High Five (BTI-TN-5B1-4) cells (Invitrogen) were grown in Grace insect cell medium (Invitrogen) and were maintained in a nonhumidified incubator at 27C as per manufacturers instructions
High Five (BTI-TN-5B1-4) cells (Invitrogen) were grown in Grace insect cell medium (Invitrogen) and were maintained in a nonhumidified incubator at 27C as per manufacturers instructions. Monoclonal and Polyclonal Antibodies Mouse anti-human methionine on 14-3-3 (Met-14-3-3) monoclonal antibody (mAb) (clone HS23) was purchased from Novus Biologicals (Littleton, CO). of nitroxoline was investigated in vivo. Results Nitroxoline inhibited MetAP2 activity in vitro (half maximal inhibitory Glyoxalase I inhibitor free base concentration [IC50] = 54.8 nM, 95% confidence interval [CI] = 22.6 to 132.8 nM) and HUVEC proliferation (IC50 = 1.9 M, 95% CI = 1.54 to 2.39 M). Nitroxoline inhibited MetAP2 activity in HUVEC in a dose-dependent manner and induced premature senescence in a biphasic manner. Nitroxoline inhibited endothelial tube formation in Matrigel and reduced microvessel density in vivo. Mice (five per group) treated with nitroxoline showed a 60% reduction in tumor volume in breast malignancy xenografts (tumor volume on day 30, vehicle vs nitroxoline, mean = 215.4 vs 86.5 mm3, difference = 128.9 mm3, 95% CI = 32.9 to 225.0 mm3, = .012) and statistically significantly inhibited growth of bladder cancer in an orthotopic mouse model (tumor bioluminescence intensities of vehicle [n = 5] vs nitroxoline [n = 6], = .045). Conclusion Nitroxoline shows promise as a potential therapeutic antiangiogenic agent. CONTEXT AND CAVEATS Prior knowledgeAngiogenesis, or the proliferation, migration, and invasion of endothelial cells to form capillaries, has an important role in tumor growth and metastasis. The type 2 methionine aminopeptidase (MetAP2) protein is usually a potential antiangiogenic target. Study designA library of 175?000 compounds was screened for MetAP2 inhibitors, and the Johns Hopkins Drug Library (JHDL) was screened for endothelial cell proliferation inhibitors among the currently used clinical drugs. Nitroxoline, an antibiotic used in many countries to treat urinary tract contamination, was the common potential hit from both screens. The drug was tested for antiangiogenic activity in Tead4 endothelial cells and in mice, and anticancer activity was assessed in mouse models of human breast malignancy and orthotopic bladder cancer xenografts. ContributionNitroxoline inhibited MetAP2 activity and endothelial cell proliferation. It also inhibited angiogenesis both in vitro and in vivo. Tumor Glyoxalase I inhibitor free base volume was reduced by 60% in breast cancer xenografts. Tumor growth and area were also statistically significantly reduced in bladder cancer xenografts. ImplicationsNitroxoline has the potential Glyoxalase I inhibitor free base for clinical use as an effective antiangiogenesis agent. LimitationsThe results in mouse models may not be predictive of clinical gains in humans. Also, a dose optimization was not done and the results in mice were obtained from a single dose. From the Editors Angiogenesis plays an important role in the development and pathogenesis of many human diseases, including cancer and rheumatoid arthritis (1C3). It requires endothelial cell proliferation, invasion across the basement membrane surrounding the existing blood vessel and differentiation to form capillary structures [reviewed in (4)]. The role of angiogenesis in tumor growth and metastasis is usually well established, since Judah Folkmans discovery of this process in the early 1970s [reviewed in (5)]. Inhibition of angiogenesis has become a promising strategy to slow the growth of tumors and to enhance the efficacy of cytotoxic anticancer drugs (6,7). The fumagillin family of Glyoxalase I inhibitor free base natural products is among the most potent small-molecule inhibitors of angiogenesis known to date, based on in vitro and preclinical studies (8). A synthetic analog of fumagillin, known as TNP-470, was tested in phase ICII clinical trials for various cancers including Kaposi sarcoma, renal cell carcinoma, brain cancer, breast cancer, cervical cancer, and prostate cancer [reviewed in (9)]. However, its in vivo instability and dose-limiting toxicity prevented TNP-470 from further clinical development (10C12). In an attempt to elucidate the molecular mechanism of inhibition of angiogenesis by fumagillin and TNP-470, we as well as others identified the type 2 methionine aminopeptidase (MetAP2) protein as the likely molecular target of these small-molecule angiogenesis inhibitors (13,14). MetAP2 is one of the three known enzymes that catalyses the removal of N-terminal initiator methionine from nascent polypeptides during protein synthesis in.