TR-644 a novel potent tubulin binding agent induces impairment of endothelial cells function and inhibits angiogenesis

Abstract

TR-644 is a novel combretastatin A-4 (CA-4) analogue endowed with potent microtubule depolymerizing activity superior to that of the lead compound and it also has high affinity to colchicines binding site of tubulin. We tested TR-644 anti-angiogenic effects in human umbilical endothelial cells (HUVEC). It showed no significant effects on the growth of HUVEC cells at concentrations below 1,000 nM, but at much lower concentrations (10-100 nM) it induced inhibition of capillary tube formation, inhibition of endothelial cell migration and affected endothelial cell morphology as demonstrated by the disruption of the microtubule network. TR-644 also increased permeability of HUVEC cells in a time dependent manner. The molecular mechanism for the anti-vascular activity of TR-644 was investigated in detail. TR-644 caused G2/M arrest in endothelial cells and this effect correlated with downregulation of the expression of Cdc25C and Cdc2Tyr15 .Moreover TR-644 inhibited VEGF-induced phosphorylation of VE-cadherin but did not prevent the VEGF-induced phosphorylation of FAK. In chick chorioallantoic membrane in vivo assay, TR-644 (0.1-1.0 pmol/egg) efficiently counteracted the strong angiogenic response induced by FGF. Also CA-4, used as reference compound, caused an antagonistic effect, but in contrast, it induced per se, a remarkable angiogenic response probably due to an inflammatory reaction in the site of treatment. In a mice allogenic tumor model, immunohistochemical staining of tumors with anti-CD31 antibody showed that TR-644 significantly reduced the number of vessel, after 24 h from the administration of a single dose (30 mg/Kg).

Different Adaptive Responses to Hypoxia in Normal and Multiple Myeloma Endothelial Cells

“…Compared with HUVEC, MMEC expressed a higher level of livin and cdc25A and a lower level of p21. This is in line with previous reports indicating that downregulation of cdc25 and upregulation of p21 expression in EC correlates with cell cycle arrest [25, 26]. With regard to livin, an inhibitor of apoptosis proteins, our observations show that its expression is positively correlated with cell survival [27].…”

Combretastatins: More Than Just Vascular Targeting Agents?

“…Furthermore, in this study, CA-4P inhibited sprout formation at concentrations that did not inhibit the proliferation of endothelial cells, confirming direct antiangiogenic properties of CA-4P. Similarly, modulation of endothelial cells by combretastatin analogs has been reported in vitro (Dupeyre et al, 2006;Kong et al, 2010;Romagnoli et al, 2010;Sanna et al, 2010;Arthuis et al, 2011;Porcù et al, 2013) and in vivo (Fortin et al, 2011;Porcù et al, 2013;Zheng et al, 2014). The molecular mechanism of the antiangiogenic properties of TR-644 CA-4 analog was linked to the inhibition of vascular endothelial growth factor-induced phosphorylation of VE-cadherin (Porcù et al, 2013).…”

“…Similarly, modulation of endothelial cells by combretastatin analogs has been reported in vitro (Dupeyre et al, 2006;Kong et al, 2010;Romagnoli et al, 2010;Sanna et al, 2010;Arthuis et al, 2011;Porcù et al, 2013) and in vivo (Fortin et al, 2011;Porcù et al, 2013;Zheng et al, 2014). The molecular mechanism of the antiangiogenic properties of TR-644 CA-4 analog was linked to the inhibition of vascular endothelial growth factor-induced phosphorylation of VE-cadherin (Porcù et al, 2013). It has also been suggested that CA-4 may exert its antiangiogenic properties by downregulation of connective tissue growth factor, a negative regulator of angiogenesis (Samarin et al, 2009).…”

“…Small interfering RNA technology demonstrated a role for the RhoA/RhoA-associated kinase signaling pathway in the CA-4-mediated inhibition of T cell migration (Pollock et al, 2014). Other synthetic combretastatatin analogs also demonstrated significant antimetastasis/migration properties (Nathwani et al, 2013;Porcù et al, 2013;Pollock et al, 2014). Importantly, CA-4 demonstrated therapeutic activity in patients with metastatic anaplastic thyroid cancer (Granata et al, 2013).…”

Blocking Blood Flow to Solid Tumors by Destabilizing Tubulin: An Approach to Targeting Tumor Growth

“…162 Other combretastatin-like compounds also inhibited cell migration and/or metastasis. 149,167,168 In particular, compound 12 reduced lymph node metastasis of LY80 Yoshida sarcoma in mice by shutting down the blood flow even in small metastases. These observations suggest that combretastatin-mediated vascular shutdown not only affects large primary tumors but also micrometastases.…”