丹参酮ⅡA抗乳腺癌细胞增殖的环氧合酶-2通路研究

Inhibitory effects of Tanshinone ⅡA on proliferation and COX-2 expression in breast cancer cells

目的探讨中药小分子单体化合物丹参酮ⅡA对乳腺癌细胞增殖及环氧合酶-2表达的影响。方法以3种乳腺癌细胞系T47D、MCF-7、MDA-MB-231作为研究材料,采用MTT法检测不同浓度与不同作用时长下丹参酮ⅡA对3种细胞增殖的影响;采用Westernblot法检测经丹参酮ⅡA处理48 h后3种细胞中环氧合酶-2的蛋白水平;分别用带有荧光素酶标记的环氧合酶-2的启动子质粒及其转录因子核因子-κB、激活蛋白-1的位点质粒转染3种细胞,采用双荧光素酶报告基因检测系统检测经丹参酮ⅡA处理48 h后3种细胞中环氧合酶-2的转录水平以及转录因子核因子-κB、激活蛋白-1的活性。结果 MTT实验结果显示丹参酮ⅡA可抑制乳腺癌细胞系T47D、MCF-7、MDAMB-231的增殖,且抑制率与其浓度和作用时长呈正相关。Westernblot检测结果显示经丹参酮ⅡA处理48 h后,3种细胞中环氧合酶-2的蛋白水平均显著降低。荧光素酶检测结果显示经丹参酮ⅡA处理48 h后,T47D和MCF-7细胞中环氧合酶-2的转录水平显著降低(P均<0.05),同时3种细胞中激活蛋白-1的活性均显著降低(P均<0.05),核因子-κB的活性仅在T47D和MCF-7细胞中明显降低(P均<0.05)。结论丹参酮ⅡA可通过抑制转录因子核因子-κB、激活蛋白-1的活性,进而抑制环氧合酶-2的表达,从而抑制乳腺癌细胞的增殖。

Objective It is to investigate the inhibitory effects of Chinese medicine small molecule compound TanshinoneⅡA on proliferation and COX-2 expression in breast cancer cells.Methods Three kinds of breast cancer cell lines T47D,MCF-7 and MDA-MB-231 were used as experimental models in vitro.The proliferation depending on the different dosage and duration of Tanshinone IIA in the 3 kinds of breast cancer cells was detected by MTT assays.Westernblot was applied to detect the protein level of COX-2 in the 3 kinds of breast cancer cells after treated with TanshinoneⅡA for 48 hours.Plasmids pCOX2-Luc,pNF-κB-Luc and pAP1-Luc were transfected into breast cancer cells respectively and the dual luciferase reporter gene assay system was used to detect the transcription level of COX-2 and the activity of NF-κB and AP-1.Results The results of MTT assays showed that TanshinoneⅡA inhibited the proliferation of breast cancer cell lines T47D,MCF-7 and MDA-MB-231,and the inhibiting rate was positively correlated with the dosage and duration of TanshinoneⅡA.Westernblot revealed that the protein level of COX-2 in the 3 kinds of breast cancer cells were markedly decreased after treated with TanshinoneⅡA for 48 hours.Luciferase assay results showed that the treatment of TanshinoneⅡA for 48 hours apparently reduced the transcription level of COX-2 in the cells T47D and MCF-7(P<0.05),and obviously inhibited the activity of AP-1 and NF-κB in the 3 kinds of breast cancer cells.Conclusion TanshinoneⅡA may inhibit the expression of COX-2 through depressing the activity of its transcription factors AP-1 and NF-κB,thereby suppress the proliferation of breast cancer cells.