mTOR信号通路调控结直肠癌LoVo/ADR细胞多药耐药性的可能机制

目的:通过雷帕霉素(rapamycin,RAPA)抑制哺乳动物雷帕霉素靶分子(mammalian target of rapamycin,mTOR)通路,观察结直肠癌多药耐药LoVo/ADR细胞对多柔比星的敏感性、自噬、凋亡及多药耐药基因1(multidrug resistance gene 1,MDR1)表达的变化,探讨mTOR通路调控结直肠癌多药耐药的可能机制.方法:多柔比星联合RAPA作用后,MTT法检测多柔比星对LoVo/ADR细胞的半数抑制浓度(half inhibitory concentration,IC50)值;多柔比星和RAPA单独或联合作用后,在透射电子显微镜和荧光显微镜下观察LoVo/ADR细胞自噬体的形成,FCM分析细胞的自噬率和细胞凋亡率；RAPA作用后,RT-PCR和免疫细胞化学法分别检测LoVo/ADR细胞中MDR1 mRNA和P-糖蛋白的表达.结果:25和50 μmol/L RAPA作用下,多柔比星对LoVo/ADR细胞的IC50值均低于不加RAPA的对照组,差异有统计学意义(P＜0.05).对照组LoVo/ADR细胞中很少或几乎看不到自噬体或点状绿色荧光分布,细胞自噬率为(2.9±0.4)％;多柔比星组和RAPA组LoVo/ADR细胞中可见自噬体形成,散在点状绿色荧光分布于细胞质及细胞核周围,细胞自噬率分别为(35.5±5.4)％和(46.7±6.7)％,与对照组比较差异有统计学意义(P＜0.05)；多柔比星联合RAPA组LoVo/ADR细胞中可见大量自噬体形成,细胞的自噬率为(73.1±7.4)％,显著高于多柔比星或RAPA单独作用组(P＜0.05).RAPA组和多柔比星组的细胞凋亡率分别为(2.06±0.43)％和(48.39±6.47)％,多柔比星组的细胞凋亡率高于对照组(2.23±0.50)％(P＜0.05)；多柔比星联合RAPA组的细胞凋亡率为(79.43±8.28)％,高于多柔比星组(P＜0.05).RAPA作用后,LoVo/ADR细胞中MDR1 mRNA和P-糖蛋白表达下调.结论:抑制mTOR通路具有逆转结直肠癌细胞多药耐药性的作用,其机制可能与RAPA促使耐药细胞自噬、凋亡及下调MDR1 mRNA的表达有关.

The possible mechanism of mTOR signaling pathway regulating multidrug resistance of colorectal cancer LoVo/ADR cells

Objective: To observe the variation of ADR (adriamycin) sensitivity, autophagy, apoptosis and the expression of MDR1 (multidrug resistance 1) gene of colorectal cancer LoVo/ADR cells after RAPA (rapamycin)-induced inhibition of mTOR (mammalian target of rapamycin) signaling pathway, and to explore the possible mechanism of mTOR signaling pathway regulating the MDR. Methods: The IC50 (half inhibitory concentration) value of ADR for LoVo/ADR cells after treatment with combination of ADR and RAPA was measured by MTT assay. The autophagosomes were observed under a transmission electron microscope and a fluorescence microscope, and the autophagic rate and apoptosis rate were measured by FCM (flow cytometry) after the LoVo/ADR cells were treated with ADR alone and RAPA alone or in combination. After RAPA treatment, the expressions of MDR1 mRNA and P-gp (P-glycoprotein) were detected by RT-PCR and immunocytochemistry, respectively. Results: The IC50 values of ADR for LoVo/ADR cells after treatment with RAPA of 25 and 50 μmol/L were lower than those for the untreated cells (P < 0.05). Autophagosomes and green fluorescence dots were rarely observed in the untreated LoVo/ADR cells, and the autophagic rate was (2.9±0.4) %; whereas in the LoVo/ADR cells treated with ADR alone and RAPA alone, the autophagosomes and green fluorescence dots which distributing in cytoplasm and around the nucleus were observed, and the autophagic rates were (35.5±5.4)% and (46.7±6.7)%, respectively, which differed significantly from those of the untreated cells (P < 0.05). The autophagosomes appeared abundantly in the LoVo/ADR cells after combined treatment with ADR and RAPA, and the autophagic rate was (73.1±7.4)%, which was significantly higher than those in the LoVo/ADR cells treated with ADR alone and RAPA alone (P < 0.05). The apoptosis rates of LoVo/ADR cells treated with RAPA and ADR alone were (2.06±0.43) % and (48.39±6.47) %, respectively. The apoptosis rate of LoVo/ADR cells treated with ADR was higher than that of the untreated cells (2.23±0.50)% (P < 0.05). The apoptosis rate of LoVo/ADR cells after combined treatment with ADR and RAPA [(79.43±8.28)%] was higher than that of the LoVo/ADR cells treated with ADR alone (P < 0.05). The expression levels of MDR1 mRNA and P-gp in LoVo/ADR cells were down-regulated after treatment with RAPA. Conclusion: MDR of colorectal cancer cells can be reversed by inhibition of mTOR signaling pathway. This effect may be associated with increased levels of autophagy and apoptosis as well as a decreased level of MDR1 mRNA expression induced by RAPA.