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研究生：	方俐晴 Fang, Li-Ching
論文名稱：	豚鼠產氣單胞菌分泌的M35金屬酶透過蛋白酶活化受體活化Akt/mTOR途徑促進胰臟癌細胞增生 The M35 metalloproteinase secreted by Aeromonas caviae promotes pancreatic cancer cell proliferation through activation of the Akt/mTOR pathway via the protease-activated receptors (PARs)
指導教授：	陳柏齡 Chen, Po-Lin
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2025
畢業學年度：	113
語文別：	英文
論文頁數：	75
中文關鍵詞：	豚鼠產氣單胞菌、條件培養基、細菌金屬酶、胰臟癌、增生、蛋白酶活化受體、Akt/mTOR 訊號傳導
外文關鍵詞：	Aeromonas caviae, conditioned medium, bacterial metalloproteinase, pancreatic cancer, proliferation, protease-activated receptors, Akt/mTOR signaling
相關次數：	點閱：6 下載：0
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中文摘要 I
Abstract II
Acknowledgment IV
Contents V
Introduction1
1. Aeromonas caviae 1
2. Pancreatic cancer 3
3. Protease-activated receptors (PARs) 5
4. Bacterial proteinase and PARs 7
5. The correlation between PARs and cancer 9
Materials and Methods 12
1. Bacterial strains 12
2. Cell culture 12
3. Polymerase chain reaction (PCR) 13
4. Preparation of conditioned medium (CM) 13
5. Plasmid transformation and selection 14
6. Expression and Purification of Recombinant M35 Protein 14
7. Cell proliferation assay 14
8. Western blot 15
9. Cytotoxicity assay 16
10. RNA isolation and Real-Time PCR 16
11. Statistical analysis 17
Results 18
1. M35 metalloproteinase may be a candidate in A. caviae conditioned medium (CM) that promotes pancreatic cancer cell proliferation. 18
2. M35 is exclusively found in clinical bile isolates of A. caviae. 20
3. The conditioned medium of A. caviae induces Akt/mTOR activation via protease-activated receptor 1 (PAR1) or protease-activated receptor 2 (PAR2). 21
4. M35 peptidase induces pancreatic cancer cell proliferation through activating Akt/mTOR signaling pathway via the combination of protease-activated receptor 1 (PAR1) and protease-activated receptor 2 (PAR2). 23
Discussion 25
References 30
Tables 37
Table 1. The primers of M35 PCR. 37
Table 2. The PCR reaction contents of primer, DNA, 2X master mix, and sterile water. The final volume of each PCR reaction was 25 μl. 38
Table 3. Primers for RT-PCR used in the study. 39
Table 4. Summary of M35 PCR of A. caviae, A. veronii, A. hydrophila, and A. dhakensis. 40
Figures 43
Figure 1. A. caviae is commonly found in patients with pancreatic cancer. 44
Figure 2. M35 peptidase may be a target for cell proliferation in A. caviae conditioned medium (CM). 49
Figure 3. M35 is exclusively found in clinical bile isolates of A. caviae. 51
Figure 4. Conditioned medium of A. caviae induces Akt/mTOR activation through protease-activated receptor 1 (PAR1) or protease-activated receptor 2 (PAR2). 56
Figure 5. M35 peptidase induces pancreatic cancer cell proliferation through activating Akt/mTOR signaling pathway via the combination of protease-activated receptor 1 (PAR1) and protease-activated receptor 2 (PAR2). 62
Figure 6. Summary of the study. 64
Supplementary 65
Figure S1. M35 mRNA expression levels were comparable in cancer and non-cancer groups. 65
Figure S2. PAR1 and PAR2 expression levels remained unchanged compared to the mock group after treatment with M35 recombinant protein. 66
                                    

1. Janda JM, Abbott SL: The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 2010, 23(1):35-73.
2. Figueras M, Hidalgo R: Aeromonas infections in humans. In., edn.; 2015: 65-108.
3. Beaz-Hidalgo R, Figueras MJ: Aeromonas spp. whole genomes and virulence factors implicated in fish disease. J Fish Dis 2013, 36(4):371-388.
4. Fernández-Bravo A, Figueras MJ: An Update on the Genus Aeromonas: Taxonomy, Epidemiology, and Pathogenicity. Microorganisms 2020, 8(1):129.
5. John N, Vidyalakshmi VB, Hatha AAM: Effect of pH and Salinity on the Production of Extracellular Virulence Factors by Aeromonas from Food Sources. J Food Sci 2019, 84(8):2250-2255.
6. González‐Serrano CJ, Santos JA, García‐López ML, Otero A: Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case. Journal of Applied Microbiology 2002, 93(3):414-419.
7. Chen YW, Su SL, Li CW, Tsai CS, Lo CL, Syue LS, Li MC, Lee CC, Lee NY, Ko WC et al: Pancreaticobiliary Cancers and Aeromonas Isolates Carrying Type Ⅲ Secretion System Genes ascF-ascG Are Associated With Increased Mortality: An Analysis of 164 Aeromonas Infection Episodes in Southern Taiwan. Front Cell Infect Microbiol 2021, 11:749269.
8. Chao CM, Lai CC, Tang HJ, Ko WC, Hsueh PR: Biliary tract infections caused by Aeromonas species. European Journal of Clinical Microbiology & Infectious Diseases 2013, 32(2):245-251.
9. Clark NM, Chenoweth CE: Aeromonas Infection of the Hepatobiliary System: Report of 15 Cases and Review of the Literature. Clinical Infectious Diseases 2003, 37(4):506-513.
10. Chan FK, Ching JY, Ling TK, Chung SC, Sung JJ: Aeromonas infection in acute suppurative cholangitis: review of 30 cases. J Infect 2000, 40(1):69-73.
11. Kimura M, Araoka H, Yoneyama A: Aeromonas caviae is the most frequent pathogen amongst cases of Aeromonas bacteremia in Japan. Scand J Infect Dis 2013, 45(4):304-309.
12. Figueras MJ: Clinical relevance of Aeromonas sM503. Reviews and Research in Medical Microbiology 2005, 16(4):145-153.
13. Igbinosa IH, Igumbor EU, Aghdasi F, Tom M, Okoh AI: Emerging Aeromonas species infections and their significance in public health. ScientificWorldJournal 31 2012, 2012:625023.
14. Lamy B, Kodjo A, Laurent F: Prospective Nationwide Study of <i>Aeromonas</i> Infections in France. Journal of Clinical Microbiology 2009, 47(4):1234-1237.
15. Song Y, Wang L-f, Zhou K, Liu S, Guo L, Ye L-y, Gu J, Cheng Y, Shen D-x: Epidemiological characteristics, virulence potential, antimicrobial resistance profiles, and phylogenetic analysis of Aeromonas caviae isolated from extraintestinal infections. Frontiers in Cellular and Infection Microbiology 2023, 13.
16. Pablos M, Remacha MA, Rodríguez-Calleja JM, Santos JA, Otero A, García-López ML: Identity, virulence genes, and clonal relatedness of Aeromonas isolates from patients with diarrhea and drinking water. European Journal of Clinical Microbiology & Infectious Diseases 2010, 29(9):1163-1172.
17. Chacón MR, Soler L, Groisman EA, Guarro J, Figueras MJ: Type III secretion system genes in clinical Aeromonas isolates. J Clin Microbiol 2004, 42(3):1285-1287.
18. Yang S, He T, Sun J, Sun S: Distinct Antimicrobial Resistance Profiling Of Clinically Important Aeromonas Spp. In Southwest China: A Seven-Year Surveillance Study. Infect Drug Resist 2019, 12:2971-2978.
19. Vincent A, Herman J, Schulick R, Hruban RH, Goggins M: Pancreatic cancer. Lancet 2011, 378(9791):607-620.
20. Kamisawa T, Wood LD, Itoi T, Takaori K: Pancreatic cancer. The Lancet 2016, 388(10039):73-85.
21. Marstrand-Daucé L, Lorenzo D, Chassac A, Nicole P, Couvelard A, Haumaitre C: Acinar-to-Ductal Metaplasia (ADM): On the Road to Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Cancer. Int J Mol Sci 2023, 24(12).
22. Dunne RF, Hezel AF: Genetics and Biology of Pancreatic Ductal Adenocarcinoma. Hematology/Oncology Clinics of North America 2015, 29(4):595-608.
23. Truong LH, Pauklin S: Pancreatic Cancer Microenvironment and Cellular Composition: Current Understandings and Therapeutic Approaches. Cancers (Basel) 2021, 13(19).
24. Maisonneuve P, Lowenfels AB: Epidemiology of pancreatic cancer: an update. Dig Dis 2010, 28(4-5):645-656.
25. Klein AP: Pancreatic cancer epidemiology: understanding the role of lifestyle and inherited risk factors. Nat Rev Gastroenterol Hepatol 2021, 18(7):493-502.
26. Bosetti C, Rosato V, Li D, Silverman D, Petersen GM, Bracci PM, Neale RE, Muscat J, Anderson K, Gallinger S et al: Diabetes, antidiabetic medications, and pancreatic cancer risk: an analysis from the International Pancreatic Cancer Case-Control Consortium. Ann Oncol 2014, 25(10):2065-2072.
27. Trikudanathan G, Philip A, Dasanu CA, Baker WL: Association between Helicobacter pylori infection and pancreatic cancer. A cumulative metaanalysis. Jop 2011, 12(1):26-31.
28. Fan X, Alekseyenko AV, Wu J, Peters BA, Jacobs EJ, Gapstur SM, Purdue MP, Abnet CC, Stolzenberg-Solomon R, Miller G et al: Human oral microbiome and prospective risk for pancreatic cancer: a population-based nested case-control study. Gut 2018, 67(1):120-127.
29. Cruz MS, Tintelnot J, Gagliani N: Roles of microbiota in pancreatic cancer development and treatment. Gut Microbes 2024, 16(1):2320280.
30. Pushalkar S, Hundeyin M, Daley D, Zambirinis CP, Kurz E, Mishra A, Mohan N, Aykut B, Usyk M, Torres LE et al: The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression. Cancer Discov 2018, 8(4):403-416.
31. Maekawa T, Fukaya R, Takamatsu S, Itoyama S, Fukuoka T, Yamada M, Hata T, Nagaoka S, Kawamoto K, Eguchi H et al: Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer. Biochem Biophys Res Commun 2018, 506(4):962-969.
32. Ren Z, Jiang J, Xie H, Li A, Lu H, Xu S, Zhou L, Zhang H, Cui G, Chen X et al: Gut microbial profile analysis by MiSeq sequencing of pancreatic carcinoma patients in China. Oncotarget 2017, 8(56):95176-95191.
33. Thomas RM, Gharaibeh RZ, Gauthier J, Beveridge M, Pope JL, Guijarro MV, Yu Q, He Z, Ohland C, Newsome R et al: Intestinal microbiota enhances pancreatic carcinogenesis in preclinical models. Carcinogenesis 2018, 39(8):1068-1078.
34. Sethi V, Kurtom S, Tarique M, Lavania S, Malchiodi Z, Hellmund L, Zhang L, Sharma U, Giri B, Garg B et al: Gut Microbiota Promotes Tumor Growth in Mice by Modulating Immune Response. Gastroenterology 2018, 155(1):33-37.e36.
35. Panebianco C, Villani A, Pisati F, Orsenigo F, Ulaszewska M, Latiano TP, Potenza A, Andolfo A, Terracciano F, Tripodo C et al: Butyrate, a postbiotic of intestinal bacteria, affects pancreatic cancer and gemcitabine response in in vitro and in vivo models. Biomedicine & Pharmacotherapy 2022, 151:113163.
36. Fulop DJ, Zylberberg HM, Wu YL, Aronson A, Labiner AJ, Wisnivesky J, Cohen DJ, Sigel KM, Lucas AL: Association of Antibiotic Receipt With Survival Among Patients With Metastatic Pancreatic Ductal Adenocarcinoma Receiving Chemotherapy. JAMA Network Open 2023, 6(3):e234254-e234254.
37. Conroy T, Hammel P, Hebbar M, Ben Abdelghani M, Wei AC, Raoul JL, Choné L, Francois E, Artru P, Biagi JJ et al: FOLFIRINOX or Gemcitabine as Adjuvant Therapy for Pancreatic Cancer. N Engl J Med 2018, 379(25):2395-2406.
38. Conroy T, Castan F, Lopez A, Turpin A, Ben Abdelghani M, Wei AC, Mitry E, Biagi JJ, Evesque L, Artru P et al: Five-Year Outcomes of FOLFIRINOX vs Gemcitabine as Adjuvant Therapy for Pancreatic Cancer: A Randomized Clinical Trial. JAMA Oncol 2022, 8(11):1571-1578.
39. Kolbeinsson HM, Chandana S, Wright GP, Chung M: Pancreatic Cancer: A Review of Current Treatment and Novel Therapies. Journal of Investigative Surgery 2023, 36(1):2129884.
40. Zhao P, Metcalf M, Bunnett NW: Biased signaling of protease-activated receptors. Front Endocrinol (Lausanne) 2014, 5:67.
41. Chandrabalan A, Ramachandran R: Molecular mechanisms regulating Proteinase-Activated Receptors (PARs). The FEBS Journal 2021, 288(8):2697- 2726.
42. Austin KM, Covic L, Kuliopulos A: Matrix metalloproteases and PAR1 activation. Blood 2013, 121(3):431-439.
43. Elmariah SB, Reddy VB, Lerner EA: Cathepsin S signals via PAR2 and generates a novel tethered ligand receptor agonist. PLoS One 2014, 9(6):e99702.
44. Dulon S, Leduc D, Cottrell GS, D'Alayer J, Hansen KK, Bunnett NW, Hollenberg MD, Pidard D, Chignard M: Pseudomonas aeruginosa elastase disables proteinase-activated receptor 2 in respiratory epithelial cells. Am J Respir Cell Mol Biol 2005, 32(5):411-419.
45. Bandara M, MacNaughton WK: Protease-activated receptor-2 activation enhances epithelial wound healing via epidermal growth factor receptor. Tissue Barriers 2022, 10(2):1968763.
46. Indrakusuma I, Romacho T, Eckel J: Protease-Activated Receptor 2 Promotes Pro-Atherogenic Effects through Transactivation of the VEGF Receptor 2 in Human Vascular Smooth Muscle Cells. Front Pharmacol 2016, 7:497.
47. Lee-Rivera I, López E, López-Colomé AM: Diversification of PAR signaling through receptor crosstalk. Cell Mol Biol Lett 2022, 27(1):77.
48. Flock T, Hauser AS, Lund N, Gloriam DE, Balaji S, Babu MM: Selectivity determinants of GPCR-G-protein binding. Nature 2017, 545(7654):317-322.
49. Ma Y, He L, Zhao X, Li W, Lv X, Zhang X, Peng J, Yang L, Xu Q, Wang H: Protease activated receptor 2 signaling promotes self-renewal and metastasis in colorectal cancer through β-catenin and periostin. Cancer Letters 2021, 521:130-141.
50. Jaber M, Maoz M, Kancharla A, Agranovich D, Peretz T, Grisaru-Granovsky S, Uziely B, Bar-Shavit R: Protease-activated-receptor-2 affects protease activated-receptor-1-driven breast cancer. Cell Mol Life Sci 2014, 71(13):2517-2533.
51. Cantrell R, Palumbo JS: The thrombin-inflammation axis in cancer progression. Thrombosis Research 2020, 191:S117-S122.
52. Covic L, Kuliopulos A: Protease-Activated Receptor 1 as Therapeutic Target in Breast, Lung, and Ovarian Cancer: Pepducin Approach. International Journal of Molecular Sciences 2018, 19(8):2237.
53. Chanakira A, Westmark PR, Ong IM, Sheehan JP: Tissue factor-factor VIIa complex triggers protease activated receptor 2-dependent growth factor release and migration in ovarian cancer. Gynecologic Oncology 2017, 145(1):167-175.
54. Warfel JM, Steele AD, D'Agnillo F: Anthrax lethal toxin induces endothelial barrier dysfunction. Am J Pathol 2005, 166(6):1871-1881.
55. Hoy B, Löwer M, Weydig C, Carra G, Tegtmeyer N, Geppert T, Schröder P, Sewald N, Backert S, Schneider G et al: Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion. EMBO Rep 2010, 11(10):798-804.
56. Gecse K, Róka R, Ferrier L, Leveque M, Eutamene H, Cartier C, Ait-Belgnaoui A, Rosztóczy A, Izbéki F, Fioramonti J et al: Increased faecal serine protease activity in diarrhoeic IBS patients: a colonic lumenal factor impairing colonic permeability and sensitivity. Gut 2008, 57(5):591-599.
57. Lourbakos A, Potempa J, Travis J, D'Andrea MR, Andrade-Gordon P, Santulli R, Mackie EJ, Pike RN: Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion. Infect Immun 2001, 69(8):5121-5130.
58. Lourbakos A, Yuan YP, Jenkins AL, Travis J, Andrade-Gordon P, Santulli R, Potempa J, Pike RN: Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity. Blood 2001, 97(12):3790-3797.
59. Whitmore SE, Lamont RJ: Oral Bacteria and Cancer. PLOS Pathogens 2014, 10(3):e1003933.
60. Nomura K, Obata K, Keira T, Miyata R, Hirakawa S, Takano K, Kohno T, Sawada N, Himi T, Kojima T: Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells. Respir Res 2014, 15(1):21.
61. Chung WO, An JY, Yin L, Hacker BM, Rohani MG, Dommisch H, DiJulio DH: Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria. Immunology Letters 2010, 131(2):113-119.
62. Nag N, Ray T, Tapader R, Gope A, Das R, Mahapatra E, Saha S, Pal A, Prasad P, Pal A: Metallo-protease Peptidase M84 from <em>Bacillus altitudinis</em> induces ROS-dependent apoptosis in ovarian cancer cells by targeting PAR-1. iScience 2024, 27(6).
63. 李國豪: 豚鼠產氣單胞菌培養過程中產生的條件培養基具有蛋白水解活性且會通過Ras/Akt 途徑促進胰臟癌細胞增殖. 台南市: 國立成功大學; 2023.
64. Søreide K, Roalsø M, Aunan JR: Is There a Trojan Horse to Aggressive Pancreatic Cancer Biology? A Review of the Trypsin-PAR2 Axis to Proliferation, Early Invasion, and Metastasis. J Pancreat Cancer 2020, 6(1):12-20.
65. Dutra-Oliveira A, Monteiro RQ, Mariano-Oliveira A: Protease-activated receptor-2 (PAR2) mediates VEGF production through the ERK1/2 pathway in human glioblastoma cell lines. Biochem Biophys Res Commun 2012, 421(2):221-227.
66. Jahan I, Fujimoto J, Alam SM, Sato E, Sakaguchi H, Tamaya T: Role of protease activated receptor-2 in tumor advancement of ovarian cancers. Annals of Oncology 2007, 18(9):1506-1512.
67. Sedda S, Marafini I, Caruso R, Pallone F, Monteleone G: Proteinase activated receptors-associated signaling in the control of gastric cancer. World J Gastroenterol 2014, 20(34):11977-11984.
68. Lidfeldt J, Bendahl PO, Forsare C, Malmström P, Fernö M, Belting M: Protease Activated Receptors 1 and 2 Correlate Differently with Breast Cancer Aggressiveness Depending on Tumor ER Status. PLoS One 2015, 10(8):e0134932.
69. Arakaki AKS, Pan WA, Trejo J: GPCRs in Cancer: Protease-Activated Receptors, Endocytic Adaptors and Signaling. Int J Mol Sci 2018, 19(7).
70. Soreide K, Janssen EA, Körner H, Baak JP: Trypsin in colorectal cancer: molecular biological mechanisms of proliferation, invasion, and metastasis. J Pathol 2006, 209(2):147-156.
71. Morris DR, Ding Y, Ricks TK, Gullapalli A, Wolfe BL, Trejo J: Protease-activated receptor-2 is essential for factor VIIa and Xa-induced signaling, migration, and invasion of breast cancer cells. Cancer Res 2006, 66(1):307-314.
72. Du X, Wang S, Lu J, Cao Y, Song N, Yang T, Dong R, Zang L, Yang Y, Wu T et al: Correlation between MMP1-PAR1 axis and clinical outcome of primary gallbladder carcinoma. Jpn J Clin Oncol 2011, 41(9):1086-1093.
73. Liao M, Tong P, Zhao J, Zhang Y, Li Z, Wang J, Feng X, Hu M, Pan Y: Prognostic value of matrix metalloproteinase-1/ proteinase-activated receptor-1 signaling axis in hepatocellular carcinoma. Pathol Oncol Res 2012, 18(2):397-403.
74. Agarwal A, Covic L, Sevigny LM, Kaneider NC, Lazarides K, Azabdaftari G, Sharifi S, Kuliopulos A: Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer. Mol Cancer Ther 2008, 7(9):2746-2757.
75. Zain J, Huang YQ, Feng X, Nierodzik ML, Li JJ, Karpatkin S: Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells. Blood 2000, 95(10):3133-3138.
76. Zhu Z, Reiser G: Signaling mechanism of protease activated receptor 1-induced proliferation of astrocytes: Stabilization of hypoxia inducible factor-1α triggers glucose metabolism and accumulation of cyclin D1. Neurochemistry International 2014, 79:20-32.
77. Guo D, Zhou H, Wu Y, Zhou F, Xu G, Wen H, Zhang X: Involvement of ERK1/2/NF-κB signal transduction pathway in TF/FVIIa/PAR2-induced proliferation and migration of colon cancer cell SW620. Tumour Biol 2011, 32(5):921-930.
78. Lee SE, Kim JM, Jeong SK, Jeon JE, Yoon HJ, Jeong MK, Lee SH: Protease activated receptor-2 mediates the expression of inflammatory cytokines, antimicrobial peptides, and matrix metalloproteinases in keratinocytes in response to Propionibacterium acnes. Arch Dermatol Res 2010, 302(10):745-756.
79. Ellenrieder V, Alber B, Lacher U, Hendler SF, Menke A, Boeck W, Wagner M, Wilda M, Friess H, Büchler M et al: Role of MT-MMPs and MMP-2 in pancreatic cancer progression. Int J Cancer 2000, 85(1):14-20.
80. Arnadottir H, Hvanndal I, Andresdottir V, Burr SE, Frey J, Gudmundsdottir BK: The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor. J Bacteriol 2009, 191(1):403-410.
81. Peach CJ, Edgington-Mitchell LE, Bunnett NW, Schmidt BL: Protease-activated receptors in health and disease. Physiol Rev 2023, 103(1):717-785.

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