Distinct Expression Pattern of TRAIL in Normal and Colon Carcinoma Cells: A Novel Chemopreventive/Therapeutic Target
KA Matkowskyj, J Liao, YT Chung, G-Y Yang. Northwestern University, Chicago, IL
Background: TRAIL is a cytotoxic protein that induces apoptosis via its death receptors. TRAIL acts following binding to one of the 4 receptor subtypes. TRAIL-R1/DR4 and TRAIL-R2/DR5 contain an intracellular death domain, while TRAIL-R3/TRID and TRAIL-R4 are decoy receptors preventing apoptosis. PPAR-γ is a nuclear receptor found in epithelial neoplasms such as colon. PPAR-γ is activated by prostaglandin J2 (PGJ2) and has been shown to induce suppression and reversal of malignant changes in colon cancer in vitro by regulation of NF-κB, c-myc, and β–catenin. Regulation of c-myc results in upregulation of DR5 and blockade of c-FLIP. Thus, the main objectives of this study were to determine TRAIL receptor expression in normal and a malignant colon cancer cell line and to determine the role of combination therapy using PGJ2 and TRAIL on colonic cell apoptosis.
Design: Expression of the 4 TRAIL receptors in the human normal colonic epithelial cell line NCM460 and colonic carcinoma cell line HT-29 were examined using a real-time PCR approach with GAPDH as a reference gene. Dose effect of TRAIL on apoptosis induction was determined by morphologic criteria and trypan blue stain. Synergistic effects of PGJ2 and TRAIL was examined by co-treatment using 3 dose combinations. All experiments were repeated (n=5) to assure statistical significance and power. Further quantitative determination of TRAIL-related pathways was also performed using western blot.
Results: Quantitative RT-PCR revealed a distinct expression pattern of TRAIL-R in normal and carcinoma cell lines; it showed an increase in death receptor and decrease in decoy receptor expression in carcinoma cells compared to normal colonic epithelial cells; DR4 was 5.5-fold and DR5 expression was 17.2-fold higher than in normal cells. In contrast, the expression of the decoy receptors TRID and TRAIL-R4 was significant lower than normal cells (0.03 and 0.622-fold, respectively). Treatment of HT-29 cells with TRAIL for 48 hours revealed ∼30% induction of apoptosis. A significant increase in apoptosis was seen in HT-29 cells co-treated with PGJ2 and TRAIL (60% induction). Further downstream regulation of the TRAIL pathway by PGJ2 was also studied.
Conclusions: A distinct expression profile of death/decoy TRAIL receptors is present between the normal and carcinoma colonic cell lines, suggesting TRAIL death receptors would be a unique therapeutic target for treatment of colonic cancer with the combination of PGJ2 and TRAIL as a novel approach to induce cancer cell death.
Wednesday, March 24, 2010 9:30 AM
Poster Session V # 215, Wednesday Morning