Combining doublecortin-like kinase silencing and vinca alkaloids results in a synergistic apoptotic effect in neuroblastoma cells.

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.

Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells.

Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response.

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

UNLABELLED: Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase ß (IKKß) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity. CONCLUSION: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis.

The cancer stem cell microenvironment and anti-cancer therapy.

PURPOSE: Tumours are composed of a heterogeneous cell population. Cancer stem cells, which make up a minor fraction of a tumour, may be the cells that initiate and sustain tumour growth. Cancer stem cells are believed to share many properties with normal stem cells that render them relatively insensitive to classical radio- and chemotherapy. CONCLUSIONS: We discuss what those (cancer) stem cell properties are and how the interactions with the microenvironment--'the niche'--control those aspects of (cancer) stem cell biology. We also describe possible strategies to target cancer stem cells in order to prevent cancers from escaping therapy.