Vandetanib对人卵巢癌细胞抑制作用机制探讨

Inhibitory effect of Vandetanib on human ovarian cancer cells and its mechanism

目的探讨Vandetanib对人卵巢浆液性乳头状腺癌细胞株SKOV3和卵巢癌顺铂耐药细胞株SKOV3/DDP的增殖抑制作用及其机制。方法采用MTT法检测体外不同时间不同浓度的Vandetanib对SKOV3及SKOV3/DDP细胞的杀伤作用,计算半数抑制浓度(IC50);流式细胞术检测药物对细胞凋亡及其周期分布变化;采用蛋白质印迹法分析凋亡基因Bcl-2、Bax及反映肿瘤细胞侵袭力的基因MMP-2的蛋白表达变化。结果与对照组相比,Vandetanib能明显抑制SKOV3和SKOV3/DDP生长,呈时间-剂量依赖性,IC50值均呈明显减小趋势。32μmol/L的Vandetanib作用SKOV3和SKOV3/DDP 72h后,抑制率分别为(43.21±0.92)%和(56.74±1.09)%,64μmol/L的Vandetanib作用SKOV3和SKOV3/DDP72h后,抑制率分别为(55.37±1.21)%和(79.20±0.39)%,耐药株SKOV3/DDP的抑制率明显高于敏感株SKOV3,差异有统计学意义,P<0.05。流式细胞术结果显示,随时间浓度的增加,两种细胞凋亡明显增加,G1期细胞增加,S期和G2期细胞减少,差异有统计学意义,P<0.05。蛋白印迹法结果显示,药物作用于SKOV3细胞株Bcl-2、MMP-2表达减少,Bax表达无明显变化;药物作用于SKOV3/DDP细胞株Bcl-2表达减少,Bax表达增加,耐药株不表达MMP-2。结论 Vandetanib对两种细胞株生长有明显的呈时间及浓度依赖性的抑制作用,可以诱导细胞凋亡并将细胞阻滞于G1期,其机制与下调Bcl-2/Bax、MMP-2表达有关,有利于降低卵巢癌细胞的侵袭力。

OBJECTIVE To discuss the inhibiting effect and the mechanism of Vandetanib on human ovarian serous papillary adenocarcinoma cell lines SKOV3 and cisplatin-resistant human ovarian cancer cell lines SKOV3/DDP.METHODS MTT assay was used to evaluate the inhibition in different concentration and time of Vandetanib treatment on SKOV3 and SKOV3/DDP cells and the half inhibitory concentration（IC50）was calculated.Flow cytometry was used to detect the changes of cell cycle distribution and apoptosis.The changes of the expressions of protein Bcl-2,Bax,MMP-2were quantified by Western blot.RESULTS Vandetanib can inhibit the growth of SKOV3/DDP and SKOV3 significantly in a time-dose-dependent manner,and IC50 showed a decreasing trend.Inhibition percentage in SKOV3 and SKOV3/DDP cells exposed to 32μmol/L Vandetanib for 72 hrespectively were（43.21±0.92）% and（56.74±1.09）%,the inhibition of 64μmol/L were（55.37±1.21）% and（79.20±0.39）%.The inhibition rate of SKOV3/DDP was higher than that of SKOV3.The result from FCM showed that with increasing concentration the apoptotic rate was significantly increased,the percentage of G1-phase cells tend to increase and S-phase and G-phase to decrease in a time and concentration of Vandetanib dependent manner（P〈0.05）.Western blot showed that there were a decrease in Bcl-2and MMP-2protein expression of SKOV3,compared with control groups,but no significant changes in expression of Bax.There were decrease in Bcl-2protein expression of SKOV3/DDP and increase in Bax that did not express MMP-2.CONCLUSIONS Vandetanib can inhibit proliferation of SKOV3 and SKOV3/DDP cells.The effect may be ascribed to inducing cell apoptosis,the G1-phase arrest and the down-regulation of Bcl-2,MMP-2expression.Vandetanib can suppress MMP-2expression,it may reduce invasion force of ovarian cencer.