PHA739358抑制乳腺癌细胞增殖、诱导凋亡的分子机制

目的 研究Aurora激酶抑制剂PHA739358抑制人乳腺癌T47D细胞增殖、诱导凋亡的分子机制.方法 用四甲基偶氮唑盐法(MTT法)评价PHA739358对T47D细胞增殖的影响;免疫荧光法观察染色体、核的变化;流式细胞术检测细胞周期阻滞和凋亡率;Western印迹检测AuroraA、pAurora A、Histone H3、p-Histone H3,周期特异性指标Cyclin B1,周期调节性指标Cdc2、Cdc25c、p-Cdc2、p-Cdc25c,凋亡诱导指标p21、p53,凋亡相关指标PARP、Bcl-2、Bax.结果 不同浓度的PHA739358处理细胞24h、48 h后,明显抑制T47D的增殖,IC50分别为(3.44±0.54) μ,mol/L、(0.21±0.67)μmol/L,核与纺锤体形态发生明显变化,G2/M期阻滞增加呈剂量依赖性,Western印迹显示Aurora A、Histone H3、Cdc2、Cdc25c无明显趋势变化,p-AuroraA、p-Histone H3、CyclinB1、Bcl-2随着浓度的增加而减少,p-Cdc2、p-Cdc25c、P21、P53、Bax、PARP随着浓度的增加而增加.流式细胞术凋亡率由0.31％ ±0.03％增加到40.6％±0.81％.结论 PHA739358抑制乳腺癌细胞T47D增殖、诱导凋亡的分子机制为乳腺癌治疗提供了新思路.

Molecular mechanism of Aurora kinase inhibitor PHA739358 in inhibited proliferation and induced apoptosis of breast cancer cells

Objective To explore the molecular mechanisms of Aurora kinase inhibitor PHA739358 in inhibited proliferation and in vitro induced apoptosis of breast cancer cells. Methods The in vitro cultured T47D cells in logarithmic growth phase were used.The effects of PHA739358 on cell proliferation were examined by MTT (methyl thiazolyl tetrazolium ) assay. The variety of nuclear and spindle morphologies was examined by immunofluorescence.And G2/M arrest was determined by flow cytometry.The morphological changes of apoptotic cells were observed by fluorescent microscope.The levels of Aurora kinase relative protein expression phosphorate-AuroraA (p-AuroraA),AuroraA,phosphorate-histone H3 (p-histone H3 ),histone H3,cell cycle-specific protein expression CyclinBl,cell cycle regulative protein expression Cdc2,Cdc25c,phosphorate-Cdc2 (p-Cdc2),phosphorate-Cdc25c (p-Cdc25c) and apoptosis relative protein expression PARP,Bcl-2 and Bax were detected by Western blot.The apoptotic rate was examined by flow cytometer.Results PHA739358 obviously inhibited the proliferation of T47D cells after a 24 h or 48 h treatment in a dose-dependent and time-dependent manner.Their IC50 values were (3.44:0.54) and ( 0.21 ± 0.67 ) μmol/L respectively. Flow cytometry showed that G2/M arrested in a dosedependent manner.PHA739358 dose-dependently and time-dependently promoted the dissection of PARP (poly (ADP-ribose) polymerase); down-regulated the expressions of Bcl-2,p-AuroraA,p-histone H3,CyclinB1 and up-regulated the levels of Bax,p-Cdc25c,p-Cdc2,P21 and P53 protein.PHA739358 had no significant effects on the expressions of AuroraA and histone H3. Flow cytometry and fluorescence microscope showed that PHA739358 significantly induced apoptosis. Flow cytometry showed the rate of apoptosis significantly increased from 0.31％ ± 0.03％ to 40.6％ ± 0.81％. Conclusion The proliferation-inhibiting and apoptosis-inducing effects of PHA739358 may provide new therapeutic approaches of breast cancer.