Phosphatase inhibitors with anti-angiogenic effect in vitro

Abstract

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.

Fig. 1

The phosphatase inhibitors NSC87877, clodronate and ibandronate impair HUVEC proliferation. ELISA results showing the inhibitory effect of 0.2 mM NSC87877, 1 mM clodronate and 0.1 mM ibandronate on HUVEC (CD31) and NHDF (dsDNA) grown in co-culture. Data are presented as relative absorbance (tested compound/milliQ water), from three to four independent experiments ± SD. Each experiment was performed in duplicate, n = 6–8.

Fig. 2

Immunostaining with CD31 antibody visualized the effects of NSC87877, clodronate and ibandronate on the endothelial cell network formation. Cells were treated with (A) milliQ water (control), (B) 0.2 mM NSC87877, (C) 1 mM clodronate, (D) 100 μM ibandronate, (E) 1 μM ibandronate and (F) 2 mM levamisole.

Fig. 4

Immunostaining with CD31 antibody visualized the effects of the PTP1B inhibitor, salubrinal, PTPi IV, PTPi II and PTPi I on the endothelial cell network formation. Cells were treated with (A) DMSO 0.1% (control), (B) 60 μM PTP1B inhibitor, (C) 10 μM PTPi IV, (D) 20 μM salubrinal, (E) 10 μM PTPi II and (F) 10 μM PTPi I.

Fig. 3

Effects of the PTP1B inhibitor, salubrinal, PTPi IV, PTPi II and PTPi I in the co-culture assay. ELISA results are illustrated when HUVEC (CD31) and NHDF (dsDNA) were treated with DMSO 0.1% (control), 60 μM PTP1B inhibitor, 20 μM salubrinal, 10 μM PTPi IV, 10 μM PTPi II and 10 μM PTPi I. Data are presented as relative absorbance (tested compound/milliQ water) from two to three independent experiments ± SD. Each experiment was performed in duplicate, n = 4–6.