醉茄素A对肺鳞状细胞癌细胞上皮间质转化及凋亡的影响

Effect of Withaferin A on epithelial-mesenchymal transition and apoptosis in cells of lung squamous cell carcinoma

目的研究醉茄素A对肺鳞状细胞癌凋亡相关蛋白的调控作用以及对细胞上皮间质转化的影响。方法体外培养肺鳞状细胞癌SK-MES-1细胞株。用终质量浓度分别为0(对照组)、5、10、20、40μg/ml的醉茄素A处理SK-MES-1细胞24h后,于倒置相差显微镜下观察细胞的一般形态结构、脱落情况等;四甲基偶氮唑盐(MTT)法检测细胞活性;流式细胞术检测细胞凋亡率,细胞免疫荧光法和反转录聚合酶链反应(RT-PCR)检测凋亡相关蛋白及基因bcl-2、bax的表达,以及E-cadherin和Vimentin的表达。结果不同浓度(0、5、10、20、40μg/ml)的醉茄素A均能抑制SK-MES-1细胞活性,各组细胞活性分别为0.62±0.05、0.42±0.04、0.33±0.06、0.21±0.03、0.17±0.04,提示抑制作用与醉茄素A浓度相关(F=386.505,P=0.005)。不同浓度(0、5、10、20、40μg/ml)醉茄素A作用SK-MES-1细胞24h后,各组细胞凋亡率分别为(0.180±0.011)%、(0.310±0.013)%、(0.500±0.021)%、(0.540±0.018)%、(0.410±0.027)%,提示醉茄素A很少引起细胞凋亡,多为坏死细胞(F=1065.78,P=0.124);免疫荧光法结果提示不同浓度醉茄素A作用SK-MES-1细胞24h后,20μg/ml醉茄素A实验组Vimentin表达较对照组减弱,荧光强度较对照组降低;E-cadherin荧光强度较对照组增强;其余浓度实验组无明显变化;而bax、bcl-2蛋白在对照组及实验组中的相对表达强度无明显改变;RT-PCR结果显示在浓度为20、40μg/ml醉茄素A组E-cadherinmRNA表达(6.7±0.6及6.4±0.9)较对照组(4.2±1.0)增加,且差异均有统计学意义(均P<0.05),同时VimentinmRNA相对表达(4.7±0.5及4.7±0.5)较对照组(7.2±0.7)减少,差异均有统计学意义(均P<0.05)。结论醉茄素A能抑制肺鳞状细胞癌细胞的生长,并抑制其上皮间质转化过程,而与凋亡及凋亡相关蛋白无明显关系。

Objective To study the regulation of Withaferin A on apoptosis-related proteins in lung squamous cell carcinoma and its effect on cell epithelial-mesenchymal transition. Methods Lung squamous cell carcinoma cell line SK-MES-1 were cultured in vitro. The SK-MES-1 cells were treated with the final mass concentration of 0 (control), 5, 10, 20, and 40 μg/ml for 28 h, and the general morphology and shedding of the cells were observed under phase contrast microscope;MTT assay was used for detection of cell viability;flow cytometry was used for detection of apoptosis;immunofluorescence and real-time fluorescent polymerase chain reaction (RT-PCR) was used for detection of apoptosis-related proteins and genes bcl-2 and bax, and expression of the epithelial marker E-cadherin and the interstitial marker Vimentin. Results Different concentrations (0, 5, 10, 20, 40 μg/ml) of Withaferin A inhibited the activity of SK-MES-1 cells with cell viability of 0.62±0.05, 0.42±0.04, and 0.33±0.06, 0.21±0.03, 0.17±0.04, respectively, which suggesting that this inhibition was related to the concentration of Withaferin A (F = 386.505, P = 0.005). After treatment with SK-MES-1 cells for 24 h at different concentrations (0, 5, 10, 20, 40 μg/ml) of Withaferin A, the apoptosis rates of each group were (0.180±0.011)%, (0.310±0.013)%, (0.500±0.021)%, (0.540±0.018)%, and (0.410±0.027)%, which suggesting that Withaferin A rarely caused apoptosis, mostly necrotic cells (F = 1 065.78, P = 0.124). In SK-MES-1 cells treated with different concentrations of Withaferin A for 24 h, the results of immunofluorescence showed that the expression of Vimentin was decreased in the experimental group at a concentration of 20 μg/ml compared with the control group, and the fluorescence intensity was lower than that of the control group, but the fluorescence intensity of E-cadherin was higher than that of the control group;the intensity did not change significantly in the experimental group with the other concentrations. While the expression levels of bax and bcl-2 proteins in the control group and the experimental group did not change significantly. RT-PCR results showed that the mRNA expression of E-cadherin (6.7±0.6 and 6.4±0.9) in the experimental group at a concentration of 20 and 40 μg/ml was significantly higher than that in the control group (4.2±1.0), and the difference was statistically significant (both P < 0.05), and the mRNA expression of Vimentin (4.7±0.5 and 4.7±0.5) was significantly lower than that in the control group (7.2±0.7), and the difference was statistically significant (both P < 0.05). Conclusion Withaferin A can inhibit the growth of lung squamous cell carcinoma cells and inhibit the process of epithelial-mesenchymal transition, but it has no obvious relationship with apoptosis and apoptosis-related proteins.