Department of Physiology, Shinshu University School of Medicine, Matsumoto, Japan.
Am J Physiol Cell Physiol. 2013 Dec 1;305(11):C1139-50. doi: 10.1152/ajpcell.00199.2013. Epub 2013 Sep 25.
To address pivotal roles of cell surface F1/FO ATP synthase in the development of acidic microenvironment in tumor tissues, we investigated effects of shear stress stimulation on the cultured human breast cancer cells, MDA-MB-231 and MDA-MB-157, or human melanoma cells, SK-Mel-1. Shear stress stimulation (0.5-5.0 dyn/cm(2)), the levels of which are similar to those produced by the interstitial flow, induced strength-dependent corelease of ATP and H(+) from the cells, which triggered CO2 gas excretion. In contrast, the same level of shear stress stimulation did not induce significant ATP release and CO2 gas excretion from the control human mammary epithelial cells (HMEC). Marked immunocytochemical and mRNA expression of cell surface F1/FO ATP synthase, vacuolar-ATPase (V-ATPase), carbonic anhydrase type IX, and ectonucleoside triphosphate diphosphohydrolase (ENTPDase) 3 were detected in MDA-MB-231 cells, but little or no expression on the HMEC. Pretreatment with cell surface F1/FO ATP synthase inhibitors, but not cell surface V-ATPase inhibitors, caused a significant reduction of the shear stress stimulation-mediated ATP release and CO2 gas excretion from MDA-MB-231 cells. The ENTPDase activity in the shear stress-loaded MDA-MB-231 cell culture medium supernatant increased significantly in a time-dependent manner. In addition, MDA-MB-231 cells displayed strong staining for purinergic 2Y1 (P2Y1) receptors on their surfaces, and the receptors partially colocalized with ENTPDase 3. These findings suggest that cell surface F1/FO ATP synthase, but not V-ATPase, may play key roles in the development of interstitial flow-mediated acidic microenvironment in tumor tissues through the shear stress stimulation-induced ATP and H(+) corelease and CO2 gas production.
为了研究细胞表面 F1/F0 ATP 合酶在肿瘤组织酸性微环境形成中的关键作用，我们研究了切应力刺激对培养的人乳腺癌细胞 MDA-MB-231 和 MDA-MB-157 或人黑色素瘤细胞 SK-Mel-1 的影响。切应力刺激（0.5-5.0 dyn/cm2）的强度与间质流产生的强度相似，诱导细胞内 ATP 和 H+的强度依赖性共释放，从而触发 CO2 气体的排出。相比之下，相同水平的切应力刺激不会引起对照人乳腺上皮细胞（HMEC）显著的 ATP 释放和 CO2 气体排出。在 MDA-MB-231 细胞中，明显的免疫细胞化学和细胞表面 F1/F0 ATP 合酶、液泡型 ATP 酶（V-ATPase）、碳酸酐酶 IX 和外核苷酸三磷酸二磷酸水解酶（ENTPDase）3 的 mRNA 表达被检测到，但在 HMEC 中几乎没有或没有表达。细胞表面 F1/F0 ATP 合酶抑制剂预处理，但不是细胞表面 V-ATPase 抑制剂预处理，可显著减少 MDA-MB-231 细胞中由切应力刺激介导的 ATP 释放和 CO2 气体排出。在切应力加载的 MDA-MB-231 细胞培养上清液中，ENTPDase 活性呈时间依赖性显著增加。此外，MDA-MB-231 细胞表面显示出强烈的嘌呤能 2Y1（P2Y1）受体染色，这些受体与 ENTPDase 3 部分共定位。这些发现表明，细胞表面 F1/F0 ATP 合酶，而不是 V-ATPase，可能通过切应力刺激诱导的 ATP 和 H+共释放和 CO2 气体产生，在间质流介导的肿瘤组织酸性微环境形成中发挥关键作用。
