A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, 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. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling 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-delta). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism.

Procaspase-2S has been reported to selectively prevent membrane blebbing and apoptotic body formation in human monocytic-like leukemic U937 cells after etoposide (VP-16) treatment (Droin et al., Oncogene 20. 260-269, 2001). Here, we show that procaspase-2S overexpressed in human B lymphoma Namalwa cells inhibits procaspase-3 processing and activation, preventing cleavage and activation of Rho GTPase-associated ROCK-1 kinase. Failure of ROCK-1 activation in Namalwa cells correlates with a sustained delay in the appearance of membrane blebbing and apoptotic body formation after VP-16 treatment. Reciprocal coimmunoprecipitation experiments indicate that procaspase-2S binds to procaspase-3, but not procaspase-2L and -9 in untreated and VP-16-treated Namalwa cells. These data suggest that procaspase-2S-mediated anti-apoptotic effects are associated with inhibition of the processing and activation of procaspase-3 in VP-16-treated cells.

Effective treatments for androgen-independent prostate cancer (AIPCa) are lacking. To address this, emerging therapeutics such as proteasome inhibitors are currently undergoing clinical trials. Inositol hexakisphosphate (IP6) is an orally non-toxic phytochemical that exhibits antitumour activity against several types of cancer including PCa. We have previously shown that treatment of PC3 cells with IP6 induces the transcription of a subset of nuclear factor-kB (NF-kB)-responsive and pro-apoptotic BCL-2 family genes. In this study, we report that although NF-kB subunits p50/p65 translocate to the nucleus of PC3 cells in response to IP6, inhibition of NF-kBmediated transcription using non-degradable inhibitor of kB (IkB)-a does not modulate IP6 sensitivity. Treatment with IP6 also leads to increased protein levels of PUMA, BIK/NBK and NOXA between 4 and 8 h of treatment and decreased levels of MCL-1 and BCL-2 after 24 h. Although blocking transcription using actinomycin D does not modulate PC3 cell sensitivity to IP6, inhibition of protein translation using cycloheximide has a significant protective effect. In contrast, blocking proteasome-mediated protein degradation using MG-132 significantly enhances the ability of IP6 to reduce cellular metabolic activity in both PC3 and DU145 AIPCa cell lines. This effect of combined treatment on mitochondrial depolarisation is particularly striking and is also reproduced by another proteasome inhibitor (ALLN). The enhanced effect of combined MG132/IP6 treatment is almost completely inhibited by cycloheximide and correlates with changes in BCL-2 family protein levels. Altogether these results suggest a role for BCL-2 family proteins in mediating the combined effect of IP6 and proteasome inhibitors and warrant further pre-clinical studies for the treatment of AIPCa.

Abstract.A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-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. Recently, in a comparative proteomics analysis performed on highlyenriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-‰ (PKC-‰).In this study, we show that the PKC-‰ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-‰ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-‰ or ASM activity reduces both the CPTmediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-‰ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment.