米非司酮对前列腺癌PC-3细胞周期及其蛋白的影响

Effect of mifepristone on cell cycle arrest and its regulators in prostate cancer cell line PC-3

目的探讨米非司酮(MIF)对前列腺癌PC-3细胞周期及其调控蛋白的影响及其作用机制。方法MTT法检测1、10、50、100μmol／L MIF作用于PC-3细胞24-120 h的吸光度(A)值,流式细胞仪检测10、50μmol／L MIF作用PC-3细胞48 h后细胞周期的变化,免疫组化法和Western blot法检测10、50μmol／L MIF处理48 h后PC-3细胞cyclin D1、bax、bcl-2蛋白表达的变化情况。结果1μmol／L MIF作用24-120 h的A值与对照组相比差异无统计学意义(P>0．05);10、50、100μmol／L组A值与对照组比较差异有统计学意义(P<0．01);MIF对PC-3细胞的抑制作用呈时间-剂量依赖性。MIF作用48 h后使PC-3细胞停滞于G1／G0期,并使此期细胞比例从对照组的27．4％增加到10μmol／L组的50．4％和50μmol／L组的59．2％,差异有统计学意义(P<0．05)。处理后PC-3细胞中bcl-2蛋白和cyclin D1蛋白表达量,与对照组比较差异有统计学意义(P<0．05);而bax表达量显著增加。结论MIF以时间-剂量依赖性方式抑制前列腺癌PC-3细胞的增殖,可能通过下调cyclin D1蛋白表达,阻止PC-3细胞G1期向S期的转换,使其停留于G1／G0期;同时降低bcl-2蛋白的表达及激活bax蛋白的表达等抑制前列腺癌PC-3细胞增殖。

Objective To investigate the effects of mifepristone（MIF） on cell cycle arrest and its regulating proteins in androgen-independent prostate cancer cell line PC-3. Methods The A values of the prostate cancer cells PC-3 in each group with various concentrations （ 1,10,50 and 100 p, mol/L） of MIF at different time intervals （24 - 120 h） were detected with MTT assay. 0.1% ethanol was used in controls. The cell cycles of the PC-3 cells treated with 10 and 50 μ mol/L of MIF for 48 h were assessed by flow cytometry （FCM） analysis, lmmunohistochemical and Western blot methods were used to determine the expression of cyclin D1, bax and bcl-2 proteins after treatment with 10 and 50 p, mol/L of MIF. Results The A values of the cancer cells treated with 1μmol/L of MIF were similar to that of controls（ P 〉 0.05） ,while those of the cells treated with 10,50 p, mol/L and 100 p, mol/L of MIF were significantly different from that of controls （P 〈 0.01 ）. MIF markedly inhibited cell proliferation of prostate cancer cells PC-3 in a dose- and time-dependent manner. FCM analysis showed the cell cycle of cancer cells treated with 10 and 50μ mol/L of MIF for 48h were blocked at G1/G0 phase,at which the cell ratio increased from 27.4% （controls） to 50.4% （10 μ mol/L MIF group） and 59.2% （50 μ mol/L MIF group）, showing significant difference between the treatment groups and controls（ P 〈 0.05 ）. Immunohistochemistry showed the bcl-2 expression decreased from 3.53 ±0.47（controls） to 2.03±0.74 and 1.83±0.44 in the cells treated with 10 and 50 μmol/L of MIF, which was significantly different from that of controls （P 〈 0.05 ） ;the expression of cyclin D1 in the cells treated with 10 and 50 μmol/L of MIF were significantly decreased to 2.66 ± 0.79 and 1.74±0.56, as compared with controls（4.27± 0.98） （P 〈 0.05 ） ; and the expression of bax increased from 1.88± 0.50 （controls） to 2.62± 0.36 （ 10 μmol/L MIF group） and 3.78 ± 0.31 （50 μmol/L MIF group）. Western blot analysis revealed the ratio of cyclin D1/β-actin decreased from 0.82± 0. 15 （controls） to 0.57 ± 0.09 （10 μmol/L MIF group） and 0.42 ±0. 13 （50 μmol/L MIF group）（P 〈0.05）. The ratio of bcl-2/β-actin in the ceils treated with 10 and 50 μmol/L of MIF significantly decreased to 0.59± 0.09 and 0. 47 ±0.09, compared with that of controls （0.78 ± 0.13 ） （ P 〈 0.05 ）. However, the ratio of bax/β-actin of 50 μmol/L MIF group （0.74± 0.11 ） significantly increased（P 〈 0. 01 ）, while the ratio of bax/β-actin of 10 μmol/L MIF group （0.63 ±0.09）was similar to that of controls （0.51 ±0.10）. Conclusions MIF can inhibit proliferation of androgen-independent prostate cancer cell line PC-3 in time- and dose-dependent manner. The inhibiting effect is through decreasing cyclin D1 protein expression,which blocks cell cycle progression in G1/G0 phase,and through down-regulating bcl-2 and increasing bax protein expression.