吡非尼酮抑制肿瘤相关成纤维细胞促进结肠癌细胞系HT29上皮间质转化的作用及机制

Effect and mechanism of pirfenidone on epithelial-mesenchymal transition induced by cancer-associated fibroblasts in HT29 colon cancer cell lines

目的:探讨吡非尼酮在肿瘤相关成纤维细胞(CAFs)诱导的HT29结肠癌细胞系侵袭、迁移、增殖、上皮-间质转化(EMT)中的作用及机制。方法:利用共培养小室将CAFs与HT29共培养后CCK-8测定HT29增殖效率;Real time-PCR和Western blot检测HT29结肠癌细胞系E-cadherin和Vimentin转录及蛋白表达水平;Transwell实验检测HT29结肠癌细胞的迁移和侵袭能力。ELISA技术检测CAFs细胞上清液中细胞因子的表达。结果:吡非尼酮抑制CAFs对HT29增殖促进作用(P<0.05);CAFs条件培养基培养HT29后,Vimentin表达水平显著升高(P<0.05),E-cadherin显著降低(P<0.05);吡非尼酮处理CAFs的条件培养基对HT29细胞中两种蛋白无明显影响;Transwell实验表明CAFs与HT29共培养后,其侵袭和迁移能力明显增强(P<0.05);ELISA结果提示CAFs分泌TGF-β1功能受吡非尼酮抑制且存在剂量关系。结论:吡非尼酮通过抑制CAFs分泌TGF-β1抑制CAFs诱导HT29结肠癌细胞系侵袭、迁移、增殖、上皮-间质转化。

Objective： To explore the effect and mechanism of pirfenidone on the invasion, migration, proliferation and epithelial-mesenchymal transition （EMT） induced by cancer-associated fibroblasts （CAFs） in HT29 colon cancer cell lines. Methods： CCK-8 was used to measure HT29 proliferation efficiency after coculture of CAFs with HT29 using co-culture chamber. The transcription and protein expression levels of E-cadherin and Vimentin in HT29 colon cancer cells were detected by Real time-PCR and Western blot, respectively. Transwell assay was used to detect the migration and invasion ability of HT29. ELISA technique was used to discover the expression of cytokines in CAFs supernatant. Results： Pirfenidone inhibited the proliferation of HT29 induced by CAFs （P＜0.05）. The expression of Vimentin was significantly increased in HT29 cells cultured in CAFs conditioned medium （P＜0.05）, while the expression of E-cadherin was significantly decreased （P＜0.05）. However, conditioned medium of pirfenidone treated CAFs had no significant effect on the expressions of the two proteins in HT29 cells （P＞0.05）. Trans-well assay showed that after co-cultured with CAFs, the invasion and migration ability of HT29 were significantly enhanced （P＜0.05）. The results of ELISA showed that the secretion of TGF-β1 by CAFs was inhibited by pirfenidone, which was dose related. Conclusion： Pirfenidone could inhibit the invasion, migration, proliferation and epithelial-mesenchymal transition of HT29 colon cancer cell lines by inhibiting the secretion of TGF-β1 by CAFs.