摘要
背景与目的:Survivin是一个抗凋亡、促增殖的双功能基因,且高表达于多种恶性肿瘤和转化组织中,使其成为抗肿瘤药物研究的新靶点。本研究观察经点突变的重组蛋白药物TATm-Survivin(T34/117A)处理乳腺癌细胞B-Cap-37后,对乳腺癌细胞B-Cap-37细胞周期和增殖的影响,检测其对癌细胞促凋亡且抑制增殖的药效并初步探讨其分子机制。方法:重叠PCR法在TATm-Survivin(T34A)基因上引入突变位点Thr117→Ala117,MTT法检测细胞增殖;流式细胞术分析细胞凋亡和细胞周期及线粒体膜电位变化;Western blot检测Aurora B激酶底物Histone H3的磷酸化水平和Caspase-3激活情况。结果:TATm-Survivin(T34/117A)蛋白对B-Cap-37细胞的增殖有显著的抑制作用;作用24 h后,细胞被阻滞在G0/G1期,G0/G1期细胞由对照组的(54.1±6.1)%显著增加到(62.7±5.1)%(P<0.05),作用48 h后,细胞被阻滞在G2/M期,G2/M细胞由对照组的(16.6±8.9)%显著增加到(24.0±6.0)%;Aurora B激酶活性通过Histone H3的磷酸化水平间接反映,蛋白药物处理48 h后p-Histone H3的灰度值由对照组的100%降低到89.8%(P<0.05);48 h时,细胞线粒体膜电位由对照组的(649.9±8.0)%显著降低到(582.9±15.6)%(P<0.05);细胞凋亡率由对照组的(4.6±0.7)%显著增加到(42.7±0.5)%(P<0.01),与对照相比,不同浓度的重组蛋白均激活了Caspase-3。结论:重组蛋白TATm-Survivin(T34/117A)对乳腺癌B-Cap-37具有抑制增殖、促凋亡作用,其与降低Aurora B激酶的活性,并激活Caspase-3相关。
Background and purpose：Survivin is a bifunctional gene in both antiapoptosis and pro-proliferation and is also highly expressed in many malignant tumors and transformed cells.This makes it an attractive target for anti-cancer drug development.This study investigated the effects of the recombinant protein TATm-Survivin（T34/117A） on cell cycles and the apoptosis of breast cancer cell B-Cap-37,as well as explored the overall molecular mechanism.Methods：Thr117 was replaced by Ala117 on TATm-Survivin（T34A） by overlapping PCR.The B-Cap-37 cells were treated by purified TATm-Survivin（T34/117A）.The MTT method was used to observe the cell proliferation process.Flow cytometry assay was used to analyze the cell cycle,apoptosis and mitochondrial membrane potential.Western blot was used to determine the phosphorylation level of Histone H3,a substrate of Aurora B kinase,as well as the expression of activated Caspase-3.Results：TATm-Survivin（T34/117A） significantly inhibited the proliferation of B-Cap-37 cells.After 24 hours of treatment,the cells arrested in the G0/G1 phase（62.7±5.1）% as compared with the control group（54.1±6.1）%（P﹤0.05）.After 48 hours,the cells significantly arrested in the G2/M phase（24.0±6.0）% as compared with the control group（16.6±8.9）%（P〈0.05）.Compared with the control group,the gray intensity in the phosphorylation levels of Histone H3 in the treated cells was significantly down to 89.8%（P〈0.05）.Apoptosis rates increased significantly,from（4.6±0.7） % in the control to（42.7±0.5）%（P〈0.01）.Meanwhile,mitochondrial membrane potential significantly decreased from（649.9±7.8） % in the control to（582.9±15.6） %（P〈0.05） while Caspase-3 was activated.Conclusion：TATm-Survivin（T34/117A） inhibits proliferation and promotes apoptosis of B-Cap-37 cells by reducing the activity of Aurora B kinase while activating Caspase-3.
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2011年第2期91-98,共8页
China Oncology
基金
国家自然科学基金资助项目(No:C0304)
