[曲古抑菌素A诱导的Bcl-2蛋白水平降低通过线粒体途径介导A549/CDDP细胞凋亡。]

[Trichostatin A Induced Bcl-2 Protein Level Decrease Mediated A549/CDDP Cells Apoptosis by Mitochondria Pathway.].

作者信息

Wu Jun, Hu Chengping

机构信息

Department of Respiratory Medicine, Xiangya Hospital, Central South University, Changsha 410008, China; Institute of Respiration, Guangdong Medical College, Zhanjiang 524023, China.

出版信息

Zhongguo Fei Ai Za Zhi. 2009 Nov 20;12(11):1143-9. doi: 10.3779/j.issn.1009-3419.2009.11.03.

DOI:10.3779/j.issn.1009-3419.2009.11.03

PMID:20723360

Abstract

BACKGROUND

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 cytometry. 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.

CONCLUSIONS

TSA induce A549/CDDP cell apoptosis by mitochondria pathway.

摘要

背景

铂类联合化疗仍然是非小细胞肺癌的标准治疗方法。然而，铂类耐药限制了临床上的进一步治疗。曲古抑菌素A（TSA）是组蛋白去乙酰化酶（HDAC）抑制剂之一。它抑制肿瘤细胞增殖并作为化学增敏剂。本研究的目的是探讨TSA对顺铂耐药人肺腺癌细胞系A549/CDDP的作用机制。

方法

采用中性红法检测细胞毒性和细胞活力。通过荧光显微镜对凋亡进行形态学评估；采用流式细胞术检测细胞周期和线粒体膜电位。此外，用Bcl-2表达载体和siRNA-bcl-2转染A549/CDDP细胞。

结果

TSA处理的A549/CDDP细胞表现出明显的细胞毒性，TSA的IC50为（446.59±27.32）nmol/L。生长曲线显示，随着TSA浓度的增加，生长率下降。（125-500）nmol/L TSA处理24小时后出现凋亡，形态学变化包括核染色质浓缩。用荧光显微镜观察荧光强度。TSA处理后，线粒体膜电位降低，细胞停滞于S期。蛋白质免疫印迹分析显示，Bcl-2水平降低，而Bax表达增加。同时，caspase-3被激活。Bcl-2的过表达可抑制TSA诱导的A549/CDDP细胞凋亡，而Bcl-2的降低增强了A549/CDDP细胞对TSA的敏感性。