Abstract LB-368: III-tubulin knockdown in endothelial cells increases the antivascular activity of chemotherapeutic drugs

Abstract

Abstract The microtubule cytoskeleton plays a crucial role in tumor angiogenesis, as evidenced by the potent anti-vascular properties of microtubule-targeting anticancer agents (MTAs). Accumulating evidence shows that the expression level of β-tubulin isotypes influences drug response and βIII-tubulin in particular has been involved in cancer cell resistance to MTAs as well as other classes of chemotherapeutic agents (1). However, the role of β-tubulin isotypes in the context of tumor angiogenesis and anti-vascular therapy remains unknown. Comparative western blot analysis revealed that human vascular endothelial cells express relatively high levels of βII- and βIII-tubulin. Using siRNA gene silencing, we effectively suppressed the expression levels of βII-and βIII-tubulin isotypes in vascular endothelial cells, by 90% and 80%, respectively. Functional analysis showed that βII-tubulin expression did not significantly influence either the capacity of endothelial cells to form vascular structures on Matrigel or the anti-vascular properties of MTAs. In contrast, knocking down the expression of βIII-tubulin in endothelial cells significantly inhibited the formation of capillary-like structures on Matrigel by 18 ± 3 % (p<0.001). Furthermore, the anti-angiogenic activity of the microtubule-stabilizing agent, paclitaxel, and the microtubule-depolymerizing agents, vinblastine and 2-methoxyestradiol (2-ME2), was significantly increased between 1.6 and 2.5-fold (p<0.05) in βIII-tubulin knockdown endothelial cells, as compared to control siRNA and βII-tubulin knockdown cells. Similarly, the vascular-disrupting activity of ENMD-1198, a new analog of 2-ME2, was also increased by 2.2-fold (p<0.05) in βIII-tubulin knockdown endothelial cells. More broadly, βIII-tubulin knockdown in endothelial cells also increased the anti-angiogenic activity of other classes of chemotherapeutic drugs, such as topoisomerase inhibitor etoposide (18 ± 1%, p<0.01) and alkylating agent cyclophosphamide (35 ± 5%, p<0.05). Altogether these results strongly suggest that βIII-tubulin may play a role in tumor angiogenesis and influence the anti-vascular activity of chemotherapeutic drugs. Further investigations are currently underway to unravel the underlying mechanisms.