8-姜酚抑制乳腺癌细胞增殖的分子机制研究

Molecular mechanism of inhibition of 8-gingerol on proliferation of breast cancer cells

目的探讨8-姜酚抑制乳腺癌细胞增殖的分子机制。方法 MTT法测定乳腺癌细胞MDA-MB-231细胞活性;流式细胞仪检测产生活性氧(reactive oxygen species,ROS)的细胞比例、细胞周期以及细胞凋亡;Western blot检测周期相关蛋白Cyclin D1和p-DCD2的表达。结果 8-姜酚能明显抑制乳腺癌MDA-MB-231和MCF-7细胞的增殖活性,差异有统计学意义(P<0.01),72 h的IC_(50)值分别为(22.06±2.93)、(35.77±5.96)μmol/L;流式细胞仪检测结果发现8-姜酚可导致乳腺癌细胞G_1期阻滞,诱导细胞产生ROS;8-姜酚作用于MDA-MB-231和MCF-7细胞24 h后G_1期细胞的百分比分别为(69.23±3.77)%、(65.87±3.76)%,与对照组G_1期的细胞比例(47.39±1.97)%、(49.17±3.52)%相比,差异具有统计学意义(P<0.01);用NAC抑制ROS后减弱了8-姜酚对乳腺癌细胞G_1期的阻滞作用,用P38抑制剂抑制P38蛋白磷酸化水平后也减弱了8-姜酚对乳腺癌细胞G_1期的阻滞作用。Western blot结果显示8-姜酚可以上调MAPK信号通路中P38蛋白的磷酸化水平;并可下调G_1期相关蛋白Cyclin D1和p-CDC2的表达,用NAC抑制ROS后减弱了8-姜酚对P38磷酸化水平上调作用的同时还减弱了8-姜酚对周期相关蛋白CyclinD1、p-CDC2的下调作用;用P38抑制剂抑制P38蛋白磷酸化水平后同样减弱了8-姜酚对周期相关蛋白CyclinD1、p-CDC2的下调作用。结论 8-姜酚具有明显的抑制肿瘤增殖活性的作用,其作用机制可能为8-姜酚通过诱导乳腺癌细胞产生ROS从而导致MAPK信号通路中P38的磷酸化激活,进而引起乳腺癌细胞的周期阻滞。

Objective To investigate the effect of cell cycle on breast cancer cells by 8-gingerol. Methods The cell viability of breast cancer cells MDA-MB-231 and MCF-7 after treated by 8-gingerol at the doses of 0, 10, 20, 30 or 40 μmol/L for 24, 48 or 72 h was assessed by MTT assay. The cell cycle, apoptosis and proportion of cells producing ROS were measured by flow cytometry. The expression of cell cycle associated proteins, Cyclin D1 and p-CDC2 were detected by Western blot analysis. Results 8-gingerol evidently inhibited the proliferation of breast cancer MDA-MB-231 and MCF-7 cells(P<0.01). The inhibitory concentration of IC50 were 22.06±2.93 and 35.77±5.96 μmol/L, respectively in the breast cancer MDA-MB-231 and MCF-7 cells after 72 h treatment with 8-gingerol. The results of flow cytometry showed that 8-gingerol can cause G1 phase arrest and induce cells to produce ROS in the breast cancer MDA-MB-231 and MCF-7 cells after treatment with 8-gingerol for 72 h. Flow cytometry found that 8-gingerol resulted in cell cycle arrest at G1 phase cells was(69.23±3.77)% and(65.87±3.76)%, respectively in the MDA-MB-231 and MCF-7 cells after 8-gingerol treatment for 24 h, significantly higher than in the cells without treatment ((47.39±1.97)%,(49.17±3.52)%, P<0.01)Pretreatment with NAC weakened the effect of 8-gingerol on G1 phase arrest. Western blot results showed that 8-gingerol resulted in the decreases of Cyclin D1 and p-CDC2 levels, and upregulated the phosphorylation level of P38. The inhibition of ROS by NAC attenuated the up-regulation of phosphorylation of P38 by 8-gingerol and the down-regulation of cyclin-related proteins Cyclin D1 and p-CDC2. The inhibition of P38 phosphorylation by P38 inhibitors also attenuated the down-regulation of cyclin-associated proteins Cyclin D1 and p-CDC2 by 8-gingerol. Conclusion 8-gingerol exhibits obviously inhibitory effects on breast cancer cell proliferation. The mechanism may be that 8-gingerol induces ROS in breast cancer cells, which leads to phosphorylation of P38 in the MAPK signaling pathway, and then leads to cell cycle arrest.