Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.

Sylvest L, Bendiksen CD, Houen G. Phosphatase inhibitors with anti-angiogenic effect in vitro. APMIS 2010; 118: 49–59.Levamisole has previously been identified as an inhibitor of angiogenesis in vitro and in vivo, but the mechanism behind the anti-angiogenic behavior has not yet been established. However, one known effect of levamisole is the inhibition of alkaline phosphatase, and this fact encouraged us to test other phosphatase inhibitors for their anti-angiogenic effects by using the same method as used to identify levamisole: an ELISA-based co-culture angiogenesis assay giving quantitative and qualitative results. Historically, intracellular phosphatases have been associated with the downregulation of signaling pathways, and kinases with their upregulation, but lately, the phospatases have also been coupled to positive signaling, which is why inhibition of phosphatases has become associated with anti-tumorigenic and anti-angiogenic effects. The results obtained in this work reveal several agents with anti-angiogenic potential and give a strong indication that phosphatase inhibition is linked to anti-angiogenic activity. An apparent disruption of endothelial tube formation was seen for seven of eight phosphatase inhibitors tested in the angiogenesis assay. By looking at the morphological results, it was seen that most of the inhibitors impaired proliferation and elongation of the endothelial cells, which still had a differentiated appearance. One inhibitor, PTP inhibitor IV, seemed to impair endothelial cell differentiation and induced the same morphology as when cells were treated with levamisole, although at a 200 times lower concentration than that of levamisole. Hence, our work points out compounds with a potential that may be of use in the search for new medical products for the treatment of malignant tumors, or other conditions where angiogenesis plays a central role.

Inhibition of angiogenesis is a promising addition to current cancer treatment strategies. Neutralization of vascular endothelial growth factor by monoclonal antibodies is clinically effective but may cause side effects due to thrombosis. Low molecular weight angiogenesis inhibitors are currently less effective than antibody treatment and are also associated with serious side effects. The discovery of new chemotypes with efficient antiangiogenic activity is therefore of pertinent interest. (S)-Levamisole hydrochloride, an anthelminthic drug approved for human use and with a known clinical profile, was recently shown to be an inhibitor of angiogenesis in vitro and exhibited tumor growth inhibition in mice. Here we describe the synthesis and in vitro evaluation of a series of N-alkylated analogues of levamisole with the aim of characterizing structure–activity relationships with regard to inhibition of angiogenesis. N-Methyllevamisole and p-bromolevamisole proved more effective than the parent compound, (S)-levamisole hydrochloride, with respect to inhibition of angiogenesis and induction of undifferentiated cluster morphology in human umbilical vein endothelial cells grown in co-culture with normal human dermal fibroblasts. Interestingly, the cluster morphology caused by N-methyllevamisole was different than the clusters observed for levamisole, and a third “cord-like” morphology resembling that of the known drug suramin was observed for an aniline-containing derivative. New chemotypes exhibiting antiangiogenic effects in vitro are thus described, and further investigation of their underlying mechanism of action is warranted.