终末糖基化产物对结肠癌SW620细胞EMT及肿瘤干细胞标志物的影响

Effects of advanced glycation end products on epithelial-mesenchymal transition and cancer stem cell associated markers in human colon cancer cell line SW620

目的:探讨终末糖基化产物(advanced glycation end products,AGEs)对结肠癌SW620细胞上皮间质转化(epithelial-to-mesenchymal transition,EMT)及肿瘤干细胞标志物CD133的影响及其作用机制。方法:用不同浓度(0、50、100、200μg/ml)的AGEs处理SW620细胞后,采用划痕实验检测细胞的迁移能力;Transwell小室检测细胞的侵袭能力;流式细胞术检测CD133^+细胞的含量;Western blotting检测AGEs受体(receptor of AGEs,RAGE)、E-cadherin、Vimentin、ERK1/2、p-ERK1/2、CD133蛋白的表达情况。结果:与对照组(0μg/ml)比较,AGEs处理组(50、100、200μg/ml)在AGEs作用后,SW620细胞24h迁移距离[(1.55±0.15)、(1.58±0.19)、(1.75±0.21)vs(0.95±0.18)mm,均P<0.05]及48 h迁移距离[(2.11±0.22)、(2.21±0.37)、(2.68±0.23)vs(1.60±0.24)mm,均P<0.05]均明显增加;穿过Matrigel胶的数量明显增加[(176±19.52)、(194±17.70)、(220±25.5)vs(125±26.06)个,均P<0.05];CD133^+细胞比例明显增加[(4.75±1.49)、(10.34±1.54)、(14.45±2.41)%vs(0.77±0.41),均P<0.05]。与对照组比较,AGEs处理组(50、100、200μg/ml)Vimentin、RAGE、p-Erk1/2、CD133蛋白表达明显增加;而ERK1/2蛋白无明显变化;E-cadherin蛋白表达明显减少。结论:AGEs可以提高结肠癌SW620细胞体外的侵袭迁移能力,促进EMT的发生,诱导肿瘤干细胞的生成。其机制可能通过AGE-RAGE受体配体的激活,上调p-ERK1/2,从而调控EMT相关蛋白的表达,促进肿瘤干细胞的生成。

Objective： To investigate the effect of advanced glycation end products（ AGEs） on epithelial-mesenchymal transition（ EMT） and cancer stem cell associated marker CD133 in human colon cancer cell line SW620 and their mechanism of actions. Methods： After the SW620 cells were treated with AGEs at different concentrations of 0,50,100,200μg /ml,migration and invasion abilities of the SW620 cells were detected by wound healing test and Transwell chamber assay,respectively,percentage of CD133~＋ cells was tested by flow cytometry,and protein expression levels of receptor of AGEs（ RAGE）,E-cadherin,Vimentin,ERK1 /2,p-ERK1 /2 and CD133 were detected by Western blotting. Results： After the treatment of AGEs,compared with the control group（ 0 μg / ml）,cellular migration distances in experiment groups（ 50,100,200 μg / ml） were significantly improved after 24 h（ [1. 55 ± 0. 15],[1. 58 ± 0. 19],[1. 75 ± 0. 21]vs [0. 95 ± 0. 18]mm,all P〈0. 05） and 48 h（ [1. 60 ± 0. 24],[2. 11 ± 0. 22],[2. 68 ± 0. 23]vs [1. 60 ± 0. 24]mm,all P〈0. 05）. In addition,treating the cells with AGEs（ 50,100,200 μg / ml） for 48 h remarkably increased number of the cells crossed Matrigel in vitro（ [176 ± 19. 52],[194 ± 17. 70],[220 ± 25. 50]vs [125 ± 26. 06],all P〈0. 05] and percentages of CD133~＋ cell（ [4. 75 ± 1. 49],[10. 34 ± 1. 54],[14. 45 ± 2. 41]% vs [0. 77 ± 0. 41]%,all P〈0. 05）. Expressions of RAGE,Vimentin,p-ERK1 /2 and CD133 proteins were significantly increased in the treatment groups comparing with control group;however,the expression of E-cadherin protein was decreased,while that of ERK1 /2 protein had not obvious change. Conclusion： AGEs could enhance migration and invasion abilities of the SW620 cells in vitro,promote occurrence of EMT,and induce tumorigenesis of cancer stem cells. Activating ligand of AGE-RAGE,up-regulating p-ERK1 /2 prtein and regulating expression of the EMT protein might be a possible mechanism for tumorigenesis of cancer stem cells.