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目的晚期肺鳞癌治疗方式相对有限。Ⅲ期临床试验提示,rh-Apo2L有望为其提供一种新颖、有效的治疗方法。本研究旨在观察rh-Apo2L、长春瑞滨及两药联合对人肺鳞癌细胞SK-MES-1的抑制增殖和促进凋亡作用,并探讨其机制。方法将不同质量浓度的rh-Apo2L、长春瑞滨干预人肺鳞癌SK-MES-1细胞,采用CCK8法检测细胞增殖;AnnexinⅤ-FITC/PI流式细胞术检测细胞凋亡;蛋白质印迹法检测细胞凋亡相关蛋白DR4、DR5和Caspase-3的表达。结果通过CCK8检测发现,rh-Apo2L对人肺鳞癌细胞SK-MES-1的体外增殖具有抑制作用,24和48h的IC50分别为28.57和8.97μg/mL;长春瑞滨对人肺鳞癌细胞SK-MES-1的体外增殖具有抑制作用,24和48h的IC50分别为27.30和7.87μg/mL。其中,1μg/mL rh-Apo2L组、1μg/mL长春瑞滨组和两药联合组(rh-Apo2L和长春瑞滨质量浓度均为1μg/mL)的24h细胞增殖抑制率分别为(15.03±1.54)%、(21.88±2.75)%和(65.11±4.09)%;0.1μg/mL rh-Apo2L组、0.5μg/mL长春瑞滨组和两药联合组(rh-Apo2L质量浓度为0.1μg/mL,长春瑞滨为0.5μg/mL)的48h细胞增殖抑制率分别为(19.01±1.12)%、(19.97±1.23)%和(84.81±3.99)%;24和48h联合组两药相互作用指数(q值)分别为1.94和2.40。细胞凋亡实验结果显示,1μg/mL rh-Apo2L组、1μg/mL长春瑞滨组和两药联合组(rh-Apo2L和长春瑞滨质量浓度均为1μg/mL)干预细胞24h的凋亡率分别为(21.76±3.13)%、(37.31±2.21)%和(74.88±3.63)%;与单药组相比较,两药联合组对SK-MES-1细胞增殖的抑制作用明显增强(P<0.001),两药联合干预24h的凋亡指数显著升高,P<0.001。蛋白印迹法检测结果发现,rh-Apo2L联合长春瑞滨在上调DR4(P=0.026)和DR5(P=0.001)表达方面具有交互作用,在增加Caspase-3活性片段表达亦具有交互作用,P=0.011。结论 rh-Apo2L与长春瑞滨均能协同抑制人肺鳞癌SK-MES-1细胞增殖并诱导细胞凋亡,其机制可能与上调SK-MES-1细胞表面的DR4、DR5蛋白表达和活化Caspase-3相关。
The purpose of treatment of advanced squamous cell carcinoma is relatively limited. Phase III clinical trials suggest that rh-Apo2L is expected to provide a novel and effective treatment. The purpose of this study was to investigate the effects of rh-Apo2L, vinorelbine and their combination on the proliferation and apoptosis of human lung squamous carcinoma cell line SK-MES-1 and its mechanism. METHODS: Human lung squamous cell carcinoma SK-MES-1 cells were treated with different concentrations of rh-Apo2L and vinorelbine. Cell proliferation was detected by CCK8 assay. Cell apoptosis was detected by AnnexinⅤ-FITC / PI flow cytometry. Expression of apoptosis related proteins DR4, DR5 and Caspase-3. Results CCK8 assay showed that rh-Apo2L could inhibit the proliferation of human lung squamous carcinoma cell line SK-MES-1 in vitro, with the IC50 values of 28.57 and 8.97 μg / mL at 24 and 48 h, respectively. The proliferation of SK-MES-1 was inhibited in vitro with IC50 of 27.30 and 7.87 μg / mL at 24 and 48 h, respectively. Among them, the inhibitory rates of proliferation of 24h cells in 1μg / mL rh-Apo2L group, 1μg / mL vinorelbine group and rh-Apo2L group and vinorelbine group were both 1μg / mL were 15.03 ± 1.54 ), (21.88 ± 2.75)% and (65.11 ± 4.09)%; 0.1μg / mL rh-Apo2L group, 0.5μg / mL vinorelbine group and the combination of two drugs (rh-Apo2L concentration of 0.1μg / mL , And vinorelbine 0.5μg / mL) for 48h were (19.01 ± 1.12)%, (19.97 ± 1.23)% and (84.81 ± 3.99)%, respectively. The two drug interaction index q values) of 1.94 and 2.40, respectively. The results of apoptosis showed that the apoptosis rate of cells was significantly inhibited by 1μg / mL rh-Apo2L, 1μg / mL vinorelbine and the combination of rh-Apo2L and vinorelbine at 1μg / mL (21.76 ± 3.13)%, (37.31 ± 2.21)% and (74.88 ± 3.63)%, respectively. Compared with the single drug group, the inhibitory effect of the two drugs on SK-MES-1 cell proliferation was significantly enhanced (P < 0.001). The apoptotic index of the two drugs combined intervention 24h increased significantly (P <0.001). Western blotting showed that rh-Apo2L combined with vinorelbine had an interaction in up-regulating the expression of DR4 (P = 0.026) and DR5 (P = 0.001), and also had an interaction in increasing the expression of Caspase-3 active fragment, P = 0.011. Conclusion Both rh-Apo2L and vinorelbine can synergistically inhibit the proliferation and induce the apoptosis of human lung squamous cell carcinoma SK-MES-1 cells. The mechanism may be related to the up-regulation of DR4 and DR5 protein expression and activation of Caspase -3 related.