浓度的增加(10～40 μmol/L),对3AO细胞的增殖抑制作用增强。手霉素或顺铂单药组以及联合用药组的裸鼠移植瘤体积均明显小于对照组,其中联合用药组裸鼠移植瘤体积明显小于手霉素或顺铂单药组(P<0.05)。各给药组裸鼠移植瘤组织中survivin、NF-κB和VEGF蛋白的表达水平均低于对照组(P<0.05),其中联合用药组又明显低于各单药组(P<0.05);各给药组caspase-3蛋白的表达水平均明显高于对照组(P<0.05)。手霉素单药组、顺铂单药组和手霉素联合顺铂组的细胞凋亡率依次升高,与对照组相比差异均有统计学意义(P<0.05);与对照组相比,G0/G1期细胞依次减少(P<0.05),G2/M期细胞依次增多(P<0.05),S期细胞变化不明显。结论:手霉素联合顺铂可明显抑制3AO细胞的体内外增殖并诱导其凋亡,其作用机制可能与下调survivin、NF-κB和VEGF以及上调caspase-3蛋白的表达有关。

Objective：To investigate the inhibition effects of manumycin combined with cisplatin（DDP） on the proliferation of human ovarian cancer cell line 3AO and the growth of transplantation tumors in nude mice,and to analyze its possible mechanisms.Methods：MTT colorimetric assay was performed to evaluate the cytotoxic effect of manumycin combined with DDP on 3AO cells.The model of nude mice bearing ovarian transplantation tumor was established.The expressions of survivin,NF-κB,vascular endothelial growth factor（VEGF） and caspase-3 proteins were detected by immunohistochemical method.The cell cycle distribution and apoptosis rate were examined by flow cytometry.Results：The proliferation of 3AO cells was inhibited by manumycin in a time-and dose-dependent manner（P0.05）.The manumycin of 10,20,40 or 60 μmol/L could strengthen the cytotoxic effect of DDP on 3AO cells.The tumor size in nude mice of each drug-treated group was significantly smaller than that of the control（untreated） group（P0.05）.Compared with the DDP alone and the manumycin alone group,the expressions of survivin,NF-κB and VEGF proteins were significantly down-regulated while the expression of caspase-3 protein was significantly up-regulated（P0.05） in the combination（manumycin plus DDP） group（P0.05）.The apoptosis rates were increased gradually in the control group,manumycin group,DDP group and the combination group.In each drug-treated group,the number of cells in G0/G1 phase was decreased significantly,while which in G2/M phase was increased significantly（P0.05）,as compared with the control group.The number of cells in S phase did not show any changes in each group.Conclusion：The combination of manumycin and DDP shows an enhanced ability to inhibit the proliferation of human ovarian cancer cells 3AO and increase their apoptosis.This effect may be associated with the down-regulation of survivin,NF-κB and VEGF expressions and the up-regulation of caspase-3 expression.