Mécanismes d'activation de la voie lysosomale durant l'apoptose chimio-induite
Apoptosis is a distinct form of regulated cell death which is essential for the development and homeostasis maintenance of multicellular animals. Apoptosis is an evolutionary conserved process involving a specific molecular pathway, known as the caspase cascade, and the different cytoplasmic organelles. A lysosomal pathway, characterized by partial rupture, labilization of lysosomal membranes (LML), and cathepsin activation in the cytoplasm, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In two independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labelling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-d). Functional experiments demonstrate that PKC-d translocation to lysosomes is required for LML, as silencing its expression with RNA interference or suppressing its activity with the inhibitor rottlerin prevents CPT-induced LLM. PKC-d translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase of ceramide (CER) content at lysosomes. The accumulation of endogenous CER at lysosomes is a critical event for CPT-induced LLM as suppressing PKC-d or ASM activity reduces both CPT-mediated CER generation at lysosomes and CPT-induced LLM.These findings reveal a novel mechanism by which PKC-d mediates ASM phosphorylation/activation and CER accumulation at lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. Taken together, these results confirm the importance of sphingolipid metabolism in the activation of the lysosomal pathway of apoptosis.
Lysosome PKC-d Apoptosis Membrane U-937 Lipid Membrane Ceramide PKC-d Sphingolipids