丙戊酸钠通过激活内源性凋亡途径诱导乳腺癌细胞凋亡的实验研究

Valproate acid sodium induces human breast cancer cell apoptosis by activating intrinsic pathway

目的探讨丙戊酸钠（VPA）诱导乳腺癌细胞凋亡的作用。方法乳腺癌细胞株MCF-7细胞分为0.75～4.0 mmoL/L VPA实验组、对照组（不加VPA）及VPA与caspase抑制剂共同作用组。Annexin V-PI双染法流式细胞术检测细胞凋亡,间接免疫荧光法定量分析及分光光度法检测caspase- 3、caspase-8、caspase-9蛋白丰度和活性,探讨VPA诱导凋亡的机制,同时检测VPA与caspase-3、caspase- 8、caspase-9特异性抑制剂协同作用后细胞凋亡的变化来加以验证。结果各浓度VPA干预MCF-7细胞48 h后,细胞凋亡率显著增加,caspase-3、caspase-9活性升高、蛋白表达明显上调,与对照组相比有显著差异（F=552.1、610.9、312.8、222.8、70.3,均P〈0.001）;而caspase-8活性及蛋白表达未见明显改变;1.5、3.0 mmol/L VPA与caspase-3、caspase-9相应特异性抑制剂共同作用组,细胞凋亡率均较VPA组显著降低（t=109.0、28.7、18.7、32.3,均P〈0.005）;VPA与caspase-8特异性抑制剂共同作用组细胞凋亡率与VPA组比较无明显改变（t=1.03、2.32,均P〉0.05）。结论VPA可通过激活caspase-9介导的内源性凋亡途径,明显诱导乳腺癌细胞凋亡,具有较好的临床应用价值。

Objective To identify the inducing effect of valproate acid sodium（VPA） on human bresat cancer cells apoptosis and study the molecular mechanisms. Methods Cultured MCF-7 cells were divided into VPA groups（ treated with 0. 75-4.0 mmol/L VPA） ,control group（ without VPA treating） and VPA plus caspase specific inhibitors group. Apoptosis rate was analyzed by flow cytometric with Annexin V/PI staining assay. The activities and protein expressions of caspase-3, caspase-8, caspase-9 were detected by spectrophotometry and indirect immunofluorescence technique respectively. Explored the mechanism of VPA inducing apoptosis, and confirmed it by detecting apoptosis changes after the cells being treated with VPA and specific inhibitors of caspase-3,caspase-8 and caspase-9. Results The apoptosis rates of MCF-7 cells increased significantly （F = 552. 1, P 〈 0. 001 ） after being treated with 0. 75-4. 0 mmol/L VPA for 48 h. The activities and protein expressions of caspase-3, caspase-9 were up-regulated significantly compared to the control group （F = 610. 9, 312. 8, 222. 8, 70. 3, P 〈 0. 001 ） ;while the activity and protein expression of caspase-8 had no difference between experimental groups and control group （ P 〉 0.05 ）. The apoptosis rates of MCF-7 cells treated with 1.5, 3.0mmol/L VPA and specific inhibitors of caspase-3, caspase-9 together were lower than those in the groups with VPA treated alone （ t = 109. 0, 28.7, 18.7, 32. 3, P 〈0. 005）. The apoptosis rate in group treated with VPA and caspase-8 specific inhibitor remained the same as that of VPA group（ t = 1.03, 2.32, P 〉 0. 05）. Conclusion VPA can induce human breast cancer cells apoptosis effectively by activating the intrinsic pathway mediated by caspase-9, and it may be used as a potential drug in clinical treatment of human breast cancer.