Extracellular adenosine induces apoptosis in Caco-2 human colonic cancer cells by activating caspase-9/-3 via A2a adenosine receptors
Extracellular adenosine induces apoptosis in Caco-2 human colonic cancer cells by activating caspase-9/-3 via A2a adenosine receptors
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DOI：
10.1007/s00535-008-2273-7
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发表时间：
2009-01-01
影响因子：
6.3
通讯作者：
Nishizaki, Tomoyuki
中科院分区：
文献类型：
--
作者：
Yasuda, Yoshiyuki;Saito, Masaru;Nishizaki, Tomoyuki
细胞外腺苷通过与细胞内腺苷摄取相关的内源性途径和随后的信号级联反应以及与腺苷受体相关的外源性途径诱导多种细胞的凋亡。为了解腺苷诱导人结肠癌Caco-2细胞凋亡的机制，采用四甲基偶氮唑蓝(MTT)比色法观察A(2a)腺苷受体pcDNA3.1对Caco-2细胞活性的影响。用碘化丙啶和膜联蛋白V流式细胞术和核小体DNA片段化分析检测细胞凋亡率。用caspase荧光试剂盒检测caspase-3、-8和-9的活性。使用DePsipher试剂盒监测线粒体膜电位。用逆转录聚合酶链式反应(RT-PCR)方法检测腺苷受体的表达。细胞外腺苷以浓度依赖(1~20 mM)和处理时间依赖(24~72 h)的方式诱导Caco-2细胞凋亡。腺苷效应可被腺苷A(2a)受体抑制剂DMPX和腺苷环化酶抑制剂SQ22536所抑制。A(2a)腺苷受体激动剂CGS21680和腺苷环化酶激活剂Forsklin模拟了腺苷的作用。A(2a)腺苷受体过表达仍可诱导Caco-2细胞死亡，腺苷进一步促进细胞死亡。腺苷破坏线粒体膜电位，激活caspase-9和caspase-3，但不激活caspase-8。胞外腺苷通过激活caspase-9及其下游效应因子caspase-3诱导Caco-2细胞凋亡，并通过A(2a)腺苷受体参与线粒体损伤。
Extracellular adenosine has been shown to induce apoptosis in a variety of cells via an intrinsic pathway linked to adenosine uptake into cells and the ensuing signaling cascades and an extrinsic pathway linked to adenosine receptors. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis of Caco-2 human colonic cancer cells.To observe cell viability, an MTT assay was carried out in Caco-2 cells untransfected or transfected with the A(2a) adenosine receptor pcDNA3.1. Apoptotic cell death was assessed with flow cytometry using propidium iodide and annexin V and internucleosomal DNA fragmentation analysis. Activities of caspase-3, -8, and -9 were measured using a caspase fluorometric assay kit. Mitochondrial membrane potentials were monitored using a DePsipher kit. Expression of adenosine receptors was examined with the reverse transcription-polymerase chain reaction (RT-PCR) method.Extracellular adenosine induced Caco-2 cell apoptosis in a concentration-dependent (1-20 mM) and treatment time-dependent (24-72 h) manner. The adenosine effect was inhibited by DMPX, an inhibitor of A(2a) adenosine receptors and SQ22536, an inhibitor of adenylate cyclase. CGS21680, an agonist of A(2a) adenosine receptors, and forskolin, an adenylate cyclase activator, mimicked the adenosine action. Caco-2 cell death was still induced by overexpressing A(2a) adenosine receptors, and adenosine further promoted the cell death. Adenosine disrupted mitochondrial membrane potentials and activated caspase-9 and -3, but not caspase-8.Extracellular adenosine induces apoptosis in Caco-2 cells by activating caspase-9 and the downstream effector caspase caspase-3 in association with mitochondrial damage via A(2a) adenosine receptors.