Angiostatin Is Directly Cytotoxic to Tumor Cells at Low Extracellular pH: A Mechanism Dependent on Cell Surface–Associated ATP Synthase

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Abstract
Angiostatin, a proteolytic fragment of plasminogen, is a potent angiogenesis inhibitor able to suppress tumor growth and metastasis through inhibition of endothelial cell proliferation and migration. Previously, we showed that angiostatin binds and inhibits F1Fo ATP synthase on the endothelial cell surface and that anti-ATP synthase antibodies reduce endothelial cell proliferation. ATP synthase also occurs on the extracellular surface of a variety of cancer cells, where its function is as yet unknown. Here, we report that ATP synthase is present and active on the tumor cell surface, and angiostatin, or antibody directed against the catalytic β-subunit of ATP synthase, inhibits the activity of the synthase. We show that tumor cell surface ATP synthase is more active at low extracellular pH (pHe). Low pHe is a unique characteristic of the tumor microenvironment. Although the mechanism of action of angiostatin has not been fully elucidated, angiostatin treatment in combination with acidosis decreases the intracellular pH (pHi) of endothelial cells, leading to cell death. We also find that, at low pHe, angiostatin and anti-β-subunit antibody induce intracellular acidification of A549 cells, as well as a direct cytotoxicity that is absent in tumor cells with low levels of extracellular ATP synthase. These results establish angiostatin as an antitumorigenic and antiangiogenic agent through a mechanism implicating tumor cell surface ATP synthase. Furthermore, these data provide evidence that extracellular ATP synthase plays a role in regulating pHi in cells challenged by acidosis. (Cancer Res 2006; 66(2): 875-82)