Growth inhibitory effect and apoptosis induced by extracellular ATP and adenosine on human gastric carcinoma cells: involvement of intracellular uptake of adenosine
Growth inhibitory effect and apoptosis induced by extracellular ATP and adenosine on human gastric carcinoma cells: involvement of intracellular uptake of adenosine
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DOI：
10.1111/j.1745-7254.2006.00342.x
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发表时间：
2006-08-01
影响因子：
8.2
通讯作者：
Ren, Lei-ming
中科院分区：
文献类型：
--
作者：
Wang, Ming-xia;Ren, Lei-ming
目的：研究三磷酸腺苷（ATP）和腺苷（ADO）对人胃癌（HGC）-27细胞的体外生长抑制和凋亡作用及其相关机制。方法：采用MTT法测定细胞活力的降低情况。吖啶橙/溴化乙锭双染细胞，在荧光显微镜下观察ATP或ADO诱导的HGC-27细胞的形态变化。通过琼脂糖凝胶电泳检测基因组DNA的核小体片段化。流式细胞术测定ATP或ADO处理后的细胞凋亡率和细胞周期分析。结果：ATP、ADO及其中间代谢物ADP和AMP以及嘌呤受体激动剂在0.3和1.0 mmol.L-1剂量下降低HGC-27细胞的细胞活力。 0.1、0.3和1 mmol/L浓度的ATP或ADO作用48 h后，HGC-27细胞的细胞周期时相分布和增殖指数（PI）值发生变化。 ATP和ADO均通过G(0)/G(1)期阻滞改变细胞周期时相的分布，并显着降低PI值。光镜下，0.3mmol.L-1 ATP或ADO作用下的肿瘤细胞出现凋亡的形态学变化； 0.1-1mmol.L-1 ATP或ADO处理的HGC-27细胞在琼脂糖凝胶电泳中出现梯状DNA片段化； ATP和ADO在0.03-1 mmol-L-1之间以剂量依赖性方式诱导HGC-27细胞凋亡。 HGC-27细胞暴露于ATP或ADO 48 h后的最大凋亡率分别为13.53%或15.9%。 ATP或ADO诱导的HGC-27细胞死亡被腺苷转运蛋白抑制剂双吡达莫(10 mmol.L-1)显着抑制，但不受P1受体广泛抑制剂氨茶碱和P2非选择性拮抗剂磷酸吡哆醛-6-偶氮苯基-2, 4-二磺酸四钠盐(30 μmol.L-1)的影响结论：细胞外ATP和ADO通过细胞内摄取ADO降低HGC-27细胞系的细胞活力，阻滞细胞周期并诱导细胞凋亡。 HGC-27 细胞中 ATP 诱导细胞凋亡的主要途径之一是分解为腺苷。
Aim: To study the growth inhibitory and apoptotic effects of adenosine triphosphate (ATP) and adenosine (ADO) on human gastric carcinoma (HGC)-27 cells in vitro and the mechanisms related to the actions of ATP and ADO.Methods: MTT assay was used to determine the reduction of cell viability. The morphological changes of HGC-27 cells induced by ATP or ADO were observed under fluorescence light microscope by acridine orange/ethidium bromide double-stained cells. The internucleosomal fragmentation of genomic DNA was detected by agarose gel electrophoresis. The apoptotic rate and cell-cycle analysis after treatment with ATP or ADO was determined by flow cytometry.Results: ATP, ADO and the intermediate metabolites, ADP and AMP, and the agonist of purinergic receptors, reduced cell viability of HGC-27 cells at doses of 0.3 and 1.0 mmol.L-1. The distribution of cell cycle phase and proliferation index (PI) value of HGC-27 cells changed when exposed to ATP or ADO at the concentrations of 0.1,0.3 and 1 mmol/L for 48 h. ATP and ADO both altered the distribution of cell cycle phase via G(0)/G(1)-phase arrest and significantly decreased PI value. Under light microscope, the tumor cells exposed to 0.3 mmol.L-1 ATP or ADO displayed morphological changes of apoptosis; a ladder-like pattern of DNA fragmentation obtained from HGC-27 cells treated with 0.1-1 mmol.L-1 ATP or ADO appeared in agarose gel electrophoresis; ATP and ADO induced the apoptosis of HGC-27 cells in a dose-dependent manner at concentrations between 0.03-1 mmol-L-1. The maximum apoptotic rate of HGC-27 cells exposed to ATP or ADO for 48 h was 13.53% or 15.9%, respectively. HGC-27 cell death induced by ATP or ADO was significantly inhibited by dipy-ridamole (10 mmol.L-1), an inhibitor of adenosine transporter, but was not affected by aminophylline, a broad inhibitor of P1 receptors and pyridoxal-phosphate-6-azophenyl-2, 4-disulphonic acid tetrasodium salt (30 mu mol.L-1), a non-selective antagonist of P2 receptors.Conclusion: Extracellular ATP and ADO reduced the cell viability, arrested cell cycle and induced apoptosis in HGC-27 cell line by intracellular uptake of ADO. One of the main routes of ATP-induced apoptosis in HGC-27 cells is through the breakdown to adenosine.