Study of TRAIL apoptosis signaling regulated by Helicobacter pylori in activation of mitochondria pathway

TRAIL (又稱為Apo2 ligand) 屬於腫瘤壞死因子家族的一員，可透過與其死亡受體結合引起細胞凋亡。TRAIL具有引發多種轉型細胞株進行細胞凋亡的能力，但是對於大部分的正常細胞則不具細胞毒殺性。在我們實驗室過去的研究中發現，人類胃上皮細胞對TRAIL具有抗性，然而胃幽門螺旋桿菌可以改變人類胃上皮細胞對TRAIL的抗性。我們認為此增加的細胞凋亡現象，是由於胃幽門螺旋桿菌經由活化caspase-8下游途徑進而造成粒線體訊息傳遞路徑活化而打破對細胞凋亡的抗性。而幽門螺旋桿菌所調控的位置是位於胃上皮細胞株內caspase-8的上游及Bid活化的下游。 在本研究中，我們的結果顯示出只有在胃幽門螺旋桿菌存在的情況下，人類胃上皮細胞株在加入TRAIL作用可以活化caspase-2。而加入caspase-2的抑制劑可以抑制幽門螺旋桿菌所引起的細胞凋亡。這樣的結果指出caspase-2在幽門螺旋桿菌所引起TRAIL的細胞凋亡途徑中是被需要的。 爲了進一步探討在TRAIL所調控的細胞凋亡當中caspase-2的作用機制，我們利用加入caspase-2的抑制劑來觀察Bid活化的情形。我們的結果顯示出，只有在幽門螺旋桿菌存在的情況下，TRAIL會引起胃上皮細胞株的Bid被切開和t-Bid產生。這樣的現象可以在加入caspase-2抑制劑所抑制。因此，在幽門螺旋桿菌所引起TRAIL細胞凋亡途徑中，caspase-2可以作用在切開Bid的上游。此外，加入caspase-8的抑制劑可以抑制caspase-2活化而加入caspase-2抑制劑卻不影響caspase-8活化。由這些結果我們認為在幽門螺旋桿菌所引起TRAIL細胞凋亡途徑中caspase-2是位於caspase-8的下游途徑。另外，我們也觀察到只有在幽門螺旋桿菌存在的情況下，TRAIL會引起Bax的轉位，暗示在幽門螺旋桿菌所引起TRAIL細胞凋亡途徑中，t-Bid可能會透過活化Bax來造成粒腺體釋放出cytochrome c。綜合以上結果顯示在幽門螺旋桿菌所引起TRAIL細胞凋亡途徑中，caspase-2可以在切開Bid上扮演調控的角色並透過Bax轉位來活化粒線體途徑。Tumor necrosis factor-related apoptosis-inducing ligand or Apo 2 ligand (TRAIL/Apo2L) is a member of the tumor necrosis factor (TNF) family of ligands capable of initiating apoptosis through engagement of its death receptors. TRAIL selectively induces apoptosis of a variety of tumor cells and transformed cells, but not most normal cells. Previous studies in our laboratory have demonstrated that human gastric epithelial cells are resistant to TRAIL. However, Helicobacter pylori can confer human gastric epithelium cells to TRAIL sensitivity. The enhanced TRAIL sensitivity by H. pylori is via activation of caspase-8 downstream pathway to activate mitochondria signaling pathway, leading to breaking apoptosis resistance. The alteration of H. pylori-sensitized TRAIL sensitivity is at the level of caspase-8 downstream pathway and upstream of Bid cleavage. In this study, our results demonstrated that caspase-2 was activated by TRAIL in human gastric epithelial cells only in the presence of H. pylori. Pre-treatment with caspase-2 inhibitor, z-VDVAD-fmk, could block H. pylori-sensitized TRAIL-mediated apoptosis in human gastric epithelium cells. This suggested that caspase-2 is required for H pylori- induced TRAIL apoptosis signaling. To further explore the regulatory mechanism of caspase-2 in TRAIL-mediated apoptosis, we investigated Bid cleavage by pretreatment with z-VDVAD-fmk. Our results showed that in the presence of H. pylori, TRAIL induced Bid cleavage and truncated Bid (t-Bid) formation in gastric epithelial cells. This phenomenon was blocked by z-VDVAD-fmk. Therefore, caspase-2 could function upstream of Bid cleavage in H. pylori-induced TRAIL apoptosis signaling. Moreover, pretreatment with caspase-8 inhibitor, z-IETD-fmk, inhibit caspase-2 processing but pretreatment with caspase-2 inhibitor, z-VDVAD-fmk have no effect on caspase-8 processing. These results suggest that caspase-2 is in the downstream pathway of caspase-8 in TRAIL apoptosis signaling. In addition, TRAIL induced Bax translocation in the presence of H. pylori, indicating that t-Bid activated Bax to promote cytochrome c release from mitochondria during H pylori-induced TRAIL apoptosis signaling. Taken together, these results show that caspase-2 can play a regulatory role on Bid cleavage and activation of mitochondria pathway via BAX translocation in H. pylori-induced TRAIL apoptosis signaling.Chapter I. Introduction Part 1. TRAIL and its receptors 1 Part 2. TRAIL-induced apoptosis signaling 2 Part 3. TRAIL sensitivity and resistance 5 Part 4. Helicobacter pylori 8 Part 5. Aims of the study 8 Chapter II. Materials and Methods Part 1. Experimental Materials 11 Part 2. Experimental Procedures 19 1. Recombinant His-TRAIL 19 2. Measurement of apoptosis 20 3. Isolation of subcellular fractions 21 4. Western Blot 23 Chapter III. Results Part 1. H. pylori induce activation of caspase-2 in TRAIL apoptosis signaling 24 Part 2. H. pylori-induced TRAIL sensitivity could be blocked by caspase-2 inhibitor, z-VDVAD-fmk 25 Part 3. Cleavage of Bid could be blocked by caspase-2 inhibitor, z-VDVAD- fmk 26 Part 4 .Caspase-2 can function downstream of caspase-8 27 Part 5 . H. pylori induce Bax translocation in AGS cells after TRAIL engagement 28 Chapter IV. Discussion Part 1. H. pylori modulate TRAIL sensitivity via Caspase-2 31 Part 2. Caspase-2 can function upstream of Bid cleavage and downstream of caspase-8 32 Part 3. Role of Bcl-2 family in TRAIL mediated apoptosis 34 Part 4. Conclusion 36 Reference 37 Figures 46 Figure1. TRAIL apoptosis signaling pathway 46 Figure2. H. pylori induce caspase-2 activation after TRAIL engagement in AGS cells 47 Figure3. H. pylori-induced TRAIL sensitivity could be blocked by caspase-2 inhibitor, z-VDVAD-fmk 48 Figure4. H. pylori-induced Bid cleavage after TRAIL engagement could be inhibited by pretreatment of caspase-2 inhibitor, z-VDVAD-fmk 50 Figure5. Pretreatment with caspase-2 inhibitor, z-VDVAD-fmk, has no significant influence on caspase-8 processing 51 Figure6. Caspase-2 processing was inhibited by pretreatment with caspase-8 inhibitor, z-IETD-fmk 52 Figure7. BAX translocation in TRAIL-induced apoptosis in AGS cells by H. pylori 53 Figure8. Bcl-2, Bcl-xL, and Mcl-1 expression in the presence or absence of H. pylori during TRAIL engagement 54 Figure9. TRAIL signaling pathway regulated by H. pylori in AGS cells 5