臺灣博碩士論文加值系統

胃幽門螺旋桿菌(Helicobacter pylori, H. pylori)是一種微厭氧的革蘭氏陰性菌，於1982年被澳洲科學家Barry Marshall以及John Warren在胃炎與胃潰瘍的病人中所發現。根據統計，世界上有50%人口曾受到胃幽門螺旋桿菌的感染，然而在這些人中，有超過80%的人口是沒有病徵的。研究指出，胃幽門螺旋桿菌與胃部一些疾病的發生相關，像是胃炎、胃潰瘍以及胃癌等。
我們實驗室先前的研究指出，胃幽門螺旋桿菌透過降解short form cellular-FLICE inhibitory protein (FLIPs)，能夠增強death-inducing signaling complex (DISC)的形成，進而促使caspase 8的活化並造成胃上皮細胞的更容易受到tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)媒介的細胞凋亡。另外，實驗室的初步實驗結果亦指出，胃幽門螺旋桿菌亦能夠透過PI3K-Akt的訊息傳遞路徑來調控TRAIL媒介的細胞凋亡。
之前的研究指出，自噬作用(Autophagy)會受到PI3K-Akt的訊息傳遞路徑的抑制。另外，研究亦指出細胞凋亡與自噬作用間有交互作用。在胃幽門螺旋桿菌的感染期間，胃幽門螺旋桿菌會誘導胃上皮細胞自噬作用的產生以及自噬作用性的細胞死亡。
在此論文中，我們探討了胃幽門螺旋桿菌如何利用自噬作用敏化TRAIL媒介胃上皮細胞之凋亡。首先，我們觀察到了胃上皮細胞在與胃幽門螺旋桿菌共培養的八個小時間，p62的表現量並沒有顯著的變化，而LC3-II的表現量則是隨著時間而增加。然而，在胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡中，我們卻發現p62有顯著降解的現象，而此現象伴隨著些許LC3-II的降解。藉於p62的降解反映的是活化的自噬流(autophagic flux)，我們進一步得探討抑制自噬作用對於胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡的影響。實驗結果指出，抑制p62抑或是Atg5的表現量能夠顯著抑制胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡，而利用chloroquine抑制後期的自噬作用則只能部分的抑制TRAIL媒介胃上皮細胞的凋亡。並且，無論是抑制自噬體(autophagosome)的形成抑或是自噬溶酶體(autolysosome)的降解，FLIPs的表現量在胃幽門螺旋桿菌敏化TRAIL媒介胃上皮細胞之凋亡中均不受影響，暗指autophagy在此細胞凋亡中所調控的訊息傳導路徑可能與FLIPs的路徑不同。
綜合以上的實驗結果，我們發現胃幽門螺旋桿菌會利用自噬作用，透過p62抑或是Atg5來敏化TRAIL媒介胃上皮細胞之凋亡，而此訊息傳導路徑不受FLIPs的影響。這些結果有助於我們理解胃幽門螺旋桿菌造成細胞凋亡的致病機制，並提供臨床治療的可能方向及目標。

Helicobacter pylori (H. pylori), a Gram-negative microaerophilic bacterium identified by Barry Marshall and John Warren in patients with gastritis and peptic ulceration, infects about 50% of the world’s population. The infection of H. pylori is associated with duodenal ulcer, peptic ulcer diseases and gastric cancers. Among the individuals infected with H. pylori, however, over 80% are asymptomatic.
Previously, our lab has demonstrated that through down-regulation of the short form of cellular-FLICE inhibitory protein (FLIPs), H. pylori sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells by enhancing death-inducing signaling complex (DISC) assembly, which in turn activates caspase-8 and its signaling cascade. Furthermore, our unpublished data indicate that upstream of FLIPs, H. pylori enhances the TRAIL-mediated apoptosis through dephosphorylating Akt.
Autophagy is a catabolic process suppressed by activation of class I PI3K through PI3K-Akt-mTOR pathway, and that crosstalk between apoptosis and autophagy has been reported previously. Moreover, autophagy is induced within human gastric epithelial cells during H. pylori infection, and the infection causes autophagic cell death.
In this thesis, we investigated how H. pylori sensitized TRAIL-mediated apoptosis in human gastric epithelial cells through autophagy. First, we analyzed the time course expression of two autophagy markers, LC3-II and p62. The results revealed no differences in the expression of p62 but increased LC3-II level within 8 hours of H. pylori co-culture. Unexpectedly, significant downregulation of p62 was only observed in AGS cells simultaneously co-cultured with H. pylori and treated with TRAIL, accompanied by slight degradation of LC3-II. Since the degradation of p62 implied active autophagic flux, we further investigated whether blockade of autophagy could reverse the H. pylori-sensitized, TRAIL-mediated apoptosis. Either Knockdown of Atg5 or p62 by siRNA indeed inhibited the H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells, while blockade of late autophagy by chloroquine only partially reversed the apoptosis. Interestingly, neither inhibition of autophagosome formation nor autolysosome degradation affected the expression of FLIPs in the H. pylori-sensitized, TRAIL-mediated apoptosis, implying that the signaling axis regulated by autophagy is independent of the FLIPs axis in the apoptosis.
Taken together, the results unveiled that H. pylori sensitizes TRAIL-mediated apoptosis through autophagy via Atg5 and p62, and that the autophagic pathway is independent of the FLIPs axis. Exploring the role of autophagy in H. pylori-sensitized, TRAIL-mediated apoptosis provides candidates for potential therapeutic targets in H. pylori-associated diseases.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract v
Introduction 1
1. Helicobacter pylori 1
2. TRAIL-mediated apoptosis and H. pylori infection 1
3. DISC and FLIPs in TRAIL-mediated apoptosis 2
4. Akt pathway in regulation of FLIPs 2
5. Autophagy and apoptosis 3
Rationales 4
Materials and Methods 5
Results 14
I. H. pylori sensitizes TRAIL-mediated apoptosis in AGS cells through downregulation of FLIPs and dephosphorylation of Akt 14
II. Active autophagic flux in H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells 14
III. Knockdown of Atg5 reversed the H. pylori-sensitized, TRAIL-mediated apoptosis 15
IV. Knockdown of p62 reversed the H. pylori-sensitized, TRAIL-mediated apoptosis 15
V. Blockade of late autophagy partially reversed the H. pylori-sensitized, TRAIL-mediated apoptosis 16
Discussion 17
I. Active autophagic flux in H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells 17
II. The effects of knockdown autophagosomal proteins on H. pylori-sensitized, TRAIL-mediated apoptosis in AGS cells 17
III. Blockade of late autophagy partially reversed the H. pylori-sensitized, TRAIL-mediated apoptosis 19
Conclusions 20
References 21
Figures 26

[1]Marshall, B., & Warren, J. R. (1984). Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. The Lancet, 323(8390), 1311-1315.
[2]Amieva, M., & Peek Jr, R. M. (2016). Pathobiology of Helicobacter pylori–induced gastric cancer. Gastroenterology, 150(1), 64-78.
[3]Bytzer, P., Dahlerup, J. F., Eriksen, J. R., Jarbøl, D. E., Rosenstock, S., & Wildt, S. (2011). Diagnosis and treatment of Helicobacter pylori infection. Dan Med Bull, 58(4), C4271.
[4]Megraud, F. (2004). H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut, 53(9), 1374-1384.
[5]Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K., ... & Goodwin, R. G. (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. immunity, 3(6), 673-682.
[6]Kayagaki, N., Yamaguchi, N., Nakayama, M., Eto, H., Okumura, K., & Yagita, H. (1999). Type I interferons (IFNs) regulate tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) expression on human T cells: a novel mechanism for the antitumor effects of type I IFNs. Journal of Experimental Medicine, 189(9), 1451-1460.
[7]Wang, J., Fan, X., Lindholm, C., Bennett, M., O''Connoll, J., Shanahan, F., ... & Ernst, P. B. (2000). Helicobacter pylori modulates lymphoepithelial cell interactions leading to epithelial cell damage through Fas/Fas ligand interactions. Infection and immunity, 68(7), 4303-4311.
[8]Wu, Y. Y., Tsai, H. F., Lin, W. C., Chou, A. H., Chen, H. T., Yang, J. C., ... & Hsu, P. N. (2004). Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells. World journal of gastroenterology: WJG, 10(16), 2334.
[9]Walczak, H., & Sprick, M. R. (2001). Biochemistry and function of the DISC. Trends in biochemical sciences, 26(7), 452-453.
[10]Sprick, M. R., Weigand, M. A., Rieser, E., Rauch, C. T., Juo, P., Blenis, J., ... & Walczak, H. (2000). FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity, 12(6), 599-609.
[11]Kischkel, F. C., Lawrence, D. A., Chuntharapai, A., Schow, P., Kim, K. J., & Ashkenazi, A. (2000). Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity, 12(6), 611-620.
[12]Bodmer, J. L., Holler, N., Reynard, S., Vinciguerra, P., Schneider, P., Juo, P., ... & Tschopp, J. (2000). TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nature cell biology, 2(4), 241
[13]Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., ... & Rimoldi, D. (1997). Inhibition of death receptor signals by cellular FLIP. Nature, 388(6638), 190.
[14]Lin, W. C., Tsai, H. F., Liao, H. J., Tang, C. H., Wu, Y. Y., Hsu, P. I., ... & Hsu, P. N. (2014). Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP. Cell death & disease, 5(3), e1109.
[15]Staal, S. P. (1987). Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proceedings of the National Academy of Sciences, 84(14), 5034-5037.
[16]Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., ... & Greenberg, M. E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. cell, 96(6), 857-868.
[17]Kodaki, T., Woscholski, R., Hallberg, B., Downward, P. R. V. J., & Parker, P. J. (1994). The activation of phosphatidylinositol 3-kinase by Ras. Current Biology, 4(9), 798-806.
[18]Oak, J. S., Chen, J., Peralta, R. Q., Deane, J. A., & Fruman, D. A. (2009). The p85β regulatory subunit of phosphoinositide 3-kinase has unique and redundant functions in B cells. Autoimmunity, 42(5), 447-458.
[19]Sarbassov, D. D., Guertin, D. A., Ali, S. M., & Sabatini, D. M. (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science, 307(5712), 1098-1101.
[20]Manning, B. D., & Toker, A. (2017). AKT/PKB signaling: navigating the network. Cell, 169(3), 381-405.
[21]Nam, S. Y., Jung, G. A., Hur, G. C., Chung, H. Y., Kim, W. H., Seol, D. W., & Lee, B. L. (2003). Upregulation of FLIPS by Akt, a possible inhibition mechanism of TRAIL‐induced apoptosis in human gastric cancers. Cancer science, 94(12), 1066-1073.
[22]Yang, Z., & Klionsky, D. J. (2010). Eaten alive: a history of macroautophagy. Nature cell biology, 12(9), 814.
[23]Mizushima, N., & Komatsu, M. (2011). Autophagy: renovation of cells and tissues. Cell, 147(4), 728-741.
[24]Yousefi, S., Perozzo, R., Schmid, I., Ziemiecki, A., Schaffner, T., Scapozza, L., ... & Simon, H. U. (2006). Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nature cell biology, 8(10), 1124.
[25]Bell, B. D., Leverrier, S., Weist, B. M., Newton, R. H., Arechiga, A. F., Luhrs, K. A., ... & Walsh, C. M. (2008). FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells. Proceedings of the National Academy of Sciences, 105(43), 16677-16682.
[26]Pyo, J. O., Jang, M. H., Kwon, Y. K., Lee, H. J., Jun, J. I., Woo, H. N., ... & Mizushima, N. (2005). Essential roles of Atg5 and FADD in autophagic cell death dissection of autophagic cell death into vacuole formation and cell death. Journal of Biological Chemistry, 280(21), 20722-20729.
[27]Young, M. M., Takahashi, Y., Khan, O., Park, S., Hori, T., Yun, J., ... & Kester, M. (2012). Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (iDISC)-mediated caspase-8 activation and apoptosis. Journal of Biological Chemistry, 287(15), 12455-12468.
[28]Jin, Z., Li, Y., Pitti, R., Lawrence, D., Pham, V. C., Lill, J. R., & Ashkenazi, A. (2009). Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling. Cell, 137(4), 721-735.
[29]Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., & Kim, D. H. (2010). mTOR regulation of autophagy. FEBS letters, 584(7), 1287-1295.
[30]Laplante, M., & Sabatini, D. M. (2009). mTOR signaling at a glance. Journal of cell science, 122(20), 3589-3594.
[31]Terebiznik, M. R., Raju, D., Vázquez, C. L., Torbricki, K., Kulkarni, R., Blanke, S. R., ... & Jones, N. L. (2009). Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells. Autophagy, 5(3), 370-379.
[32]Zhu, P., Xue, J., Zhang, Z. J., Jia, Y. P., Tong, Y. N., Han, D., ... & Tang, B. (2017). Helicobacter pylori VacA induces autophagic cell death in gastric epithelial cells via the endoplasmic reticulum stress pathway. Cell death & disease, 8(12), 3207.
[33]Kimura, S., Noda, T., & Yoshimori, T. (2007). Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy, 3(5), 452-460.
[34]Radoshevich, L., Murrow, L., Chen, N., Fernandez, E., Roy, S., Fung, C., & Debnath, J. (2010). ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell, 142(4), 590-600.
[35]Mukhopadhyay, S., Das, D. N., Panda, P. K., Sinha, N., Naik, P. P., Bissoyi, A., ... & Bhutia, S. K. (2015). Autophagy protein Ulk1 promotes mitochondrial apoptosis through reactive oxygen species. Free Radical Biology and Medicine, 89, 311-321.
[36]Greenfield, L. K., & Jones, N. L. (2013). Modulation of autophagy by Helicobacter pylori and its role in gastric carcinogenesis. Trends in microbiology, 21(11), 602-612.
[37]Liu, W. J., Ye, L., Huang, W. F., Guo, L. J., Xu, Z. G., Wu, H. L., ... & Liu, H. F. (2016). p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation. Cellular & molecular biology letters, 21(1), 29.