Abstract C216: Small molecule antitubulin drugs that evade multiple drug resistance mechanisms.

Background: A new library of small molecules with structural features resembling combretastatin analogs was synthesized and two analogues were chosen by the NCI for screening. Both agents exhibited activity in all NCI-60 panel cell lines with GI 50 values Methods: Biochemical assays to assess the interaction with tubulin were carried out using purified tubulin. Immunofluorescence staining for beta-tubulin was also evaluated in PC3 cells following drug treatment. Vinblastine and docetaxel were used to demonstrate tubulin destabilizing and stabilizing modes of action, respectively. Cytotoxicity assays (72 hour GI 50 ) were carried out in PC3, DU145, LNCaP and C4/2 prostate cancer cells and in PC3-DR cells (docetaxel resistant PC3 cells). To assess the effect of P-gp, we used NCI/ADR-Res ovarian cancer cells and L-MDR1 porcine epithelial cells and their non-expressing parent lines. Assays were performed in 96-well plates using AlamarBlue cell viability dye. Cell-cycle analysis was performed using propidium iodide staining and flow activated cell sorting. Western blot analyses were used to assess the effect of drug treatment on apoptotic pathway and on cell cycle proteins. Results: The GI 50 in all cell lines was in the 1–10 nM range with no apparent resistance in P-gp expressing cells or in docetaxel resistant PC3-DR cells. The lead combretastatin analog showed a concentration dependent antitubulin interaction with a mode of action similar to vinblastine. This suggests that these compounds act by interfering with the microtubule polymerization process. Immunofluorescence staining for beta-tubulin showed diffuse staining patterns with drug increased concentration suggesting the lack of microtubule formation. Cell-cycle analysis demonstrated a G2/M cell cycle accumulation after 24-hr treatment with drug. Interestingly, there was no drug dependent effect on the number of cells in the sub-G1 fraction suggesting that the mode of cell death may be different than apoptosis. This was corroborated with the lack of caspase activation as well as the lack of cleaved-PARP. A concentration dependent p-Bcl2 was noted, however, suggesting that the drug interferes with the anti-apoptotic role of Bcl-2. Nuclear staining with Hoechst 33342 and DAPI demonstrated the presence of multinucleated cells suggesting that mitotic catastrophe could be the primary mode of cell death. This was further corroborated by the concentration dependent centrosome overduplication and presence of multiple spindle poles. Conclusion: These exquisitely potent antitubulin agents appear to overcome multiple modes of drug resistance including P-glycoprotein, p-53 deregulation, and Bcl-2. Further studies are ongoing to verify the exact molecular mechanism mediating cell death and to determine the in-vivo activity of these novel compounds. 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 C216.