Research Report 1: The role of differentiation in the regulation of migration and invasion in glioblastoma

De begeleider en/of auteur heeft geen toestemming gegeven tot het openbaar maken van de scriptie. The supervisor and/or the author did not authorize public publication of the thesis.

Comparative proteomics analysis of caspase-9-protein complexes in untreated and cytochrome c/dATP stimulated lysates of NSCLC cells

Apoptosis is a process of cellular suicide executed by caspases. impaired activation of caspase-9 may contribute to chemoresistance in cancer. Activation of caspase-9 occurs after binding to Apaf-1 and formation of the apoptosome in the presence of cytochrome c/(d)ATP. We used a proteomics approach to identify proteins in caspase-9-protein complexes in extracts derived from NSCLC cells with(out) cytochrome c/dATP. Using co-immunoprecipitation, one-dimensional gel electrophoresis and tandem mass spectrometry, 38 proteins were identified of which 24 differential interactors. The differential interactors can be functionally assigned to cytoskeletal (re)organization and cell motility, catalytic activity, and transcriptional processes and apoptosis. The interaction of caspase-9 with Apaf-1 was confirmed and acetylserotonin-O-methyltransferase-like protein was identified as a candidate substrate of caspase-9. Novel interactors were found including galectin-3, swiprosin-1 and the membrane-cytoskeleton linkers Ezrin/Radixin/Moesin. Co-immunoprecipitation and Western blot experiments confirmed the interaction of caspase-9 with several identified binding partners. A large number of cytoskeletal proteins associated with unprocessed caspase-9 may indicate a scaffold function of this structure and/or may act as caspase substrates during apoptosis. Together, our results indicate that proteomic analysis of the caspase-9-associated protein complexes is a powerful exploratory approach to identify novel caspase substrates and/or regulators of caspase-9-dependent apoptosis. (C) 2009 Elsevier B.V. All rights reserve

Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells.

We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway