华蟾素对TGF-β1诱导人结肠癌细胞上皮-间质化的实验研究

Experiment research of cinobufacini on TGF-β1-induced epithelial-to-mesenchymal transition in human colorectal carcinoma cells

目的研究华蟾素在体外对肿瘤坏死因子-β1(TGF-β1)诱导的人结肠癌细胞上皮-间质化(EMT)的作用。方法将体外培养的人结肠癌细胞株(SW480)分为:(1)正常对照组;(2)TGF-β1(10ng/mL)单独处理组;(3)TGF-β1(10ng/mL)+华蟾素(2.5、5、10、20、40、80mg/mL)共同处理组,并在体外培养48h。CCK8检测细胞增殖抑制情况,利用倒置相差显微镜观察各处理组细胞形态学变化,Transwell小室检测细胞侵袭与迁移能力,实时荧光定量PCR(QRT-PCR)和Western blot分别检测E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)在mRNA及蛋白水平表达的变化。结果 (1)与TGF-β1(10ng/mL)单独处理组比较,TGF-β1(10ng/mL)+华蟾素(10、20、40、80mg/mL)共同处理组对SW480具有显著的增殖抑制作用(P<0.05)。(2)与正常对照组相比,TGF-β1单独处理组、TGF-β1(10ng/mL)+华蟾素(2.5mg/mL)共同处理组呈现典型的间质化表型,TGF-β1(10ng/mL)+华蟾素(5mg/mL)共同处理组则呈现经典的上皮表型。(3)TGF-β1(10ng/mL)+华蟾素(2.5、5mg/mL)共同处理组与TGF-β1单独处理组相比侵袭和迁移能力均明显减弱(P<0.05)。(4)QRT-PCR和Western blot结果显示TGF-β1单独处理组与正常对照组比较,Vimentin表达水平明显增强,E-cadherin表达显著减弱。TGF-β1(10ng/mL)+华蟾素(2.5、5mg/mL)共同处理组与TGF-β1单独处理组、对照组相比Vimentin表达水平显著降低,E-cadherin表达显著增强。结论华蟾素可抑制TGF-β1诱导的人结肠癌细胞SW480的增殖,其机制可能与促进E-钙黏蛋白表达增强,同时使波形蛋白表达减弱,从而与抑制TGF-β1诱导的EMT过程有关。

Objective To investigate the effects of cinobufacini on TGF-β1-induced epithelial-to-mesenchymal transition of human colorectal carcinoma cells in vitro. Methods The cultured colorectal carcinoma cell line（SW480） was divided into the control group,TGF-β1 （10 ng/mL） individual treatment group and co-treatment groups with TGF-β1 （10 ng/mL） 4- cinobufacini （2.5,5, 10,20,40,80 mg/mL）, which were cultured in vitro for 48 h. The proliferation of the cells were measured by CCK8 assay. The mor- phological changes were observed by inverted phase contrast microscope. The ability of cell invasion and migration was detected by Transwell assay. The mRNA and protein expressions of E-cadherin and Vimentin were detected with QRT-PCR and Western Blot. Results （1）Compared with the TGF-β1 （10 ng/mL） individual treatment group,TGF-β1 （10 ng/mL） ; cinobufacini （10,20,40, 80 mg/mL） co-treatment groups significantly had significantly proliferation inhibitory effect on SW480 （P〈0.05）. （2） Compared with the normal control group,TGF-β1 individual treatment group and TGF-β1 （10 ng/mL） 4- cinobufacini（2.5 mg/mL） group exhibited classical mesenchymal phenotype,while the TGF-β1 （10 ng/mL） ＋cinobufacini （5 mg/mL） co-treatment group showed classical epithelial phenotype. （3） The ability of invasion and migration in the TGF-β1（10 ng/mL）＋ cinobufacini（2.5,5 mg/mL） co-treatment group were significantly weakened compared with the TGF-β1 individual treatment group （P〈0.01）. （4） QRT-PCR and Western Blot results indicated that compared with the normal control group, the Vimentin expression in the in the TGF-β1 indi- vidual treatment group was significantly increased and the E-eadherin expression was significantly decreased. Furthermore, com- pared with the TGF-β1 individual treatment group and control group,the Vimentin expression level in the TGF-β1 （10 ng/mL） ＋einobufacini （2.5,5 mg/mL） groups was significantly decreased and E-cadherin expression was significantly increased. Conclusion Cinobufacini can inhibit TGF-β1-induced cell proliferation in human colorectal carcinoma SW480 cells, and its mechanism may be related with promoting E-cadherin expression increase, meanwhile decreasing the vimentin expression, thus inhibiting the EMT process induced by TGF-β1.