Microtubule-destabilizing agents, such as vinca alkaloids (VAs), are part of the treatment currently applied in patients with high-risk neuroblastoma (NB). However, development of drug resistance and toxicity make NB difficult to treat with these drugs. In this study we explore the combination of VAs (vincristine or vinblastine) with knockdown of the microtubule-associated proteins (MAPs) encoded by the doublecortin-like kinase (DCLK) gene using siRNAs. We examined the effect of VAs and DCLK knockdown on the microtubule network by immunohistochemistry. We performed dose-response studies on cell viability and proliferation. By combining VA with DCLK knockdown we observed a strong reduction in the EC50 to induce cell death: up to 7.3 fold reduction of vincristine and 21.1 fold reduction of vinblastine. Using time-lapse imaging of phosphatidylserine translocation and a TUNEL-based assay we found a significant increase of apoptosis by the combined treatment. Induction of caspase-3 activity, as detected via cleavage of Ac-DEVD-AMC, showed a 3.3 to 12.0 fold increase in the combined treatment. We detected significant increases in caspase-8 activity as well. Moreover, multi-drug dose-effect calculated using the median effect method showed a strong synergistic inhibition of proliferation and induction of apoptosis at most of the different combined concentrations of siRNAs and VAs. Together, our data demonstrate that silencing of DCLK sensitizes NB cells to VAs resulting in a synergetic apoptotic effect.
- Received October 7, 2011.
- Revision received March 27, 2012.
- Accepted March 28, 2012.
- The American Society for Pharmacology and Experimental Therapeutics