Inhibition of endothelial cell function in Vitro and angiogenesis in Vivo by docetaxel (taxotere): Association with impaired repositioning of the microtubule organizing center

A number of cancer chemotherapeutic drugs designed to have cytotoxic actions on tumor cells have recently been shown to also have antiangiogenic activities. Endothelial cell migration and proliferation are key components of tumor angiogenesis, and agents that target the microtubule cytoskeleton can interfere with these processes. In this study, the effect on endothelial cell functions of the microtubule-stabilizing drugs Taxotere and Taxol were evaluated in three in vitro assays: a chemokinetic migration assay, an angiogenesis factor-mediated chemotactic migration assay, and a three-dimensional Matrigel tubule formation assay, using rat fat pad endothelial cells (RFPECs) and/or human umbilical vein endothelial cells (HUVECs). Taxotere was active in all three assays at concentrations that were not cytotoxic and did not inhibit endothelial cell proliferation. In the RFPEC chemokinetic migration and in vitro tubule formation assays, the IC₅₀ values were approximately 10 ^-9 M for both Taxotere and Taxol. HUVEC migration, however, was more sensitive to Taxotere, with an observed IC₅₀ of 10^-12 M in a chemokinetic assay. In a Boyden chamber assay, HUVEC chemotaxis stimulated by either of two angiogenic factors, thymidine phosphorylase or vascular endothelial growth factor, was inhibited by Taxotere with an IC₅₀ of 10^-11 M and was ablated at 10^-9 M. Taxotere was also up to 1000-fold more potent than Taxol in inhibiting either chemokinetic or chemotactic migration. When the microtubule cytoskeleton was visualized using immunofluorescence staining of α-tubulin, there were no gross morphological changes observed in HUVECs or RFPECs treated with Taxotere at concentrations that inhibited endothelial cell migration but not proliferation. The effects of Taxotere on migration were associated with a reduction in the reorientation of the cell's centrosome, at concentrations that did not affect gross microtubule morphology or proliferation. Reorientation of the centrosome, which acts as the microtubule organizing center, in the intended direction of movement is a critical early step in the stabilization of directed cell migration. These data indicate that endothelial cell migration correlates more closely with changes in microtubule plasticity than with microtubule gross structure. The antiangiogenic activity of Taxotere in vivo was assessed in a Matrigel plug assay. In this assay, the angiogenic response to fibroblast growth factor 2 was inhibited in vivo by Taxotere with an ID₅₀ of 5.4 mg/kg when injected twice weekly over a 14-day period, and angiogenesis was completely blocked in mice that received 10 mg/kg Taxotere. The in vivo data further suggested that Taxotere had selectivity for endothelial cell migration and/or microvessel formation because infiltration of inflammatory cells into the Matrigel plug was much less sensitive to inhibition by Taxotere. In conclusion, Taxotere is a potent and potentially specific inhibitor of endothelial cell migration in vitro and angiogenesis in vitro and in vivo.