Time sequence of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin treatment is responsible for a complex pattern of synergistic cytotoxicity

The combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin resulted in a greater cytotoxicity than could be accounted for by the addition of the cytotoxic effects of the agents alone. In this study, we hypothesized that the synergistic interaction between the two modalities can be changed when both the sequence and the time interval between the two treatments are varied. To test the hypothesis, human head-and-neck squamous-cell carcinoma (HNSCC)-6 cells were either pretreated with 0.01 - 0.5 mu g/ml TRAIL for various times (0-24 h) followed by treatment with 5 mu g/ml cisplatin or pretreated with 5 mu g/ml cisplatin for various times (0-24 h) followed by treatment with 0.5 pg/ml TRAIL. In latter case, the synergistic effect was gradually increased when the time interval between the two treatments was increased. In former case, a maximal synergy occurred within 0-4 h of pretreatment with TRAIL. However, the synergistic effect was gradually decreased when the time interval between the two treatments was increased. Data from immunoblotting analysis reveal that a similar pattern emerged for the PARP cleavage and caspase activation. The synergistic effect is not associated with DR4, DR5, FADD, and FLIPL. Interestingly, a complex pattern of synergistic interaction between TRAIL and cisplatin is related to the cleavage of FLIPS. Although overexpression of FLIPS protected cells from FLIPS cleavage and apoptotic death, blockage of FLIPS cleavage by replacing ASP(39) and Asp(42) residues with alanine did not further enhance FLIPS-mediated protection. Taken together, FLIPS cleavage reflects apoptotic damage, but it does not cause apoptosis.