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Monomethyl selenium–specific inhibition of MMP‐2 and VEGF expression: Implications for angiogenic switch regulation

Previous work suggested that antiangiogenic activity may be a novel mechanism contributing to the cancer chemopreventive activity of selenium (Se). Because methylselenol has been implicated as an in vivo active chemopreventive Se metabolite, experiments were conducted to test the hypothesis that this metabolite pool might inhibit the expression of matrix metalloproteinase‐2 (MMP‐2) by vascular endothelial cells and of vascular endothelial growth factor (VEGF) by cancer epithelial cells, two proteins critical for angiogenesis and its regulation. In human umbilical vein endothelial cells (HUVECs), zymographic analyses showed that short‐term exposure to methylseleninic acid (MSeA) and methylselenocyanate (MSeCN), both immediate methylselenol precursors, decreased the MMP‐2 gelatinolytic activity in a concentration‐dependent manner. In contrast, Se forms that enter the hydrogen selenide pool lacked any inhibitory effect. The methyl Se inhibitory effect on MMP‐2 was cell dependent because direct incubation with Se compounds in the test tube did not result in its inactivation. Immunoblot and enzyme‐linked immunosorbent assay analyses showed that a decrease of the MMP‐2 protein level largely accounted for the methyl Se–induced reduction of gelatinolytic activity. The effect of MSeA on MMP‐2 expression occurred within 0.5 h of exposure and preceded MSeA‐induced reduction of the phosphorylation level of mitogen‐activated protein kinases (MAPKs) 1 and 2 (∼3 h) and endothelial apoptosis (∼25 h). In addition to these biochemical effects in monolayer culture, MSeA and MSeCN exposure decreased HUVEC viability and cell retraction in a three‐dimensional context of capillary tubes formed on Matrigel, whereas comparable or higher concentrations of selenite failed to exert such effects. In human prostate cancer (DU145) and breast cancer (MCF‐7 and MDA‐MB‐468) cell lines, exposure to MSeA but not to selenite led to a rapid and sustained decrease of cellular (lysate) and secreted (conditioned medium) VEGF protein levels irrespective of the serum level (serum‐free medium vs. 10% fetal bovine serum) in which Se treatments were carried out. The concentration of MSeA required for suppressing VEGF expression was much lower than that needed for apoptosis induction. Taken together, the data support the hypothesis that the monomethyl Se pool is a proximal Se for inhibiting the expression of MMP‐2 and VEGF and of angiogenesis. The data also indicate that the methyl Se–specific inhibitory effects on these proteins are rapid and primary actions, preceding or independent of inhibitory effects on mitogenic signaling at the level of MAPK1/2 and on cell growth and survival. Mol. Carcinog. 29:236–250, 2000. © 2000 Wiley‐Liss, Inc.