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Regulation of TRAIL-induced Apoptosis of Gastric Adenocarcinoma

Author: WuPing
Tutor: ZhangLinJie；ZhangXuDong
School: Anhui Medical University,
Course: Immunology
Keywords: gastric adenocarcinoma apoptosis TRAIL
CLC: R735.2
Type: Master's thesis
Year: 2008
Downloads: 176
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Abstract

Background & Objective: Gastric cancer is one of the most common cancers and most frequent causes of cancer-related deaths in the world. Intrinsic, as well as acquired, resistance to chemotherapy remains a major problem in the treatment of this disease. It is, therefore, of great importance to develop new, patient-tailored, treatment stategies for gastric cancer patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts through the pro-apoptotic TRAIL-R1, -R2 receptors in tumor cells without harming normal cells and now is tested in clinical trials as a novel anti-cancer agent. However, not all human gastric cancer cell lines are sensitive to TRAIL. To optimize treatment of gastric cancer patients, and possibly tailor it to individual patients, further insight in the TRAIL apoptosis pathway is needed. This paper will dissect the apoptotic pathway induced by TRAIL in gastric adenocarcinoma, and discuss possible mechanisms underlying TRAIL resistance.Methods: Apoptotic cells were determined by the propidium iodide method using flow cytometry. Effect of caspase inhibitors on TRAIL-induced apoptosis were carried out by pretreating the cells with pan-caspase inhibitor (zVAD-fmk),caspase-8 inhibitor (z-IETD-fmk),or caspase-9 inhibitor (z-LEHD-fmk) before adding TRAIL. TRAIL- induced activation of Pro-caspase-3, -8, -9 and cleavage of Bid, PARP were conducted by Western blot analysis. The changes in mitochondrial membrane potential (Δψm) were measured by uptake of JC-1 using flow cytometry. The expression of Bcl-2 family members, Bcl-2, Bcl-XL and Mcl-1, were measured by Western blot analysis. TRAIL-R1, -R2, -R3, -R4 cell surface expression were evaluated by flow cytometry. And the expression of XIAP and Survivin before and after treatment of TRAIL were also conducted by Western blot analyses.Results: TRAIL induced apoptosis of gastric adenocarcinoma cells in a dose- and time-dependent manner. It appeared that BGC-823 were more sensitve to TRAIL-induced apoptosis than SGC-7901. The rates of apoptotic cells were 59.9 % and 24.3 %, respectively, after treatment with TRAIL (100μg/L) for 24 h. Caspase inhibitors blocked TRAIL-induced apoptosis nearly completely. caspase-3, -9 activation and PARP cleavage were detected early after exposure to TRAIL. The mitochondrial membrane potential (Δψm) decreased in response to TRAIL. The expression of the antiapoptotic proteins Mcl-1 were down-regulated at 24 h. In contrast, no changes in the overall protein levels of Bcl-2 and Bcl-XL were observed even at 24 h after treatment. The cell surface expression of death receptors, TRAIL-R1 and -R2, on the BGC-823 cell line were 97.87 % and 99.42 %, while on the SGC-7901 cell line, 7.03 % and 95.31 %, respectively. The expression of TRAIL-R3 and -R4 decoy receptors were very low. TRAIL induced equivalent cleavage of caspase-8 and Bid in TRAIL-sensitive and -resistant gastric cancer cell lines that express TRAIL death receptors. XIAP was down-regulated in TRAIL-sensitive cell line (BGC-823) during TRAIL-induced apoptosis, while there was no change in TRAIL-resistant cell line (SGC-7901). Moreover, no changes were observed in Survivin expression in the two cell lines.Conclusions: Induction of apoptosis of gastric adenocarcinoma cells by TRAIL is caspase-dependent. TRAIL induces both receptor-mediated and mitochondrial intrinsic pathways. Sensitivity of gastric adenocarcinoma cells to TRAIL-induced apoptosis may be regulated by expression of two death-induing receptors, especially TRAIL-R1, on the cell surface. TRAIL-induced apoptosis appeared to be determined at the level of the effector caspase-3, XIAP protects gastric cancer cells from TRAIL-indued apoptosis.

Regulation of TRAIL-induced Apoptosis of Gastric Adenocarcinoma

Abstract

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