Role of mitochondria activation in Helicobacter pylori-sensitized TRAIL-mediated apoptosis in human gastric epithelial cells

TRAIL會引起釵h轉型細胞株的細胞凋亡，而非大部分的原始細胞。目前發現一些腫瘤細胞株會藉由細胞內的調控分子產生對TRAIL的抗拒性，因而細胞對TRAIL的感受性是受到調控的。在我們實驗室過去的研究中發現，人類胃上皮細胞會受到胃幽門螺旋桿菌的影響，而對TRAIL敏感度增加造成細胞凋亡；我們認為此增加的細胞凋亡現象，是由於胃幽門螺旋桿菌改變胃上皮細胞內訊息傳遞的路徑而造成。 為了探討受到胃幽門螺旋桿菌感染後，TRAIL引起胃上皮細胞凋亡的訊息傳導路徑，我們利用人類幽門螺旋桿菌與胃上細胞株共同培養的系統。而在實驗室過去的實驗發現此細胞凋亡現象可被caspase-3、 caspase-8和caspase-9的抑制因子所抑制。因此我們探討這些分子活化的情形，發現在沒有人類幽門螺旋桿菌存在的情況下，caspase-8可被TRAIL引起的訊息活化；但是只有在人類幽門螺旋桿菌存在的情況下，caspase-3和caspase-9才會被TRAILTRAIL引起的訊息活化。因此我們認為人類幽門螺旋桿菌影響胃上皮細胞，介於上游caspase-8 與 caspase-9之間，讓下游caspase-3 活化使得胃上皮細胞對TRAIL的敏感度增加造成細胞凋亡。此外，我們也觀察在細胞凋亡的過程中，可能參與粒線體活化的分子cytochrome c 的釋放以及調控粒線體的分子Bid活化的情形；由結果發現，在有幽門螺旋桿菌存在的情況下，TRAIL會引起胃上皮細胞株內的粒線體釋放cytochrome c 以及Bid活化的情形。因此我們進一步建立會大量表現Bcl-2的胃上皮細胞株，發現其在幽門螺旋桿菌存在的情況下，可恢復對TRAIL抗拒性。由於在幽門螺旋桿菌存在的情況下，TRAIL會引起胃上皮細胞株內Bid活化；而利用表現突變Bid的胃上皮細胞株則有一半的細胞恢復對TRAIL的抗拒性。由此結果我們認為幽門螺旋桿菌調控在胃上皮細胞株內Bid的活化步驟，藉此活化粒線體，讓胃上皮細胞對TRAIL敏感性增加。Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various transformed cell lines but not in most primary cells. The sensitivity to TRAIL is under regulation and the resistance to TRAIL in some tumor cells lines have been demonstrated to be controlled by intracellular regulators. Previous studies in our laboratory have demonstrated that human gastric epithelial cells were sensitized to Helicobacter pylori (H. pylori), conferring susceptibility to TRAIL-mediated apodosis. The enhanced TRAIL sensitivity by H. pylori is via altering intracellular signaling pathway in human gastric epithelial cells. In order to investigate signaling pathway of TRAIL-mediated apoptosis in gastric epithelial cell during H. pylori infection, we used an in vitro co-culture system to study the gastric epithelial cell apoptosis after interaction with H. pylori. Our previous study had shown that H. pylori-induced TRAIL-mediated apoptosis in gastric epithelial cells could be blocked by caspase-3, -8 or -9 inhibitors. In this study, our results demonstrated that caspase-8 was activated induced by TRAIL in human gastric epithelial cells either in the absence or presence of H. pylori. In contrast, activation of caspase-3 and -9 in gastric epithelial cells were induced by TRAIL only in the presence of H. pylori. These results indicate that H. pylori induces TRAIL-mediated apoptosis in gastric epithelial cells through a pathway involving the sequential induction of apical caspase-8 activity, effector-cascade and effector caspase-3 activity. The alteration of TRAIL signal transduction by H. pylori seems to be regulated at the level of caspase-8 downstream, and caspase-9 upstream pathway. Besides, we also examined the cytochrome c release and activation of regulatory molecules, Bid, in mitochondria activation. Our results showed that in the presence of H. pylori, TRAIL induces cytochrome c release and Bid cleavage in gastric epithelial cells. We further generated Bcl-2-overexpressing cell line and in which the H. pylori-induced TRAIL sensitivity could be reversed by overexpression of Bcl-2. Moreover, Bid cleavage after TRAIL engagement was observed only in the presence of H. pylori and expression of exogenous DN-Bid in AGS cells partially inhibit H. pylori-sensitized TRAIL-mediated apoptosis. These results indicated that mitochondria activation is required for H. pylori-sensitized TRAIL-mediated apoptosis and the regulation of TRAIL apoptosis signaling is at the level of Bid activation.Abstract i Abstract (Chinese) iii Chapter I. Introduction Part 1. Apoptosis 1 Part 2. TRAIL and its receptors 4 Part 3. TRAIL-induced apoptosis signaling 5 Part 4. TRAIL sensitivity and resistance 6 Part 5. Helicobacter pylori 7 Part 6. Aims of the study 7 Chapter II. Materials and Methods Part 1. Experimental Materials 9 Part 2. Experimental Procedures 17 a. Recombinant His-TRAIL 17 b. Measurement of apoptosis 18 c. Isolation of subcellular fractions 19 d. Western Blot 20 e. DN-Bid construction 21 f. Transfection 22 Chapter III. Results Part 1. H. pylori induces activation of casepase cascade in TRAIL apoptosis signaling 24 Part 2. Cytochrome C release 26 Part 3. Inhibition of apoptosis by bcl-2 overexpression 27 Part 4. Cleavage of BID in the presence of H. pylori 28 Chapter IV. Discussion Part 1. H. pylori modulates the TRAIL apoptosis signaling pathway 31 Part 2. Mitochondria pathway is regulated for H. pylori-induced TRAIL sensitivity 32 Part 3. Regulators of TRAIL signaling 35 Part 4. Conclusion 38 Reference 39 Figures 47 Figure1. Activation of caspase-8 in TRAIL-induced signaling in AGS cells 47 Figure2. H. pylori induces activation of caspase-3 in TRAIL signaling 48 Figure3. Activation of caspase-9 in TRAIL-induced apoptosis signaling in AGS cells in the presence of H. pylori 50 Figure4. TRAIL induces cytochrome c release from mitochondria in AGS cells in the presence of H. pylori 52 Figure5. Overexpression of Bcl-2 inhibits TRAIL-induced apoptosis mediated by H. pylori in AGS cells 53 Figure6. TRAIL induces Bid cleavage in AGS cells in the presence of H. pylori 56 Figure7. DN-Bid partially inhibits TRAIL-mediated apoptosis induced by H. pylori in AGS cells 57 Figure8. The death signaling cascade in H. pylori-sensitized TRAIL-mediated apoptosis 5