Activity of Plasma Membrane V-ATPases Is Critical for the Invasion of MDA-MB231 Breast Cancer Cells
Activity of Plasma Membrane V-ATPases Is Critical for the Invasion of MDA-MB231 Breast Cancer Cells
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DOI：
10.1074/jbc.m114.611210
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发表时间：
2015-02-06
影响因子：
4.8
通讯作者：
Forgac, Michael
中科院分区：
文献类型：
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作者：
Cotter, Kristina;Capecci, Joseph;Forgac, Michael
液泡 (H+)-ATP 酶 (V-ATP 酶) 是 ATP 驱动的质子泵家族，它将 ATP 水解与质子跨膜易位结合起来。先前的研究表明 V-ATP 酶与癌细胞侵袭有关。有人提出，V-ATP酶通过定位于质膜并引起细胞外空间酸化来参与侵袭。为了验证这一假设，我们采用了两种不同的方法来特异性抑制质膜 V-ATP 酶。首先，我们用膜嵌入的 V-ATPase c 亚基的 V5 标记构建体稳定转染高度侵袭性的 MDA-MB231 细胞，从而允许 V5 表位的细胞外表达。我们使用体外基质胶测定评估了添加针对 V5 表位的单克隆抗体对 V-ATPase 介导的质子跨质膜易位和侵袭的影响。添加抗 V5 抗体会导致转染细胞而非对照（未转染）细胞中的胞质酸化以及跨质膜的 V-ATP 酶依赖性质子通量减少。这些结果证明抗V5抗体抑制转染细胞中质膜V-ATP酶的活性。添加抗V5抗体也抑制了转染（但未转染）细胞的体外侵袭。其次，我们利用了生物素结合形式的特定 V-ATP 酶抑制剂巴弗洛霉素。当与链霉亲和素结合时，该化合物不能穿过质膜。将该化合物添加到 MDA-MB231 细胞中也能抑制体外侵袭。这些研究表明质膜V-ATP酶在乳腺癌细胞的侵袭中发挥重要作用。
The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. Previous studies have implicated V-ATPases in cancer cell invasion. It has been proposed that V-ATPases participate in invasion by localizing to the plasma membrane and causing acidification of the extracellular space. To test this hypothesis, we utilized two separate approaches to specifically inhibit plasma membrane V-ATPases. First, we stably transfected highly invasive MDA-MB231 cells with a V5-tagged construct of the membrane-embedded c subunit of the V-ATPase, allowing for extracellular expression of the V5 epitope. We evaluated the effect of addition of a monoclonal antibody directed against the V5 epitope on both V-ATPase-mediated proton translocation across the plasma-membrane and invasion using an in vitro Matrigel assay. The addition of anti-V5 antibody resulted in acidification of the cytosol and a decrease in V-ATPase-dependent proton flux across the plasma membrane in transfected but not control (untransfected) cells. These results demonstrate that the anti-V5 antibody inhibits activity of plasma membrane V-ATPases in transfected cells. Addition of the anti-V5 antibody also inhibited in vitro invasion of transfected (but not untransfected) cells. Second, we utilized a biotin-conjugated form of the specific V-ATPase inhibitor bafilomycin. When bound to streptavidin, this compound cannot cross the plasma membrane. Addition of this compound to MDA-MB231 cells also inhibited in vitro invasion. These studies suggest that plasma membrane V-ATPases play an important role in invasion of breast cancer cells.