Induction Of Apoptosis In HT29 Cells Based On A Combination Of Three Drugs Involved In The Death Receptor Pathway

TRAIL can specifically induce apoptosis of cancer cells,but not normal cells.Unfortunately,most of cancer cells are resistant to the TRAIL treatment.In this study,a strategy based on the combination of three agents that target at different key proteins involved in the death receptor signaling pathway was explored.The Peptide-H-1 a designed peptide antagonist targeting at cFLIP-FADD interaction,IG107 an antibody of death receptors and AT406 an inhibitor of IAP,were used in our combination treatments.A TRAIL-resistant cell line of HT29 was used in this study.After up to 72 h treatments,Rocaglamide an inhibitor of the protein expression of c-FLIPs,Peptide-H-1,IG107 and AT406 used alone,did not cause significantly apoptosis of HT29 up to the concentrations of 1,600 nM,1,600 nM,7?g/mL,and 3?g/mL,respectively.The double combination treatments of AT406 with Rocaglamide,Peptide-H-1 or IG107 induced significantly apoptosis of HT29.After cells treated with above double-agents combination for 48 h,the apoptotic rates of HT29 cells were reached up to 38%,40% and 60%,respectively.The apoptotic rate reached up to 52%,51% and 72%,respectively after the 72 h treatments with double agents.Finally,results from combination treatments with three agents(AT403,Peptide-H-1 and IG107)revealed that HT29 cells shrinked and their morphology severely deformed,and most of cells were lysed.The apoptotic rate reached to 63-70% when cells were treated with the combined three agents for 48 h.After 72 hours treatment with the combined three agents,the apoptosis rate reached to 43-50% even at the lowest concentration of IG107 used and reached up to 75-82% at higher concentrations of IG107.The experiments also proved that no changes in morphological,toxic or apoptotic effects in normal diploid cell lines of KMB17 and MRC-5 treated with the combination of three drugs for 48 hours.Meanwhile,we have studied the mechanisms of the combination of three agents leading to apoptosis of HT29 cells.The expression of DR5 receptor,Caspase-8,c-FLIP,Caspase-3 and Cleaved PARP involved in the HT29 apoptotic signaling pathway were determined by Western Blotting.The results showed that there were significant effects on the expression levels of those proteins of the HT29 cells treated with the triple combination of AT406,Peptide-H-1 and IG107.The HT29 cells treated with three agents increased the levels of DR receptor,Caspase 8,Caspase 3 and cleaved PARP,consistent with the activation of the apoptotic signaling.These cells treated with three agents decreased the level of c-FLIP,consistent with the inhibition of the anti-apoptotic factors in the pathway.Triple combination treatment of AT406,peptide-H-1 and IG107 promoted the apoptotic signal transmission step by step in cancer cells and eventually led to the apoptosis and the death of HT29 cancer cells.This study demonstrates that the combination of three agents based on the death receptor signaling pathway can induce apoptosis of TRAIL-resistant HT29 tumor cells and is not cytotoxic to normal cells.It provides a good theoretical and practical basis for future animal experiments and related clinical research.