TAM诱导MA782细胞凋亡与Cyclin D1、CDK4、TGF-β1的关系

A study on relations of MA782 cell apoptosis induced by TAM to cyclin D1、CDK4 and TGF-β1

目的 :探讨Tamoxifen(TAM )诱导ER(- )小鼠乳腺癌细胞MA782细胞凋亡及其分子作用机制。方法 :TAM体外作用于MA782细胞 ,用MTT法检测细胞增殖活性、流式细胞仪检测细胞凋亡和细胞周期分布 ,免疫组化检测cyclinD1、CDK4、TGF β1蛋白表达 ,并用病理图像分析软件进行半定量分析。 结果 :TAM在体外能明显抑制MA782细胞生长 ,诱导细胞凋亡 ,下调cyclinD1、CDK4表达 ,上调TGF β1表达。免疫组化半定量分析显示 6μmol/L作用 72小时后 ,cyclinD1、CDK4蛋白平均灰度值分别从 132 .5± 0 .0 2、10 7.2± 0 .0 1下降至 12 6 .18± 0 .0 3、76 .2 1± 0 .0 3,10 μmol/L作用 72小时后分别为 73.5 6± 0 .0 2、72 .0 3± 0 .0 1,与对照组相比差异极其显著性 (P <0 0 1)。MA782细胞TGF β1平均灰度值为 5 9.72± 0 .0 2 ,2 μmol/L作用 4 8小时后无明显变化 ,72小时后上升到97 1± 0 .0 3,6、10 μmol/L作用 4 8小时后平均灰度值分别为 83.2± 0 .0 4、96 .83± 0 .0 2 ,72小时后分别上升到12 1 75± 0 .0 3、139.0 1± 0 .0 5 ,与对照组相比差异极其显著 (P <0 .0 1)。结论 :TAM诱导MA782细胞凋亡其分子作用机制可能与下调cyclinD1、CDK4蛋白表达 ,上调TGF β1蛋白表达有关。

Purpose:To study if tamoxifen (TAM) can induce growth arrest and apoptosis of ER negative MA782 mouse breast cancer cell line and to explore the molecular mechanisims. Methods:MA782 cells were cultured in RPMI 1640 medium with TAM.The proliferative activity of cells was detected by MTT methods, and cells apoptosis by flowcytometric methods. The expression of cyclin D1, CDK4 and TGF β1 proteins was detected by immunohistochemical methods, the semi quantification of protein expression was analyzed by pathological image analysis software.Results:TAM can induce growth arrest and apoptosis of cells. ICC results showed that MA782 cells were ER negative. There was no change of cell cycle regulators in cells with 2 μmol/L TAM. After 48、72h with 6 μmol/L or 10 μmol/L TAM, the level of cyclin D1 proteins decreased from 132.5±0.02 to 129.67±0.03、126.18±0.03(6 μmol/L) and 109.1±0.01、73.56±0.02(10 μmol/L),CDK4 proteins decreased from 107.2±0.01 to 91.23±0.02、76.21±0.03(6 μmol/L)and 83.52±0.02、72.03±0.01(10 μmol/L), while TGF β1 proteins increased from 59.72±0.02 to 83.2±0.04、121.75±0.03(6 μmol/L)and 96.83±0.02、139.01±0.05(10 μmol/L),The difference was significant( P <0.01).Conclusions:Down regulation of cyclin D1, CDK4 proteins and up regulation of TGF β1 protein may be one of the mechanisms through which TAM induced growth inhibition and apoptosis of MA782 cell . [