盐霉素通过TGFβ1/Smad信号通路抑制MMP-2、9降低CD44+/CD24-/low表型乳腺癌干细胞迁移和侵袭能力  

Salinomycin reduces migration and invasion of CD44^+/CD24^(-/low) phenotype breast cancer stem cells by suppressing MMP-2/9 expression through TGFβ1/Smad pathway

作　　者：尹燕雪 李传伟[1] 于爱莲[2] 李玲玉[1] 曹露[1] 王学春[1] 

机构地区：[1]山东省泰山医学院病理学教研室,泰安271000 [2]山东省泰山医学院微生物学教研室,泰安271000

出　　处：《临床与实验病理学杂志》2016年第3期262-267,共6页Chinese Journal of Clinical and Experimental Pathology

基　　金：山东省科技攻关项目资助课题(2009GG20002047);山东省医药卫生科技发展计划(2011HD015)

摘　　要：目的分析盐霉素对CD44^+ /CD24^(-/low)表型乳腺癌干细胞迁移和侵袭能力的影响,并初步探讨其影响机制。方法通过有血清和无血清培养技术培养人乳腺癌MCF-7细胞株,收集两种细胞,对其CD24、CD44标志物进行荧光染色,采用流式细胞仪检测CD44^+ /CD24^(-/low)亚群细胞的比例;盐霉素培养MCF-7细胞株和CD44^+ /CD24^(-/low)表型乳腺癌干细胞,MTT法筛选出引起CD44^+ /CD24^(-/low)表型乳腺癌干细胞细胞凋亡低于IC50的浓度;Transwell技术检测MCF-7和CD44^+ /CD24^(-/low)表型乳腺癌干细胞的迁移和侵袭能力,以筛选出的浓度诱导CD44^+ /CD24^(-/low)表型乳腺癌干细胞,以排除盐霉素对该细胞增殖抑制作用的影响,Transwell技术检测该细胞迁移和侵袭能力的变化;Western blot技术检测TGFβ1、Smad2、Smad3、p-Smad2、pSmad3、MMP-2、MMP-9蛋白水平的变化。结果 CD44^+ /CD24^(-/low)表型乳腺癌干细胞在无血清培养和有血清培养中的比例分别为(86.93±0.53)%和(19.98±0.62)%(P<0.01),CD44^+ /CD24^+ 表型细胞的比例分别是(12.68±0.59)%和(79.90±0.57)%(P<0.01);MTT法筛选出低于IC50的浓度是1、3、5、7μmol/L;Transwell技术检测CD44^+ /CD24^(-/low)表型乳腺癌干细胞迁移和侵袭能力明显高于MCF-7细胞株,并随盐霉素浓度的上升呈下降趋势(P<0.05)。Western blot技术检测TGFβ1、Smad2、Smad3、p-Smad2、p-Smad3、MMP-2、MMP-9蛋白水平下调(P<0.01)。结论盐霉素可能通过TGFβ1/Smad信号通路下调MMP-2、MMP-9蛋白水平,从而降低CD44^+ /CD24^(-/low)表型乳腺癌干细胞迁移和侵袭能力。Purpose To investigate the effects of salinomycin on the migration and invasion of CIM4 ＋/CD24＋,ow phenotype breast cancer stem cells, and to explore its possible mechanism. Methods MCF-7 human breast cancer cell line was cultured by serum-free culture and serum culture technology, the proportion of CD44 ＋/CD24-/low phenotype ceils was detected by the flow cytometry. MCF-7 and CD44 ＋/CD24 /low subpopulation cells were cultured by salinomycin, to select the concentration under IC50 about cell apoptosis of CD44 ＋/CD24-/Low subpopulation cells. Migration and invasion were compared between MCF-7 and breast cancer stem cells by TraM- swell assay, CD44＋/CD24-/low subpopulation cells were cultured by the selecting concentration of salinomycin. The expression of TGFβ1, Smad2, Smad3, p-Smad2, p-Smad3, MMP-2, MMP-9 was detected by Western blot. Results The proportion of CD44 ＋/ CD24 -/low phenotype cells in the suspended microsphere cells and the adherent cells was （86. 93 ± 0. 53） % and （ 19. 98 ± 0. 62） %, respectively （P 〈 0. 01 ）, the proportion of CD44 ＋/CD24 ＋ phenotype ceils was （ 12. 68 ± 0. 59 ） % and （79. 90± 0. 57 ） %, respec- tively （P 〈 0. 01 ）. The selecting concentrations of salinomycin were 1, 3, 5, and 7 p, mol/L. The migration and invasion of breast cancer stem ceils were significantly higher than that of MCF-7 and reduced with the increasing of the concentration （ P 〈 0. 05 ）. The expression of TGFβ1, Smad2, Smad3, p-Smad2, p-Smad3, MMP-2 and MMP-9 was significantly decreased with increasing of the con- centration （P 〈 0. 01 ）. Conclusion Salinomycin may reduce migration and invasion of CD44 ＋/CD24 -/lowphenotype in breast cancer stem cells by suppressing MMP-2/9 expression through TGFβ1/Smad pathway.

关 键 词：乳腺肿瘤 乳腺癌干细胞 CD44+/CD24-/low表型干细胞 盐霉素 迁移能力 侵袭能力 

分 类 号：R737.9[医药卫生—肿瘤]