title: Combining doublecortin-like kinase silencing and vinca alkaloids results in a synergistic apoptotic effect in neuroblastoma cells.
abstract: Microtubule-destabilizing agents, such as vinca alkaloids (VAs), are part of the treatment currently applied in patients with high-risk neuroblastoma (NB). However, the 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 encoded by the doublecortin-like kinase (DCLK) gene by using short interference RNA (siRNA). 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 EC(50) 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 terminal deoxynucleotidyl transferase dUTP nick-end labeling-based assay, we found a significant increase of apoptosis by the combined treatment. Induction of caspase-3 activity, as detected via cleavage of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, showed a 3.3- to 12.0-fold increase in the combined treatment. We detected significant increases in caspase-8 activity as well. Moreover, the multidrug dose effect calculated by using the median effect method showed a strong synergistic inhibition of proliferation and induction of apoptosis at most of the combined concentrations of siRNAs and VAs. Together, our data demonstrate that the silencing of DCLK sensitizes NB cells to VAs, resulting in a synergetic apoptotic effect.
authors.affiliation: Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands.
authors.forename: Carla S
authors.initials: CS
authors.lastname: Verissimo
authors.forename: Sou
authors.initials: S
authors.lastname: Cheng
authors.forename: Jordi C
authors.initials: JC
authors.lastname: Puigvert
authors.forename: Yu
authors.initials: Y
authors.lastname: Qin
authors.forename: Anne
authors.initials: A
authors.lastname: Vroon
authors.forename: Judith
authors.initials: J
authors.lastname: van Deutekom
authors.forename: Leo S
authors.initials: LS
authors.lastname: Price
authors.forename: Erik H J
authors.initials: EH
authors.lastname: Danen
authors.forename: Bob
authors.initials: B
authors.lastname: van de Water
authors.forename: Carlos P
authors.initials: CP
authors.lastname: Fitzsimons
authors.forename: Erno
authors.initials: E
authors.lastname: Vreugdenhil
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D000089742:Doublecortin-Like Kinases; D004357:Drug Synergism; D020868:Gene Silencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009447:Neuroblastoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010718:Phosphatidylserines / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D014747:Vinblastine / Q000494:pharmacology; D014748:Vinca Alkaloids / Q000494:pharmacology*; D014750:Vincristine / Q000494:pharmacology
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D010718:Phosphatidylserines; D014748:Vinca Alkaloids; D014750:Vincristine; D014747:Vinblastine; C117347:DCLK1 protein, human; D000089742:Doublecortin-Like Kinases; C485525:Dclk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; D053181:Caspase 8