Abstract 3275: Effects of combination of microtubule depolymerizing peptides and vinblastine in breast cancer.

Abstract

Abstract Background: Stathmin is a major cytosolic protein that promotes microtubule depolymerization. Recent crystallographic data indicate that both stathmin & the chemotherapeutic agent, vinblastine, act together by binding simultaneously on free tubulin. Stathmin was shown to mediate vinblastine activity by increasing the affinity of vinblastine for tubulin. We had previously demonstrated that intracellular delivery of short stathmin-like peptide(s) could inhibit the malignant proliferation of breast cancer (BC) cells by disrupting microtubule assembly. Here, we asked whether the anti-proliferative effects of stathmin-like peptides could be further enhanced by vinblastine. Methods: The peptide(s) that we used consisted of a short stathmin-like domain that was fused to a TAT transduction domain & a hemagglutinin epitope tag to track the peptide intracellularly. Since stathmin could be phosphorylated & inactivated intracellularly by p34cdc2 kinase, we also used a mutant peptide in which Ser-16 was mutated to an alanine to prevent its inactivation by phosphorylation. As a control, we used a similar peptide in which the stathmin-like domain was scrambled. Results: We tested the effects of these peptides in combination with vinblastine in different BC cell lines (MDA-MB-231, T47D, & SKBR-3). The rate of proliferation of untreated cells or cells exposed to control peptide was not affected by 2 nM concentration of vinblastine & was modestly inhibited at 5 nM concentration. When cells were exposed to wild type (WT) stathmin-like peptide & vinblastine, the rate of proliferation was further decreased at 2 nM concentration & nearly suppressed at 5 nM concentration of vinblastine. In contrast, cells exposed to mutant (MT) peptide showed complete cessation of growth when combined with 2 nM or 5 nM vinblastine. Thus, exposure to MT peptide seemed to result in greater sensitization of BC cells to the growth inhibitory effects of vinblastine. Similarly, cells exposed to either WT or MT peptide showed a 22% & 39% reduction in the colony formation respectively. When cells were exposed to either WT or MT peptide in combination with vinblastine, the clonogenicity was further reduced to 47% & 73%, respectively. This suggests that the peptides can inhibit anchorage independent growth, which is markedly enhanced by vinblastine. We next analyzed the ability of the designed peptides to induce apoptosis either as a single agent or in combination with vinblastine. TUNEL analysis of control cells showed essentially no apoptosis. When the cells were exposed to either WT or MT peptides, there was a 13% & 24% increase in the fraction of apoptosis respectively. However, combination of vinblastine with either WT or MT peptide significantly increased the fraction of apoptotic cells to 54% & 76%, respectively. These data suggest that the anti-tumor activity of stathmin-like peptides is markedly enhanced by vinblastine in BC cells in vitro. Citation Format: Ishan Purani, Arlene Tejada, Sucharita J. Mistry. Effects of combination of microtubule depolymerizing peptides and vinblastine in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3275. doi:10.1158/1538-7445.AM2013-3275