臺灣博碩士論文加值系統

黑色素瘤是一種具有高致死性的侵襲性癌症，為了尋找新的抗癌劑，從蘭嶼肉桂中分離出的isokotomolide A和secokotomolide A是對人類黑色素瘤的潛在生物活性劑。細胞增生試驗測定顯示經過isokotomolide A和secokotomolide A處理的正常人體皮膚細胞有高生存力。在B16F10，A2058，MeWo和A375黑色素瘤細胞中進一步驗證了兩者都具有強烈的抗黑素瘤作用。傷口癒合試驗呈現出其優異的抗黑色素瘤遷移效應。透過吖啶橙染色和西方點墨法和定量即時聚合酶鏈鎖反應，證實其誘導自噬的效果。我們透過使用annexin V-FITC / PI雙重染色，去驗證凋亡現象。同時也利用其誘導細胞週期停滯和造成DNA損傷。蛋白質表現證實caspase激活被誘導。並進一步做活體實驗，透過組織病理學分析染色去驗證有抑制腫瘤細胞生長的效果。在這項研究中，我們證實了isokotomolide A和secokotomolide A能通過早期的自噬作用和後期的凋亡作用來誘導黑色素瘤細胞死亡。

Melanoma is an aggressive cancer with high lethality, to find new anticancer agent, we identified isokotomolide A and secokotomolide A isolated from Cinnamomum kotoense to be potential bioactive agents against human melanoma but none anti-oxidant. Cell proliferation assay displayed isokotomolide A and secokotomolide A treated in the normal human skin cells showed high viabilities. It also verified both two of them possess strong anti-melanoma effect in dose-dependent manners, especially on B16F10, A2058, MeWo and A375 cells. Wound healing assay presented their excellent anti-migratory effects. Through AO staining and Western blot, we confirmed the autophagy induced by treatment. By using annexin V- FITC/PI double-stain, the apoptosis was confirmed. They can also induce cell cycle arrest and DNA damage. Western blot demonstrated caspase cascade activation were induced. To further evaluate in vivo experiments, through histopathological staining to verify the inhibition of tumor cell growth. Within this study, we confirmed isokotomolide A and secokotomolide A induce melanoma cells death via early autophagy and late apoptosis process.

Contents
中文摘要 i
Abstract ii
Contents iii
List of figures vi
List of tables vii
1. Introduction 1
2. Materials and methods 4
2.1 Materials and reagents 4
2.2. 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH) radical scavenging activity 4
2.3. Metal chelating activity 5
2.4. Reducing power 5
2.5. Extraction and isolation of compounds 6
2.6. Cell cultures 7
2.7. Cell proliferation assay 8
2.8. Autophagic vacuoles detection by acridine orange staining 8
2.9. Annexin V-FITC/PI binding assay to analyze apoptosis 9
2.10. DNA damage and cell cycle analysis 10
2.11. Western blot analysis 10
2.12. RNA isolation and extraction 11
2.13 Quantitative real time polymerase chain reaction 12
2.14. Cell migration assay 13
2.15. Animal Material 14
2.16. Xenograft tumor assay 14
2.17. Histopathological analyses of xenografted tumor 15
2.18. Statistical analysis 15
3. Results 17
3.1. Antioxidant activity of isokotomolide A and secokotomolide A 17
3.2. Anti-proliferative effects of isokotomolide A and secokotomolide A 20
3.3. The formation of autophagic vacuoles in isokotomolide A and secokotomolide 21
A treated B16F10 cells 21
3.4. The autophagy related protein and mRNA expressions 23
3.5. Isokotomolide A and secokotomolide A cause apoptotic cell death on B16F10 cells 26
3.6. Isokotomolide A and secokotomolide A induce DNA damage and cell cycle arrest 27
3.7. Examining the apoptoic related protein and mRNA expression 29
3.8. Cell migration inhibited by isokotomolide A and secokotomolide A 31
3.9. Histopathological analyses 32
3.10. Immunohistochemical analyses 33
4. Discussion 35
5. Conclusion 41
6. Reference 42
7. Background 50

Reference
Chen B. H., Chang H. W., Huang H. M., Chong I. W., Chen J. S., Chen C. Y. and Wang H. M., The antiproliferative effect of C2-ceramide on lung cancer cells through apoptosis by inhibiting Akt and NFκB, Journal of Agricultural and Food Chemistry, 2011, 59, 2284-2290.
Chen C. Y., Cheng K.C., Chang A. Y., Lin Y. T., Hseu Y. C.,and Wang H. M., 10-Shogaol, an Antioxidant from Zingiber officinale for Skin Cell Proliferation and Migration Enhancer. International Journal of Molecular Sciences, 2012, 13(2), 1762–1777.
Chen Y. T., Kao C. J., Huang H. Y., Huang S. Y., Chen C. Y., Lin Y.S., Wen Z.H. and Wang H. M., Astaxanthin reduces MMP expressions, suppresses cancer cell migrations, and triggers apoptotic caspases of in vitro and in vivo models in melanoma, Journal of Functional Foods, 2017, 31, 20-31.
Chen Y., Huang J. Y., Lin Y. C., Lin I. F., Lu Y. R., Liu L. H., Wang H. M., Antioxidative and Anti-melanoma Effects of Various Tea Extracts via a Green Extraction Method, Journal of Food Quality, 2018, 5156073.
Chen C. Y., Hsu Y. L., Chen Y. Y., Hung J. Y., Huang M. S., Kuo P. L., Isokotomolide A, a new butanolide extracted from the leaves of Cinnamomum kotoense, arrests cell cycle progression and induces apoptosis through the induction of p53/p21 and the initiation of mitochondrial system in human non-small cell lung cancer A549 cells, European Journal of Pharmacology, 2007, 574, 94-102.
Chen C. H., Lo W. L., Liu Y. C. and Chen C. Y., Chemical and Cytotoxic Constituents from the Leaves of Cinnamomum kotoense, Journal of Natural Products, 2006, 69, 927-933.
Chen J., The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression, Cold Spring Harbor Perspectives in Medicine 2016, 6, a026104.
Chen Q., Kang, J., Fu C., The independence of and associations among apoptosis, autophagy, and necrosis. Signal Transduction and Targeted Therapy. 2018.
Chang C. T., Hseu Y. C., Varadharajan T., Lin K. Y., Way T. D., Korivi Mallikarjuna, Liao J. W., Yang H. L., Chalcone flavokawain B induces autophagic-cell death via reactive oxygen species-mediated signaling pathways in human gastric carcinoma and suppresses tumor growth in nude mice, Archives of Toxicology, 2017, 91, 3341–3364.
Chang C. T., Hseu Y. C., Thiyagarajanb V., Huang H. C., Hsu L. S., Huang P. J., Liu J. Y., Liao J. W., Yang H. L., Antrodia salmonea induces G2 cell-cycle arrest in human triple-negative breast cancer (MDA-MB-231) cells and suppresses tumor growth in athymic nude mice, Journal of Ethnopharmacology, 2017, 196, 9-19.
Chou H. L., Fong Y., Lin H. H.,Tsai E. M., Chen Y. F., Chang W. T., Wu C. Y., Wang H. M., Huang H. W., Chiu C. C., An acetamide derivative as a camptothecin sensitizer for human non-small-cell lung cancer cells through increased oxidative stress and JNK activation, Oxidative Medicine and Cellular Longevity, 2016, 9128102.
Chou H. Y., Lee C., Pan J. L., Wen Z. H., Huang S. H., Lan C. W., Wang H. M.,Enriched Astaxanthin Extract from Haematococcuspluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts. International Journal of Molecular Sciences, 2016 ,17(6), 955.
Davey R. J., A. Westhuizen van der and Bowden N. A., Metastatic melanoma treatment: Combining old and new therapies, Critical Reviews in Oncology/Hematology, 2016, 98, 242-253.
Denton D., Xu T., Kumar S., Autophagy as a pro-death pathway, Immunology & Cell Biology, 2015, 93, 35-42.
Dvorankova B., Szabo P., Kodet O., Strnad H., Kolar M., Lacina L., Krejci E., Nanka O., Sedo A. and Smetana K., In vitro characterisation of melanoma progression in a melanoma skin equivalent model, Protoplasma, 2017, 254, 1143-1150.
Farooqi A. A., R. N. Li, Huang H. W., M. Ismail, S. S. F. Yuan, H. M. Wang, J. R. Liu, J. Y. Tang, H. W. Chang, Natural Products Mediated Regulation of Oxidative Stress and DNA damage in Ultraviolet Exposed Skin Cells, 2014, Current Pharmaceutical Biotechnology, 16(12), 1078-1084.
Hersey P., Zhang X. D., How melanoma cells evade trail-induced apoptosis, Nature Reviews Cancer. 2001,1,142-150.
Jing K., Lim K. Why is autophagy important in human diseases? Experimental And Molecular Medicine, 2012, 44: 69-72.
Jhang J., Lu J. Ong, C., Hsu C., Lin J., Liao J. and Yen G., Hypouricemic effects of Mesona procumbens Hemsl. through modulating xanthine oxidase activity in vitro and in vivo, Food & Function., 2016, 7, 4239
Klionsky D. J., Eskelinen E. L, Deretic V., Autophagosomes, phagosomes, autolysosomes, phagolysosomes, autophagolysosomes... Wait, I'm confused, Autophagy, 2014, 10(4), 278, 403-413.
Kuschal C., Thoms K. M., Schubert S., Schafer A., Boeckmann L., Schon M. P. and Emmert S., Skin cancer in organ transplant recipients: effects of immunosuppressive medications on DNA repair, Experimental Dermatology, 2012, 21, 2-6.
Li C. L., Chen C.Y., Kuo C. H., Lin Y. S., Wang H. M. , 36H: A Novel Potent Inhibitor for Antimelanogenesis, Oxidative Medicine and Cellular Longevity, 2018, 6354972.
Li P. H., Chiu Y. P., Shih C. C., Wen Z. H., Ibeto L. K., Huang S. H., Chiu C. C., Ma D. L., Leung C. H., Chang Y. N., Wang H. M., Biofunctional Activities of Equisetum ramosissimum Extract: Protective Effects against Oxidation, Melanoma, and Melanogenesis, Oxidative Medicine and Cellular Longevity, 2016, 2853543.
Li P. H., Liu L. H., Chang C. C., Gao R.; Leung C. H., Ma D. L., Wang H. M., Silencing Stem Cell Factor Gene in Fibroblasts to Regulate Paracrine Factor Productions and Enhance c-Kit Expression in Melanocytes on Melanogenesis. International Journal of Molecular Sciences. 2018, 19(5), 1475
Liu B, Oltvai, Zoltan N., Bayir Hulya, Silverman, Gary A.; Pak Stephen C., Perlmutter David H., Bahar Ivet, Quantitative assessment of cell fate decision between autophagy and apoptosis. Scientific Reports. 2017 ,7, 17605. 1-14..
Oppermann M., Geilen C. C., Fecker L. F., Gillissen B., Daniel P. T., Eberle J., Caspase-independent induction of apoptosis in human melanoma cells by the proapoptotic Bcl-2-related protein Nbk / Bik, Oncogene. 2005 ,24, 7369–7380.
Shimizu S., Yoshida T., Tsujioka M., Arakawa S., Autophagic Cell Death and Cancer, International Journal of Molecular Sciences, 2014, 15, 3145-3153.
Ivanov V. N., Bhoumik A., Ronai Z., Death receptors and melanoma resistance to apoptosis, Oncogene. 2003, 22, 3152–3161.
Tait S. W., Green D. R., Mitochondrial regulation of cell death, Cold Spring Harbor Perspectives in Biology, 2013, 5.
Tang M. K. S.,Yue P. Y. K, Ip P. P., Huang R. L., Lai H. C., Cheung, A. N. Y.,. Soluble E-cadherin promotes tumor angiogenesis and localizes to exosome surface. Nature Communications, 2018, 9, 2270.
Wang C. C., Huang S. Y., Huang S. H., Wen Z. H., Huang J. Y., Liu W. S., Wang H. M., A synthetic biological secondary metabolite, LycogenTM, produced and extracted from Rhodobacter sphaeroides WL-APD911 in an optimizatioal scale-up strategy, Food Science and Human Wellness, 2017, 6(4), 195-201.
Wang H. M., Chen C. Y. and Wu P. F., Enhancements of Skin Cell Proliferations and Migrations via 6-Dehydrogingerdione, Journal of Agricultural and Food Chemistry, 2014, 62, 1057-1065.
Wang H. M., Chen C. C., Huynh P., Chang J. S., Exploring the potential of using algae in cosmetics, Bioresource Technology, 2015 ,184, 355-362
Wang H. M., Kao C. L., Liu C. M., Li W. J., Yeh H. C., Li H. T.,Kuo C. N., Chen C. Y., Chemical Constituents of the Roots of Pluchea indica. Chemistry of Natural Compounds, 2017, 53(4).
Wang H. M., Kao C. L., Li W. J., Li H. T., Chen C. Y., Two new phenylalkanoids from the rhizomes of Zingiber officinale, Chemistry of Natural Compounds, 2018, 54(1), 3-9.
Wu H., X. Che, Q. Zheng, A. Wu, K. Pan, A. Shao, Q. Wu, J. Zhang and Y. Hong, Caspases: A Molecular Switch Node in the Crosstalk between Autophagy and Apoptosis, International Journal of Biological Sciences, 2014, 10, 1072-1083.
Wu P. F., Chiu C. C., Chen C. Y., Wang H. M., 7‐Hydroxydehydronuciferine induces human melanoma death via triggering autophagy and apoptosis, Experimental Dermatology, 2015, 24, 930-935.
Wu J. C., Tsai H. E., Liu G. S., Wu C. S., Tai M. H., Autophagic cell death participates in POMC-induced melanoma suppression, Cell Death Discoveryvolume, 2018,11.
Yang J., Yao S., JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1, International Journal of Molecular Sciences, 2015, 16, 25744-25758.