We have previously shown that the death receptor ligand TRAIL induces an increase in intracellular C16-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C16-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C16-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may play a role in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

Tumor necrosis factor -related apoptosis -inducing ligand ( TRAIL / Apo2L ) has been shown to induce apoptosis in malignant cells without harming normal cells. To determine the antitumor potential of TRAIL against prostate cells, we undertook a comprehensive study that included eight prostate cancer cells lines ( CWR22Rv1, Du145, DuPro, JCA -1, LNCaP, PC -3, PPC -1, and TsuPr1 ) and primary cultures of normal prostate epithelial cells ( PrEC ). Cells were tested for susceptibility to soluble TRAIL in the presence or absence of the chemotherapeutic agent doxorubicin. TRAIL was also delivered by an adenoviral vector. Our results reveal that Du145, DuPro, LNCap, TsuPr1, and PrEC were resistant to 100 ng / mL TRAIL. JCA -1 and PPC -1 were slightly sensitive ( 20% killing ) and PC -3 and CWR22Rv1 exhibited the highest sensitivity to TRAIL ( 30% and 50% killing, respectively ). The combination of 10 ng / mL TRAIL with doxorubicin resulted in 60 -80% cytotoxicity in seven of eight prostate cancer cells. TRAIL -mediated apoptosis involved cleavage of Bid, caspase -3, and PARP, and required caspase -8 and -9 activity. Full -length TRAIL delivered by an adenoviral vector ( AdTRAIL -IRES -GFP ) killed prostate cancer cell lines and PrEC without requisite doxorubicin cotreatment. Therefore, expression of the transgene from a tissue -specific promotor would make gene therapy with AdTRAIL -IRES -GFP a possibility.

The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study, we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat, but not the individual drugs, rapidly increased reactive oxygen species (ROS), Ca 2+, and ceramide levels in gastrointestinal tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation, and cell killing was also blocked by quenching of induced Ca 2+ levels or by inhibition of PP2A.