Vázquez-Cuevas Francisco G, Martínez-Ramírez Angélica S, Robles-Martínez Leticia, Garay Edith, García-Carrancá Alejandro, Pérez-Montiel Delia, Castañeda-García Carolina, Arellano Rogelio O
Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Boulevard Juriquilla 3001, Juriquilla Querétaro, CP 76230, México.
J Cell Biochem. 2014 Nov;115(11):1955-66. doi: 10.1002/jcb.24867.
P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration (Ca(2+)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a Ca(2+) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability.
P2X7是一种嘌呤能受体通道；ATP对其激活会引发一系列广泛的细胞活动，从细胞凋亡到生存信号。在此，对人卵巢癌细胞（OCA）中的P2X7表达及功能进行了研究，并通过免疫组织化学分析了非癌患者和癌症患者的活检样本。健康组织中的卵巢表面上皮高水平表达P2X7，且在整个癌变过程中持续存在。使用细胞系SKOV-3和CAOV-3研究P2X7在OCA中的功能。在SKOV-3细胞中，2'(3')-O-(4-苯甲酰苯甲酰)腺苷-5'-三磷酸（BzATP）对P2X7的选择性刺激导致细胞内Ca(2+)浓度（Ca(2+)）呈剂量依赖性增加，但未引起细胞死亡。相反，BzATP增加了磷酸化ERK和AKT（pERK和pAKT）的水平，其半数有效浓度（EC(50)）分别为44±2和1.27±0.5μM；10μM BzATP在15分钟内引发最大效应，并持续120分钟。有趣的是，培养基中存在腺苷三磷酸双磷酸酶（apyrase）时，pERK和pAKT的基础水平降低，强烈提示这是一种内源性的、ATP介导的现象。因此：（i）机械刺激细胞会使Ca(2+)增加，而apyrase可消除这种增加；（ii）如通过用于线粒体活性的MTS检测法所测，apyrase导致培养物活力下降；（iii）用10μM AZ10606120（一种特异性P2X7拮抗剂）孵育以及用显性负性P2X7突变体E496A转染，均分别将细胞活力降低至对照培养物的70.1±8.9%和76.5±5%。这些观察结果表明，P2X7活性通过ATP外流自动诱导；这增加了pERK和pAKT水平，从而对细胞活力产生正反馈。
