1. Silencing of doublecortin-like (DCL) results in decreased mitochondrial activity and delayed neuroblastoma tumor growth
Carla S Verissimo, et al. PLoS One. 2013 Sep 26;8(9):e75752. doi: 10.1371/journal.pone.0075752. eCollection 2013.
Doublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblastoma (NB) cell proliferation. We have investigated whether the anti-proliferative effect of DCL knockdown is linked to reduced mitochondrial activity. We found a delay in tumor development after DCL knockdown in vivo in doxycycline-inducible NB tumor xenografts. To understand the mechanisms underlying this tumor growth retardation we performed a series of in vitro experiments in NB cell lines. DCL colocalizes with mitochondria, interacts with the mitochondrial outer membrane protein OMP25/ SYNJ2BP and DCL knockdown results in decreased expression of genes involved in oxidative phosphorylation. Moreover, DCL knockdown decreases cytochrome c oxidase activity and ATP synthesis. We identified the C-terminal Serine/Proline-rich domain and the second microtubule-binding area as crucial DCL domains for the regulation of cytochrome c oxidase activity and ATP synthesis. Furthermore, DCL knockdown causes a significant reduction in the proliferation rate of NB cells under an energetic challenge induced by low glucose availability. Together with our previous studies, our results corroborate DCL as a key player in NB tumor growth in which DCL controls not only mitotic spindle formation and the stabilization of the microtubule cytoskeleton, but also regulates mitochondrial activity and energy availability, which makes DCL a promising molecular target for NB therapy.
2. Polo-like kinase 1 inhibition sensitizes neuroblastoma cells for vinca alkaloid-induced apoptosis
Sebastian Czaplinski, Manuela Hugle, Valerie Stiehl, Simone Fulda Oncotarget. 2016 Feb 23;7(8):8700-11. doi: 10.18632/oncotarget.3901.
High polo-like kinase 1 (PLK1) expression has been linked to poor outcome in neuroblastoma (NB), indicating that it represents a relevant therapeutic target in this malignancy. Here, we identify a synergistic induction of apoptosis by the PLK1 inhibitor BI 2536 and vinca alkaloids in NB cells. Synergistic drug interaction of BI 2536 together with vincristine (VCR), vinblastine (VBL) or vinorelbine (VNR) is confirmed by calculation of combination index (CI). Also, BI 2536 and VCR act in concert to reduce long-term clonogenic survival. Importantly, BI 2536 significantly enhances the antitumor activity of VCR in an in vivo model of NB. Mechanistically, BI 2536/VCR co-treatment triggers prolonged mitotic arrest, which is necessary for BI 2536/VCR-mediated apoptosis, since pharmacological inhibition of mitotic arrest by the CDK1 inhibitor RO-3306 significantly reduces cell death. Prolonged mitotic arrest leads to phosphorylation-mediated inactivation of BCL-2 and BCL-XL as well as downregulation of MCL-1, since inhibition of mitotic arrest by RO-3306 also prevents phosphorylation of BCL-2 and BCL-XL and MCL-1 downregulation. This inactivation of antiapoptotic BCL-2 proteins promotes activation of BAX and BAK, cleavage of caspase-9 and -3 and caspase-dependent apoptosis. Engagement of the mitochondrial pathway of apoptosis is critically required for BI 2536/VCR-induced apoptosis, since ectopic expression of a non-degradable MCL-1 phospho-mutant, BCL-2 overexpression or BAK knockdown significantly reduce BI 2536/VCR-mediated apoptosis. Thus, PLK1 inhibitors may open new perspectives for chemosensitization of NB.
3. Silencing of the microtubule-associated proteins doublecortin-like and doublecortin-like kinase-long induces apoptosis in neuroblastoma cells
Carla S Verissimo, et al. Endocr Relat Cancer. 2010 Apr 21;17(2):399-414. doi: 10.1677/ERC-09-0301. Print 2010 Jun.
Doublecortin-like kinase-long (DCLK-long) and doublecortin-like (DCL) are two splice variants of DCLK gene. DCL and DCLK-long are microtubule-associated proteins with specific expression in proliferative neural progenitor cells. We have tested the hypothesis that knockdown of DCL/DCLK-long by RNA interference technology will induce cell death in neuroblastoma (NB) cells. First, we analyzed the expression of DCL and DCLK-long in several human neuroblastic tumors, other tumors, and normal tissues, revealing high expression of both DCL and DCLK-long in NB and glioma. Secondly, gene expression profiling revealed numerous differentially expressed genes indicating apoptosis induction after DCL/DCLK-long knockdown in NB cells. Finally, apoptosis was confirmed by time-lapse imaging of phosphatidylserine translocation, caspase-3 activation, live/dead double staining assays, and fluorescence-activated cell sorting. Together, our results suggest that silencing DCL/DCLK-long induces apoptosis in NB cells.
