依曲替酸诱导上皮鳞癌细胞凋亡及相关信号途径的改变

Acitretin induces apoptosis and changes of relative signaling pathway in epidermoid carcinoma cell line A431

目的:研究依曲替酸诱导上皮鳞癌细胞凋亡及其分子机制。方法:用MTT法观察依曲替酸对A 431细胞的生长影响,电镜观察细胞形态,流式细胞仪检测凋亡峰的出现,A nnex in-V染色证实凋亡的发生。同时用逆转录PCR法观察,经依曲替酸作用不同的时间后,A 431细胞中信号传导和转录激活因子3(STAT 3)、细胞周期蛋白D 1(CyclinD 1)、p42/44丝裂原激活的蛋白激酶(p42/44MAPK)mRNA表达的改变;用蛋白印迹术,研究经依曲替酸作用不同的时间对A 431细胞中磷酸化STAT 3(p-STAT 3)、CyclinD 1蛋白表达的影响。结果:①随着依曲替酸作用时间的延长及剂量的增加,对A 431细胞增殖的抑制作用增加。流式细胞仪检测显示,亚G1期凋亡峰明显增加,电镜观察和A nnex in-V染色证实了凋亡的存在。②依曲替酸能明显抑制A 431细胞中STAT 3、CyclinD 1 mRNA表达的改变,随着作用时间的延长,抑制作用增强,P<0.05;并下调p-STAT 3、CyclinD 1蛋白的表达;下调p42/44MAPK mRNA的表达。③经依曲替酸作用后,A 431细胞中CyclinD 1 mRNA表达的下降与STAT 3 mRNA表达的下降呈正相关,P<0.05;p-STAT 3、CyclinD 1蛋白也同步下调,P<0.05;而CyclinD 1 mRNA表达的下降与p42/44MAPK mRNA的下调无相关性。结论:①依曲替酸具有抗上皮鳞癌细胞生长及诱导上皮鳞癌细胞凋亡作用。②依曲替酸抗上皮鳞癌细胞生长和诱导肿瘤细胞凋亡的机制,可能主要是通过调节JAK/STAT 3信号途径而实现的。

Objective. To study the effects of Acitretin on growth inhibition and apoptosis of epidermoid carcinoma cell line A431 and its molecular mechanisms. Methods. A431 cells were treated with Acitretin at the concentration of 10^-5mol/L in different time intervals. The inhibition of cell growth was determined by MTT method, morphological changes were observed by electron microscopy, apoptosis was assessed by flow cytometry and Annexin-V staining. The mRNA expression levels of STAT3, cyclinD1 and p42/44MAPK were detected by reverse transcriptase polymerase chain reaction （RT-PCR）. The protein expression levels of P-STAT3 and CyclinD1 were observed by Western blot in A431 cells. Results： （1）Acitretin inhibited the growth of A431 cells in vitro in a dose- and time-dependent manner. Morphological changes revealed characteristics of cell apoptosis. Flow cytometry showed more sub-G1 phase in A431 cells and more cells positively stained with Annexin-V. （2）Acitretin significantly inhibited the expression of STAT3 and CyclinD1 mRNA in A431 cells in vitro in a time-dependent manner （P〈0.05）. The p-STAT3 and CyclinD1 protein levels were down regulated. The Acitretin could also down regulate the p42/4MAPK mRNA in A431 cells. （3）After incubation with Acitretin,the mRNA level of CyclinD1 in A431 cells was positively correlated with that of STAT3 （P〈0. 05）. The protein level of CyclinD1 was also positively correlated with that of p-STAT3 （P〈0.05）. However, there was no correlation between mRNA levels of CyclinD1 and p42/44MAPK. Conclusion： （1）Acitretin plays an inhibitory role in the tumor cell growth and induces the cell apoptosis in A431 cells. （2）The regulation of the Jak/STAT3 signaling pathway may play an important role in inducing growth inhibition and apoptosis by Acitretin in A431 cells.