羟基喜树碱联合奥沙利铂诱导人大肠癌耐药细胞株LoVo/5-FU的凋亡机制

Combination of Hydroxycamptothecine with oxaliplatin in induction of drug-resistant colorectal cancer cell line LoVo/5-FU and mechanism of apoptosis

目的:体外观察羟基喜树碱(HCPT)联合奥沙利铂(L-OHP)对人大肠癌耐药细胞株LoVo/5-FU增殖抑制及诱导凋亡作用,并初步探讨其诱导凋亡方面机制。方法:MTT法测定HCPT联合L-OHP对LoVo/5-FU细胞增殖抑制作用,并根据中效原理判断两药合用时的效应。流式细胞仪观察两药对LoVo/5-FU细胞的凋亡诱导作用。半定量RT-PCR方法检测单药及联合作用后X染色体连锁的凋亡抑制蛋白(XIAP)mRNA表达变化。结果:HCPT联合L-OHP对LoVo/5-FU细胞有明显的抑制增殖作用,细胞增殖的抑制率与药物浓度呈正相关。在抑制率(fa)>0.18时合用指数(CI)<1,两药表现为协同作用。HCPT浓度分别为0.02、0.04和0.08 mg/L。作用48 h后,LoVo/5-FU细胞凋亡率分别为5.92±0.58、9.93±0.62和3.76±0.45。当0.04 mg/L HCPT联合L-OHP作用48 h后,其凋亡率为18.44±0.57,联合组与单药组比较差异有统计学意义,P<0.05。耐药细胞LoVo/5-FU较亲本细胞LoVo高表达XIAP mRNA(0.88±0.03vs0.11±0.02,P<0.01),两药联合作用后XIAP mRNA降低明显为0.21±0.03,P<0.01。结论:HCPT联合L-OHP能够协同抑制LoVo/5-FU细胞增殖,其机制可能与诱导细胞凋亡有关,抑制XIAP表达发挥作用。

OBJECTIVE： To investigate the possible mechanism of proliferation inhibition and apoptosis induced by hydroxycamptothecine（HCPT） and oxaliplatin（L-OHP） on cell line LoVo/5-FU in vitro.METHODS： Proliferation inhibition induced by HCPT and L-OHP on cell line LoVo/5-FU was tested by MTT method.Flow cytometry observed the role of HCPT as a single drug and in combination with L-OHP to induce the apoptosis in LoVo/5-FU cell line.Semi quantitative RT-PCR test was done to determine the expression of mRNA of X-linked inhibitor of apoptosis protein（XIAP） in LoVo/5-FU and LoVo cells after using HCPT and L-OHP as individual drug and in combination.RESULTS：HCPT combined with L-OHP significantly inhibited LoVo/5-FU cell proliferation,cell proliferation inhibition rate was positively correlatedwith drug concentration.When fa〉0.18,CI1,the two drugs showed synergy.When HCPT concentrations were 0.02,0.04 and 0.08 mg/L,the apoptosis rates of LoVo/5-FU cells were 5.92±0.58,9.93±0.62,3.76±0.45 after 48 h.Use concentration of HCPT 0.04 mg/L combined with L-OHP,the apoptosis rate were 18.44±0.57.Single drug groups vs combination group was statistically significant（P0.05）.Compared with LoVo,the LoVo/5-FU had high expression of XIAP mRNA（（0.88±0.03） vs（0.11±0.02）,P〈0.01）,two drugs combined significantly reduced XIAP mRNA for 0.21±0.03（P〈0.01）.CONCLUSION： HCPT combined with L-OHP can actedsynergistically inhibit the cell proliferation of LoVo/5-FU cell line,which may be related to inhibit the expression of XIAP,thus inducing apoptosis.