Laulimalide, a natural product isolated from marine sponges, is a microtubule- stabilizing agent that binds to the tubulin polymer at a site that is distinct from that of the taxoids. Laulimalide has been shown to be active against tumor cells that were resistant to the taxanes due either to overexpression of P-glycoprotein or due to mutations in the β-tubulin gene. In addition to their effects on tumor cell proliferation and apoptosis, microtubule-binding agents have also been shown to be highly potent inhibitors of angiogenesis, and this could contribute to their anti-tumor actions. In the present study we found that laulimalide inhibited human umbilical vein endothelial cell (HUVEC) tubule formation and VEGF-induced HUVEC migration in vitro. Laulimalide inhibited migration with an IC50 of 10 pM, comparable to that of taxotere. Concentrations of laulimalide that were 400-fold higher were required to inhibit HUVEC proliferation. Laulimalide and taxotere had minimal effect on HUVEC migration when used individually at a concentration of 0.1 pM, however, when combined at this concentration, migration was inhibited by 70%. Possible mechanism(s) by which laulimalide inhibited VEGF-induced HUVEC migration were explored. VEGF’s actions are mediated by the tyrosine phosphorylation and activation of its receptors followed by signaling events which include the activation of integrins, the phosphorylation of proteins involved in cell migration signaling including focal adhesion kinase (FAK) and paxillin, and the recruitment of growth factor receptors, integrins and downstream effectors into newly formed focal adhesions. Similar to taxotere, laulimalide showed no effect on the extent of VEGF-induced Tyr951 phosphorylation of the VEGF receptor FLK1/KDR (VEGFR-2) in HUVEC. Also similar to taxotere, low concentrations of laulimalide substantially blocked subsequent downstream events, including the phosphorylation of the Tyr397 and Tyr407 residues of FAK, and the Tyr31 phosphorylation of paxillin. When compared to taxotere, however, laulimalide had a weaker inhibitory effect on the VEGF-induced association of VEGFR-2 with the α5β1 integrin. Also in contrast to taxotere, laulimalide potently caused a reduction in the constitutive levels of phosphorylated paxillin, in addition to blocking its phosphorylation in response to VEGF. In conclusion, while both taxotere and laulimalide inhibited integrin-associated signaling pathways which mediated VEGF-induced cell migration, their actions on the signaling cascade appeared not to be identical. Thus taxotere had a larger inhibitory effect on the association of the VEGF receptor with integrins, while laulimalide seemed to have a greater effect on the level of paxillin phosphorylation. These complementary actions could account for their apparent synergistic effect on HUVEC migration.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]