CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced cytochrome c release

Death ligands such as CD95 ligand (CD95L) or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) induce apoptosis in rad iochemotherapy-resistant human malignant glioma cell lines. The death-signa ling TRAIL receptors 2 (TRAIL-R2/death receptor (DR) 5) and TRAIL-R1/ DR4 w ere expressed more abundantly than the non-death-inducing (decoy) receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in 12 human glioma cell lines. Four of the 12 cell lines were TRAIL/Apo2L-sensitive in the absence of a protein synth esis inhibitor, cycloheximide (CHX), Three of the 12 cell lines were still TRAIL/ Apo2L-resistant in the presence of CHX, TRAIL-R2 expression predicte d sensitivity to apoptosis. Coexposure to TRAIL/Apo2L and cytotoxic drugs s uch as topotecan, lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, CCNU) or temozolomide resulted in synergistic killing. Synergistic killing was more often observed in cell lines retaining wild-type p53 activity (U87 MG, LN-229) than in p53 mutant cell lines (LN-18, T98G, U373MG). Drug expos ure resulted in enhanced TRAIL-R2 expression, but decreased TRAIL-R4 expres sion in U87MG cells. Ectopic expression of dominant-negative p53(V135A) abr ogated the drug-induced changes in TRAIL-R2 and TRAIL-R4 expression, but ha d no effect on synergy. Thus, neither wildtype p53 function nor changes in TRAIL receptor expression were required for synergy, In contrast, synergy r esulted possibly from drug-induced cytochrome c release from mitochondria, serving as an amplifier of the TRAIL/Apo2L-mediated cascade of caspase acti vation. These data provide novel insights into the role of the TRAIL/Apo2L system in malignant gliomas and illustrate that TRAIL/Apo2L-based immunoche motherapy may be an effective therapeutic strategy for these lethal neoplas ms.