Abnormal Expression Of Procaspase-8 Inhibits TRAIL-induced Apoptosis In Tumor Cells

TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) is a member of the family of TNF-a-like cytokines and potential as an agent for tumor treatment due to its anti-tumor apoptosis selectivity and its nontoxicity for normal cells. AT-406 is a pan antagonist of inhibitor of apoptosis proteins (IAPs). Rocaglamide a natural product isolated from Aglaia species was previously shown to be a translational inhibitor of de novo synthesis of c-FLIP (Cellular FADD-Like (FLICE)-Inhibitory Protein). In the accompanied study, we have demonstrated that most peripheral solid tumor cells are sensitive to the apoptotic effects of triple combination (ART) of AT-406 (A), Rocaglamide (R) and TRAIL (T). We found that the above ART triple combination is very effective in inducing apoptosis for most peripheral solid tumor cells, but ineffective in a breast cancer cell line MCF-7, glioma cells(U251, U373, U87), and normal pulp cells. In this study, we further demonstrated that in addition to the high levels of expression of c-FLIP and IAPs, the expression of procasepase8, an upstream key protein of apoptosis pathway, is extremely low in these cells resistant to ART triple combination treatment. Up-regulation of procasepase8 sensitized these cells to the apoptotic effect to ART triple combination treatment.In this study, we used MTT cell viability assay for the apoptosis effect and Western Blots analysis of proteins in the TRAIL-induced extrinsic pathway of apoptosis to investigate the apoptotic effects of ART triple combination.Results showed that the breast cancer cells MCF-7, glioma cells and normal pulp cells all showed high resistance to the ART triple combination treatment. Western Blots protein analysis revealed the very low levels of expression of procaspase-8 in these cells, but high levels of expression of other proteins in the extrinsic apoptosis pathways including DR4/5, FADD, c-FLIP, and XIAP. Dup697, a COX-2 inhibitor and valproic acid (VA), a histone deacetylase inhibitor were used respectively in these cells for pretreatment to up-regulate the expression of procaspase-8 and sensitized these cells to the apoptotic effects of the ART triple combination treatment.In conclusion, our data in this and the accompanied. study suggest that the ART triple combination of TRAIL/a pan antagonist of IAPs/a c-FLIP inhibitor can effectively induce apoptosis of most peripheral solid tumor cancer cells. There are some solid tumor cells and most glioma cells are still resistant to the ART triple combination treatment. Up-regulation of procaspase-8 sensitized these cells to the ART triple combination treatment. Normal pulp cells were also resistant to the ART triple combination treatment and became sensitive after pretreatment to up-regulate the expression of procaspase-8. This demonstrated that our ART triple combination treatment has nontoxicity for normal cells. Low level expression of procaspase-8 may be the reason of it.Therefore, we consider the evasion of apoptosis in these tutrror cells including human normal Pulp cell can be caused by the loss of procaspase-8, which is obligatory in TRAIL apoptosis process.