Abstract A4: Antiangiogenic effect of low-dose paclitaxel is mediated by RhoA inactivation: A potential new mechanism.
Authors: Lim Siong Meng , Perdomo Jose , Galettis Peter , Links Matthew 
Published: June 2012

Abstract
Introduction: Paclitaxel, a cytotoxic agent that destroys cancer cells by stabilizing the microtubule, inhibits tube formation by human umbilical vein endothelial cells (HUVEC) at subtoxic concentrations (≤10nM). The clear separation of the antiangiogenic effect of paclitaxel from its cytotoxicity suggests the possibility of alternative mechanisms of action. Understanding the mechanisms will help materialize the combination use of antiangiogenic cytotoxic agents with targeted therapeutic agents to obtain a synergistic antiangiogenic effect in cancer treatment. Our previous findings indicated that low dose paclitaxel can inhibit RhoA activation in HUVEC and this is strongly correlated to the antiangiogenic effect of paclitaxel. We therefore postulate that RhoA, a member of the Rho GTPases family which regulates cytoskeleton reorganization and branching of blood vessels, may be the critical link underlying the antiangiogenic effect of low dose paclitaxel. Aims: The present study aims to assess the causal effect of RhoA inactivation against the antiangiogenic effect of low dose paclitaxel through molecular and pharmacological approaches.
Methods: Plasmid DNA expressing fusion proteins of enhanced green fluorescent protein (EGFP) with either wild-type RhoA (WT-RhoA) or its constitutively active mutant (V14-RhoA) were constructed in-house and confirmed by sequencing. Protein expression was evaluated using fluorescent microscopy, flow cytometry and Western Blot analysis. Transfection of HUVEC was facilitated by the Amaxa's Nucleofector Technology (Lonza). The antiangiogenic effect of paclitaxel (0.1–100nM) was examined through the 24 h inhibition of tube formation by HUVEC transfected with our plasmid constructs. The pharmacological study involved the simultaneous use of 5μM 1-oleoyllysophosphatidic acid (LPA), a RhoA activator, with low dose paclitaxel in the 24 h tube formation assay.
Results: Transfection of HUVEC with WT-RhoA and V14-RhoA yielded EGFP fluorescence in 52 and 68% of the HUVEC, respectively. The antiangiogenic effect of 5, 10 and 100nM paclitaxel against HUVEC expressing V14-RhoA was reduced by 7, 56 and 6%, respectively when compared to WT-RhoA. Failure in RhoA inactivation led to modest attenuation of the antiangiogenic effect of paclitaxel. Subsequent investigation using LPA showed significant reversal of the antiangiogenic effect of low dose paclitaxel. Tube inhibition in the presence of LPA was attenuated by 89 and 42% for HUVEC treated with 0.1 and 10nM paclitaxel, respectively.
Conclusions: The antiangiogenic effect of low dose paclitaxel is dependent, to a great extent, upon RhoA inactivation. We propose in-depth studies exploring the possible effect of the interaction of RhoA and other related proteins against the therapeutic effect of low dose paclitaxel. The antiangiogenic effect of paclitaxel may also be enhanced by combination with targeted therapy that inhibits RhoA activation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A4.