標準的癌症療法本身俱有局限性也面對許多挑戰，並且可能導致癌症療法的無效結果，進而導致腫瘤的複發，耐藥性腫瘤細胞的出現或更嚴重的情況即癌症患者的死亡。我們希望從基礎了解抗癌肽作為治療藥物的進一步發展的基本作用方式。在這項研究中，我們證明了抗菌肽將其作用轉變為抗癌肽的功效和適用性，根據研究因爲癌細胞的表面和細菌表面都富含陰離子所以將惡性細胞作為抗菌肽的新靶標，並試圖瞭解抗菌肽的抗癌活性。首先，我們第一步先使用MTT分析法針對各種癌細胞株去測試Histatin 5 衍生肽及其衍生抗菌胜肽對癌細胞和正常細胞的選擇性和抗癌活性。完成抗癌活性測定後，在進入與免疫原性細胞死亡相關的實驗之前，我們確定設計的抗菌肽的時間殺滅動力學和半抑制濃度。先天性免疫系統的危險信號稱為DAMPs，主要介導ICD的免疫原性，因此，我們想要研究Histatin 5 衍生肽及其衍生抗菌胜肽在所選的細胞株中經過處理後誘導DAMPs即損傷或死亡相關的分子模式的能力。

The standard cancer therapy had the limitation on its own and it may cause the ineffective results for the cancer therapy and therefore resulting in the reoccurrence of tumour, drug-resistance tumour cells or the death of the patients. We wish to understand the basic mode of action of anticancer peptide preliminarily the further development as therapeutic drug. In this study, we demonstrate the efficacy and applicability of antimicrobial peptide turning its role into the anticancer peptide to considering malignant cells as the target as the surface of cancerous cells were reported to be anionic as the bacteria surface. We then tested the selectivity and anticancer activity of the Histatin 5 derived peptide and all of its derivative peptides were being replaced using MTT assay against various cancer cell lines for the first step. Following the anticancer activity assay, the time killing kinetic of the peptides and the half inhibitory concentration were determined before entering the experiments related to immunogenic cell death. The danger signals for innate immune system called damage-associated DAMPs mainly mediate the immunogenic characteristics of ICD, thus we try to investigate the ability of the Histatin 5 derived peptide and its derivative peptides in inducing the damage-associated molecular patterns in the selected cell line after the treatment.

中文摘要 ii
Abstract iii
Acknowledgement iv
List of Contents v
Abbreviation vii
List of Figures viii
Figure 1 The cancer-immunity cycle viii
Figure 2 Immune escape in the cancer-immunity cycle viii
Figure 3 Five of the peptides which is P-113, Phe-P-113, Nal-P-113, Bip-P-113 and Dip-P-113 were tested in different concentrations against human red blood cells (hRBC) for the haemolysis determination viii
Figure 4 Cytotoxicity assay of P-113 peptide and its derivatives (Phe-P-113, Nal-P-113, Bip-P-113 and Dip-P-113) on human fibroblast cells (HFW) viii
Figure 5 Anticancer activities of P-113, Phe-P-113, Nal-P-113, Bip-P-113 and Dip-P-113 against various cell lines were determined using MTT cytotoxicity assay viii
Figure 6 Time killing analysis of the selected Nal-P-113, Bip-P-113 and Dip-P-113 peptides against PC 9 cell line viii
Figure 7 The ATP secretion in PC 9 after treating with Nal-P-113, Bip-P-113 and Dip-P-113 for desired time points was measured viii
Figure 8 ROS release after the treatment with 2x IC50 of Nal-P-113, Bip-P-113 and Dip-P-113 using DCFDA cellular ROS detection kit viii
Figure 9 The cytochrome c secretion were quantified using ELISA method viii
Figure 10 The HMGB1 protein secretion from cell lysate to supernatant after the incubation with Nal-P-113, Bip-P-113 and Dip-P-113 viii
List of Tables ix
Table 1 Sequences of the anticancer peptides used in the study ix
Table 2 Structural properties of aromatic amino acid side chains ix
Table 3 Half inhibitory concentration of P-113 and all of its derivative peptides against selected 5 cancerous cell lines. ix
Chapter 1 Introduction 1
Cancer Trends 1
Current Method of Treatments 2
Cross Over from Antimicrobial Peptide to Anticancer Peptide 3
Immunogenic Cell Death 5
P-113 Peptide and its Derivative Peptides 8
Aim 10
Chapter 2 Materials and Methods 11
Peptide Preparation 11
Reagents 12
Cell Culture 13
Cell Viability Determination 14
Half Maximal Inhibitory Concentration Determination 14
Kinetic Analysis 15
Detection of Extracellular HMGB1 15
Cellular ROS Measurement 17
Luciferin-Luciferase Detection of Extracellular ATP 18
Detection of Extracellular Cytochrome C 19
Statistical Analysis 20
Chapter 3 Results 21
Cell Viability Determination 21
Half Inhibitory Concentration 22
Kinetic Analysis 22
Detection of Extracellular HMGB1 23
DCFDA Cellular ROS Detection 24
Luciferin-Luciferase Detection of Extracellular ATP 24
Detection of Extracellular Cytochrome C 25
Chapter 4 Discussion 27
Chapter 5 Conclusion 30
Figures 31
Tables 41
References 43

1. Cancer, I.A.f.R.o. Estimated number of new cases from 2020 to 2040. 2020 [cited 2020 25 November 2020]; Available from: https://gco.iarc.fr/tomorrow/en/dataviz/isotype.
2. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2019. CA Cancer J Clin, 2019. 69(1): p. 7-34.
3. Henley, S.J., et al., Annual report to the nation on the status of cancer, part I: National cancer statistics. Cancer, 2020. 126(10): p. 2225-2249.
4. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2020. CA Cancer J Clin, 2020. 70(1): p. 7-30.
5. International Agency for Research on Cancer, W.H.O. Estimated number of new cases from 2020 to 2040. 2020 [cited 2020 December 29]; Available from: https://gco.iarc.fr/tomorrow/en/dataviz/isotype?cancers=15&single_unit=50000.
6. Nouri Rouzbahani, F., et al., Immunotherapy a New Hope for Cancer Treatment: A Review. Pak J Biol Sci, 2018. 21(3): p. 135-150.
7. National Cancer Institute. Types of Cancer Treatment. 2020 [cited 2020 22 November]; Available from: https://www.cancer.gov/about-cancer/treatment/types.
8. Perez-Herrero, E. and A. Fernandez-Medarde, Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy. Eur J Pharm Biopharm, 2015. 93: p. 52-79.
9. Chidambaram, M., R. Manavalan, and K. Kathiresan, Nanotherapeutics to overcome conventional cancer chemotherapy limitations. J Pharm Pharm Sci, 2011. 14(1): p. 67-77.
10. Li, J., et al., The pharmaceutical multi-activity of metallofullerenol invigorates cancer therapy. Nanoscale, 2019. 11(31): p. 14528-14539.
11. Dubos, R.J., STUDIES ON A BACTERICIDAL AGENT EXTRACTED FROM A SOIL BACILLUS : I. PREPARATION OF THE AGENT. ITS ACTIVITY IN VITRO. J Exp Med, 1939. 70(1): p. 1-10.
12. Li, Y., et al., Overview on the recent study of antimicrobial peptides: Origins, functions, relative mechanisms and application. Peptides, 2012. 37(2): p. 207-215.
13. Qin, Y., et al., From Antimicrobial to Anticancer Peptides: The Transformation of Peptides. Recent Pat Anticancer Drug Discov, 2019. 14(1): p. 70-84.
14. Liscano, Y., J. Oñate-Garzón, and J.P. Delgado, Peptides with Dual Antimicrobial-Anticancer Activity: Strategies to Overcome Peptide Limitations and Rational Design of Anticancer Peptides. Molecules, 2020. 25(18).
15. Gaspar, D., A.S. Veiga, and M.A. Castanho, From antimicrobial to anticancer peptides. A review. Front Microbiol, 2013. 4: p. 294.
16. Haney, E.F., S.C. Mansour, and R.E. Hancock, Antimicrobial Peptides: An Introduction. Methods Mol Biol, 2017. 1548: p. 3-22.
17. Inoue, H. and K. Tani, Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments. Cell Death Differ, 2014. 21(1): p. 39-49.
18. Serrano-del Valle, A., et al., Immunogenic Cell Death and Immunotherapy of Multiple Myeloma. Frontiers in Cell and Developmental Biology, 2019. 7(50).
19. Kazama, H., et al., Induction of Immunological Tolerance by Apoptotic Cells Requires Caspase-Dependent Oxidation of High-Mobility Group Box-1 Protein. Immunity, 2008. 29(1): p. 21-32.
20. Pitt, J.M., G. Kroemer, and L. Zitvogel, Immunogenic and Non-immunogenic Cell Death in the Tumor Microenvironment. Adv Exp Med Biol, 2017. 1036: p. 65-79.
21. Kroemer, G., et al., Immunogenic Cell Death in Cancer Therapy. Annual Review of Immunology, 2013. 31(1): p. 51-72.
22. Li, W., et al., Targeting photodynamic and photothermal therapy to the endoplasmic reticulum enhances immunogenic cancer cell death. Nature Communications, 2019. 10(1): p. 3349.
23. Garg, A.D., et al., Immunogenic cell death. Int J Dev Biol, 2015. 59(1-3): p. 131-40.
24. Rivera Vargas, T. and L. Apetoh, Danger signals: Chemotherapy enhancers? Immunol Rev, 2017. 280(1): p. 175-193.
25. Zhao, X., et al., Inducing enhanced immunogenic cell death with nanocarrier-based drug delivery systems for pancreatic cancer therapy. Biomaterials, 2016. 102: p. 187-97.
26. Aaes, T.L. and P. Vandenabeele, The intrinsic immunogenic properties of cancer cell lines, immunogenic cell death, and how these influence host antitumor immune responses. Cell Death & Differentiation, 2020.
27. Chen, Daniel S. and I. Mellman, Oncology Meets Immunology: The Cancer-Immunity Cycle. Immunity, 2013. 39(1): p. 1-10.
28. Tang, S., et al., Mechanisms of immune escape in the cancer immune cycle. International Immunopharmacology, 2020. 86: p. 106700.
29. Kumar, V., et al., The Nature of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment. Trends in immunology, 2016. 37(3): p. 208-220.
30. Rothstein, D.M., et al., Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5. Antimicrob Agents Chemother, 2001. 45(5): p. 1367-73.
31. Di Giampaolo, A., et al., P-113 peptide: New experimental evidences on its biological activity and conformational insights from molecular dynamics simulations. Biopolymers, 2014. 102(2): p. 159-67.
32. Giacometti, A., et al., In vitro activity of the histatin derivative P-113 against multidrug-resistant pathogens responsible for pneumonia in immunocompromised patients. Antimicrob Agents Chemother, 2005. 49(3): p. 1249-52.
33. Fernández de Ullivarri, M., et al., Antifungal Peptides as Therapeutic Agents. Frontiers in Cellular and Infection Microbiology, 2020. 10(105).
34. Yu, H.Y., et al., Easy strategy to increase salt resistance of antimicrobial peptides. Antimicrob Agents Chemother, 2011. 55(10): p. 4918-21.
35. Chu, H.L., et al., Boosting salt resistance of short antimicrobial peptides. Antimicrob Agents Chemother, 2013. 57(8): p. 4050-2.
36. Chih, Y.H., et al., Dependence on size and shape of non-nature amino acids in the enhancement of lipopolysaccharide (LPS) neutralizing activities of antimicrobial peptides. J Colloid Interface Sci, 2019. 533: p. 492-502.
37. Wu, C.L., et al., Antimicrobial Peptides Display Strong Synergy with Vancomycin Against Vancomycin-Resistant E. faecium, S. aureus, and Wild-Type E. coli. Int J Mol Sci, 2020. 21(13).
38. Haug, B.E., M.L. Skar, and J.S. Svendsen, Bulky aromatic amino acids increase the antibacterial activity of 15-residue bovine lactoferricin derivatives. J Pept Sci, 2001. 7(8): p. 425-32.
39. Herbst, R.S., D. Morgensztern, and C. Boshoff, The biology and management of non-small cell lung cancer. Nature, 2018. 553(7689): p. 446-454.
40. Chen, X., et al., A novel antimicrobial peptide, Ranatuerin-2PLx, showing therapeutic potential in inhibiting proliferation of cancer cells. Biosci Rep, 2018. 38(6).
41. Hsiao, Y.C., et al., Anticancer activities of an antimicrobial peptide derivative of Ixosin-B amide. Bioorg Med Chem Lett, 2013. 23(20): p. 5744-7.
42. Neshani, A., et al., Epinecidin-1, a highly potent marine antimicrobial peptide with anticancer and immunomodulatory activities. BMC Pharmacol Toxicol, 2019. 20(1): p. 33.
43. Kang, S.J., H.Y. Ji, and B.J. Lee, Anticancer activity of undecapeptide analogues derived from antimicrobial peptide, brevinin-1EMa. Arch Pharm Res, 2012. 35(5): p. 791-9.
44. Mauseth, B., et al., The Novel Oncolytic Compound LTX-401 Induces Antitumor Immune Responses in Experimental Hepatocellular Carcinoma. Mol Ther Oncolytics, 2019. 14: p. 139-148.
45. Chu, H.L., et al., Novel antimicrobial peptides with high anticancer activity and selectivity. PLoS One, 2015. 10(5): p. e0126390.
46. Swinney, D.C., Molecular Mechanism of Action (MMoA) in Drug Discovery. 2011. p. 301-317.
47. Eike, L.M., et al., The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma. PLoS One, 2016. 11(2): p. e0148980.
48. Dahm, F., et al., Mitochondrially targeted ceramide LCL-30 inhibits colorectal cancer in mice. Br J Cancer, 2008. 98(1): p. 98-105.
49. Moreira, J.d.V., et al., The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect. Metabolites, 2016. 6(4): p. 33.
50. Zhang, M., et al., Allicin Decreases Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Umbilical Vein Endothelial Cells through Suppression of Mitochondrial Dysfunction and Activation of Nrf2. Cell Physiol Biochem, 2017. 41(6): p. 2255-2267.
51. An, H.M., et al., Di-Huang-Yi-Zhi herbal formula attenuates amyloid-beta-induced neurotoxicity in PC12 cells. Exp Ther Med, 2017. 13(6): p. 3003-3008.
52. Alnuaimi, A.D., et al., Oral Candida colonization in oral cancer patients and its relationship with traditional risk factors of oral cancer: a matched case-control study. Oral Oncol, 2015. 51(2): p. 139-45.
53. Zhang, C.C., et al., The HMGB1 protein sensitizes colon carcinoma cells to cell death triggered by pro-apoptotic agents. Int J Oncol, 2015. 46(2): p. 667-76.
54. Andersson, U., H. Yang, and H. Harris, Extracellular HMGB1 as a therapeutic target in inflammatory diseases. Expert Opin Ther Targets, 2018. 22(3): p. 263-277.
55. Krysko, D.V., et al., Immunogenic cell death and DAMPs in cancer therapy. Nature Reviews Cancer, 2012. 12(12): p. 860-875.
56. Srinivas, U.S., et al., ROS and the DNA damage response in cancer. Redox Biol, 2019. 25: p. 101084.
57. Deng, H., et al., Targeted scavenging of extracellular ROS relieves suppressive immunogenic cell death. Nat Commun, 2020. 11(1): p. 4951.
58. Tang, D., et al., High-mobility group box 1, oxidative stress, and disease. Antioxidants & redox signaling, 2011. 14(7): p. 1315-1335.
59. Zamaraeva, M.V., et al., Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase. Cell Death Differ, 2005. 12(11): p. 1390-7.
60. Elmore, S., Apoptosis: a review of programmed cell death. Toxicologic pathology, 2007. 35(4): p. 495-516.
61. Garrido, C., et al., Mechanisms of cytochrome c release from mitochondria. Cell Death & Differentiation, 2006. 13(9): p. 1423-1433.