摘要
背景与目的以铂类为基础的联合化疗是非小细胞肺癌目前首选的治疗方案,然而由于铂类药物的毒副作用和耐药的发生,限制了铂类药物的广泛应用。曲古抑素A(Trichostatin A,TSA)是一种组蛋白去乙酰化酶(histone deacetylase,HDAC)抑制剂,能诱导肿瘤细胞生长抑制、分化、凋亡,并可以增强多种肿瘤细胞对铂类药物敏感性。本研究探讨TSA诱导耐顺铂人肺腺癌A549/CDDP细胞株凋亡的作用及机制。方法中性红法测定TSA对A549/CDDP细胞毒性,荧光显微镜观察细胞DNA的变化,流式细胞仪分析细胞周期及线粒体膜电位的变化,分别转染Bcl-2表达质粒过表达Bcl-2和利用siRNA干扰Bcl-2表达。Western blot分析凋亡相关蛋白的变化。结果TSA对A549/CDDP细胞的IC50是(446.59±27.32)nmol/L,随着TSA浓度升高A549/CDDP细胞生长率明显下降。细胞凋亡在浓度为(125-500)nmol/LTSA处理后24h出现。凋亡细胞主要表现为核染色质固缩,荧光染色增强。流式细胞仪检测发现TSA阻断细胞于S期,线粒体膜电位降低。Western blot结果显示,TSA处理细胞后,Bcl-2蛋白水平下降,而Bax表达上调,同时观察到caspase-3被激活。转染Bcl-2表达质粒可以抑制TSA诱导的细胞凋亡,而沉默Bcl-2表达可以增加细胞对TSA的敏感性。结论TSA可能通过线粒体途径诱导A549/CDDP细胞凋亡。
Background and objective The use of platinum-based combination chemotherpy remains the standard treatment for non-small cell lung cancer.However,the resistance to platinum limits further treatment clinically.Trichostatin A（TSA） is one of histone deacetylase（HDAC） inhibitors.It inhibits tumor cell proliferation and acts as a chemosensitizer.The aim of this study is to investigate the action mechanism of TSA on cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP.Methods Cytotoxicity and cell viability was assayed by Neutral Red method.Morphologic assessment of apoptosis was determined by fluorescence microscope;cell cycle and mitochondrial membrane potential were detected by flow cy-tometry.In addition,A549/CDDP cells were transfected with Bcl-2 expression Vector and siRNA-bcl-2.Results A549/CDDP cells treated with TSA showed apparently cytotoxicity,IC50 of TSA was（446.59±27.32） nmol/L.The growth curve showed the ratio of growth decreased with the increase of concentration of TSA.The apoptosis appeared 24 hours after treated by（125-500） nmol/L TSA,morphologic changes including nuclear chromatin condensation.Fluorescence strength was observed with fluorescence microscope.Treated by TSA,mitochondrial membrane potential was decreased and cells were arrested at S phase.Western blotting analyses showed that the levels of Bcl-2 decreased,while expression of Bax increased.Simultaneously caspase-3 was activated.Over expression of Bcl-2 can inhibit TSA-induced A549/CDDP cell apoptosis,while the decrease of Bcl-2 enhanced the sensitivity of A549/CDDP cell to TSA.Conclusion TSA induce A549/CDDP cell apoptosis by mitochondria pathway.
出处
《中国肺癌杂志》
CAS
2009年第11期1143-1149,共7页
Chinese Journal of Lung Cancer
