隐丹参酮对人乳腺癌MCF-7细胞周期调控因子表达的影响

Effect of cryptotanshinone on the expression of cell cycle regulatory factors in human breast cancer MCF-7 cells

目的研究隐丹参酮(CTS)对人乳腺癌MCF-7细胞增殖和细胞周期的影响并探讨其作用机制。方法将MCF-7细胞分为8组:空白组、对照组和实验-Ι,-Ⅱ,-Ⅲ,-Ⅳ,-Ⅴ,-Ⅵ组。空白组予以空白培养基溶液;对照组予以5.4μmol·L^(-1)表柔比星溶液;实验Ι,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ组分别予以含1.5,3,6,12,24,48μmol·L^(-1)隐丹参酮的培养基溶液。作用24,48,72 h后,用CCK-8法检测隐丹参酮对MCF-7细胞增殖的抑制作用;用流式细胞术PI单染法检测MCF-7细胞周期时相的分布;用免疫蛋白印记法检测周期调控相关蛋白表达的变化。结果隐丹参酮能抑制MCF-7细胞的增殖,且具有时间和剂量的依赖性。空白组和实验-Ⅳ,-V,-Ⅵ组作用24 h的存活率分别为(100.12±1.15)%,(62.59±2.16)%,(48.43±2.24)%,(45.96±1.36)%;这4组作用48 h的存活率分别为(100.24±2.18)%,(50.41±1.87)%,(41.32±1.06)%,(37.74±1.14)%;这4组作用72 h的存活率分别为(100.18±2.46)%,(42.06±1.13)%,(38.62±1.08)%,(32.18±1.35)%;实验-Ⅳ,-V,-Ⅵ组与空白组相比,差异均有统计学意义(均P<0.01)。隐丹参酮可影响MCF-7细胞周期时相分布,引起G2/M期阻滞,并诱导细胞早期凋亡。隐丹参酮能明显下调Cyclin B1和CDK1蛋白的表达,上调p53、p21cip1和p27kip1蛋白的表达。结论隐丹参酮对MCF-7细胞有显著增殖抑制作用,并影响细胞周期时相的分布,可能与p53蛋白的上调和Cyclin B1蛋白的下调及CDK1激酶活性的抑制有关。

Objective To investigate effect of cryptotanshinone（ CTS）on the proliferation and cell cycle of human breast cancer MCF-7 cells,and explore the mechanism. Methods The MCF-7 cells were divided into 8 groups： blank group,control group,and experimental-I,-Ⅱ,-Ⅲ,-Ⅳ,-V,-Ⅵ groups. Blank group was given a blank medium solution; control group was given epirubicin at dose of 5. 4 μmol·L-（-1）; experimental-I,-Ⅱ,-Ⅲ,-Ⅳ,-V,-Ⅵ groups were given cultured medium solution containing CTS 1. 5,3,6,12,24,48 μmol·L-（-1）,respectively. After 24,48,72 h,the proliferation of cells was measured by CCK-8（ cell counting Kit-8）. The distribution of MCF-7 cell cycle phase was detected by flow cytometry with PI single staining methods. The related protein expression of cell cycle regulation was detected byWestern blotting. Results The results showed that CTS strongly inhibited the proliferation of MCF-7 cells,which were time and dose-dependent. The cell survival rates of blank group and the experimental-Ⅳ,-V,-Ⅵ groups after 24 h were（ 100. 12 ± 1. 15） %,（ 62. 59 ± 2. 16） %,（ 48. 43 ± 2. 24） %,（ 45. 96 ± 1. 36） %,respectively; after 48 h,cell survival rates in the 4 groups were（ 100. 24 ± 2. 18） %,（ 50. 41 ± 1. 87） %,（ 41. 32 ± 1. 06） %,（ 37. 74 ± 1. 14） %,respectively; after 72 h,cell survival rates in the 4 groups were（ 100. 18 ± 2. 46） %,（ 42. 06 ± 1. 13） %,（ 38. 62 ± 1. 08） %,（ 32. 18 ± 1. 35） %,respectively. CTS groups had statistically significant differences compared with the blank group（ all P〈0. 01）. CTS affected the phase distribution of MCF-7 cell cycle,caused G2/M phase arrest,and induced the early apoptosis. The expression of Cyclin B1 and CDK1 proteins were down-regulated,and the expression of p53,p21 cip1 and p27 kip1 proteins were up-regulated significantly by CTS.Conclusion CTS had a significant value-added inhibition and regulated the cell cycle phase distribution on the MCF-7 cells,and its mechanism may be related to the up-regulation of p53 protein,the down-regulation of Cyclin B1 protein,inhibition of the kinase activity of CDK1.