姜黄素对转化生长因子-β1诱导的乳腺癌MDA-MB-231细胞基质金属蛋白酶-9表达及其侵袭能力的影响

Effect of curcumin on matrix metallopeptidase-9 expression and invasion ability of human breast cancer MDA-MB-231 cells induced by transforming growth factor-β1

目的研究姜黄素对转化生长因子-β1(Transforming growth factor-beta 1,TGF-β1)诱导的人乳腺癌MDA-MB-231细胞基质金属蛋白酶-9(Matrix metallopeptidase-9,MMP-9)表达及其侵袭能力的影响,探讨姜黄素在防治TGF-β1诱导的乳腺癌侵袭转移中可能的作用机制。方法采用CCK-8法检测姜黄素对MDA-MB-231细胞的细胞毒性作用。将MDA-MB-231细胞分为阴性对照组(无血清RPMI1640培养基/DMSO)、TGF-β1处理组(无血清RPMI1640培养基/DMSO+10 ng/ml TGF-β1),姜黄素+TGF-β1处理组(无血清RPMI1640培养基+5、7.5、10μmol/L姜黄素+10 ng/ml TGF-β1)。处理24 h后,采用侵袭小室试验观察细胞的侵袭能力;处理不同时间后,采用Western blot法检测细胞MMP-9、p-Smad2、p-ERK1/2以及p-p38的表达,明胶酶谱法检测各组细胞上清液中MMP-9的活性变化;以ERK特异性抑制剂PD98059、p38特异性抑制剂SB202580、姜黄素和/或TGF-β1处理细胞48 h,采用Western blot和明胶酶谱法检测MMP-9的表达。结果低浓度姜黄素(≤10μmol/L)对MDA-MB-231细胞无明显的细胞毒性作用;姜黄素呈剂量依赖性明显减少了TGF-β1诱导的细胞穿膜数量(P<0.001);姜黄素可显著抑制TGF-β1诱导的MDA-MB-231细胞MMP-9、p-Smad2、p-ERK1/2及p-p38蛋白的表达,且呈浓度、时间依赖性(P<0.05);姜黄素随浓度的增加,对TGF-β1诱导的MDA-MB-231细胞MMP-9活性的抑制作用明显增强(P<0.05);PD98059抑制MMP-9蛋白表达及活性的作用与姜黄素相似,而SB202580对MMP-9无明显影响。结论姜黄素可能通过TGF-β/Smad、TGF-β/ERK信号通路下调TGF-β1诱导的MMP-9的表达及其活性,从而抑制肿瘤的侵袭能力。

Objective To evaluate the effect of curcumin on the expression of matrix metallopeptidease-9 （MMP-9） and invasion ability of human breast cancer MDA,MB-231 cells induced by transforming growth factor-β1（TGF-β1）, and investigate the potential action mechanism of curcumin in prevention and treatment of TGF-β1-induced invasion and metastasis of breast cancer. Methods The toxic effect of curcumin on MDA-MB-231 cells was determined by CCK-8 method. The cells were divided into five groups. The ceils in negative control and TGF-β1 control groups were treated with treated with serum-free RPMI1640 medium/DMSO and serum-free RPMI1640 medium/DMSO ＋ 10 ng/ml TGF-β1, while those in three test groups with serum-free RPMI1640 medium ＋ 10 ng/ml TGF-131 ＋ curcumin at dosages of 7, 7.5 and 10 μmol/L, respectively. The invasion ability of cells was checked by transwell chamber 24 h after treatment. The expression levels of MMP-9, p-Smad2, p-ERK1/2 and p-p38 in the ceils after treatment for various hours were determined by Western blot, while the activity of MMP-9 in cell supernatant by gelatin zymography. The cells were treated with ERK-specific inhibitor PD98059, p83-specific inhibitor SB202580, cureumin and/or TGF-β1 for 48 h, and determined for MMP-9 expression by Western blot and gelatin zymography. Results Curcumin at a low concentration （not more than 10 μmol/L） showed no cytotoxicity to MDA-MB-231 cells. However, curcumin significantly decreased the count of transmembrane MDA-MB-231 cells, in a dose-dependent mode （P 〈 0. 001）. Curcumin showed significantly dose- and time-dependent inhibitory effect on expressions of MMP-9, p-Smad2, p-ERK1/2 and p-p38 in MDA-MB-231 cells induced by TGF-β1 （P 〈 O. 05）. The inhibitory effect on TGF-β1-induced MMP-9 activity in MDA-MB-231 cells was enhanced significantly with the increasing concentration of curcumin （P 〈 0. 05）. The inhibitory effect of PD98059 on expression and activity of MMP-9 induced by TGF-β1 was similar to that of curcumin. However, SB202580 showed no significant effect on MMP-9. Conclusion Curcumin might down-regulate TGF-β1-induced expression and activity of MMP-9 through TGF-β / Smad and TGF-β / ERK signaling pathway, thus inhibit the invasion ability of tumor.