臺灣博碩士論文加值系統

檳榔引發的口腔癌在許多領域都很常見，包括在台灣。順鉑 (Cisplatin) 是一種用於治療口腔癌的的基礎的化療藥物。然而近年來許多研究指出順鉑會有抗藥性的產生。蟲草素 (3'-脫氧腺苷) (Cordycepin) 為一從冬蟲夏草中分離出的純化合物，已被證明具有抗腫瘤的特性。最近的研究表示，合併使用化療藥物或許可以對抗抗藥性的產生。因此，我們研究了在不同口腔癌的細胞株 (OC3，OEC-M1和Fadu) 中蟲草素以及順鉑的抗癌作用。結果發現，蟲草素（100 μM），順鉑（300 μM和600 μM）單獨處理，以及合併使用 (蟲草素100 μM合併順鉑300 μM或600 μM)24小時後，在形態學變化下可觀察出三個細胞株皆會傾向細胞凋亡。在細胞存活試驗 (MTT assay)中，所有細胞株隨著蟲草素以及順鉑劑量的增加（蟲草素10 μM至1000 μM，順鉑100 μM至1000 μM）存活率顯著下降 (p〈0.05)，同時可觀察出蟲草素（100 μM）與順鉑（300μM和600 μM）合併處理達到加乘的效果。流式細胞儀檢測中，細胞百分比在蟲草素組別中相較於對照組在subG1週期顯著增加了2〜3倍。蟲草素及順鉑（300 μM）合併處理組的subG1期細胞比例顯著增加至20％。在細胞機制研究中，蟲草素及順鉑誘導caspase-8，caspase-9，caspase-3以及PARP的裂解，同時在合併處理組別中可觀察出表現量較單獨處理組別更高。在MAPKs途徑研究中，合併處理會活化JNK，ERK和p38的表現。綜合以上結果可得知，順鉑和蟲草素會藉由活化外在/內在caspase和MAPKs途徑引發人類口腔癌細胞株的細胞凋亡，高度表明了蟲草素及順鉑的組合可能具有潛在的加乘性抗癌效果。

Betel quid-related oral cavity cancer is common in certain areas, including Taiwan. Cisplatin is a platinum-based chemotherapy drug used to treat oral cancer. However, the development of drug-resistance to cisplatin has been reported recently. Cordycepin (3’–deoxyadenosine), a pure compound isolated from Cordyceps sinensis, has been demonstrated to have anti-tumor properties. Recent studies illustrate that combining chemotherapy agents may counteract drug resistance. Hence, we investigated the anticancer effect of cordycepin and cisplatin on different oral cancer cell lines; OC3, OEC-M1 and Fadu. Results demonstrated that cordycepin (100 μM), cisplatin (300 μM and 600 μM) and combined treatments (cordycepin 100 μM with cisplatin 300 μM or 600 μM ) caused cell death morphological changes after 24 hour treatment among 3 cell lines. In viability test, all three cell lines surviving rate significantly decreased as the dosage of cordycepin and cisplatin increased (10 μM to 1000 μM of cordycepin and 100 μM to 1000 μM of cisplatin), and the synergistical effect could be observed in cordycepin (100 μM) with cisplatin (300 μM and 600μM, respectively) treatments. In flow cytometry assay, percentage of subG1 phase cells in cordycepin group significantly increased by 2~3 times than control group. The percentage of subG1 phase cells in cordycepin and cisplatin (300 μM) co-treatment group significantly increased to 20%. In cellular mechanism study, both cordycepin and cisplatin induced cleavage of caspase-8, -9, and -3 and cleavage of poly ADP-ribose polymerase (PARP), and co-treatments induced more expression compared to cisplatin or cordycepin alone treatment among 3 cell lines. In the MAPKs pathway, co-treatments activated more expression of JNK, ERK and p38 than alone treatment among 3 cell lines. In conclusion, cisplatin and cordycepin possess synergistically/addictively apoptotic effect by activating the expression of extrinsic/intrinsic caspase and MAPK pathways in human oral cavity cancer cell lines, which highly suggest that the combination of cordycepin and cisplatin might be a potential anti-cancer drug compared to the single agent chemotherapy.

ABSTRACTS
Chinese abstract …………………………………………i
English abstract …………………………………………ii
ACKNOWLEDGEMENTS …………………………………………iv
TABLE OF CONTENTS …………………………………………v
LIST OF FIGURES …………………………………………vi
INTRODUCTION …………………………………………1
METERIALS AND METHODS
Chemicals …………………………………………5
Cells and cell culture …………………………………………5
MTT cell viability test …………………………………………6
Morphology study …………………………………………7
Flow cytometry analysis …………………………………………7
Immunoblotting Analysis …………………………………………8
Statistics …………………………………………8
RESULTS
Effects of cordycepin and/or cisplatin on cell viability of oral cavity cancer cells…………………………………………10
Effect of cordycepin and/or cisplatin on morphological change in oral cavity cancer cells………………11
The analysis of cell cycle under cordycepin and/or cisplatin influence in oral cavity cancer cells………12
Modulation of the effect on extrinsic and intrinsic pathways of apoptosis………………………………………………………13
Effect on the regulation of MAPK pathway……………………………16
DISCUSSION …………………………………………18
REFERENCES …………………………………………43

Reference
Adelstein DJ, Li Y, Adams GL, Wagner H Jr, Kish JA, Ensley JF, et al. An intergroup phase III comparison of standard radiation therapy and two schedules of concurrent chemoradiotherapy in patients with unresectable squamous cell head and neck cancer. J Clin Oncol. 21(1): 92–98, 2003.
Aggarwal A, Misro MM, Maheshwari A, Sehgal N. Differential modulation of apoptotic gene expression by N-acetyl-L-cysteine in Leydig cells stimulated persistently with hCG in vivo. Mol Cell Endocrinol. 348(1): 155–164, 2012.
Aissat N, Le Tourneau C, Ghoul A, Serova M, Bieche I, Lokiec F et al. Antiproliferative effects of rapamycin as a single agent and in combination with carboplatin and paclitaxel in head and neck cancer cell lines. Cancer Chemother Pharmaco. 62(2): 305-313, 2008.
Alberts K, Johnson A, Lewis J, Raff M, Roberts, Walter P. Chapter 18: Apoptosis: Programmed Cell Death Eliminates Unwanted Cells. Molecular Biology of the Cell (textbook). 5th ed. New York: Garland Science, 1115.
Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science. 281(5381): 1305-1308, 1998.
Blair BG, Larson CA, Safaei R, Howell SB. Copper transporter 2 regulates the cellular accumulation and cytotoxicity of cisplatin and carboplatin. Clin Cancer Res. 15(13): 4312-4321, 2009.
Boulikas T, Vougiouka M, Recent clinical trials using cisplatin, carboplatin and their combination chemotherapy drugs (review). Oncol Rep. 11(3):559-595, 2004.
Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S et al. Role of Poly (ADP-ribose) Polymerase (PARP) Cleavage in Apoptosis – caspase 3 resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem. 274(33): 22932-22940, 1999.
Brozovic A, Osmak M. Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin resistance. Cancer lett. 251(1): 1-16, 2007.
Chan LP, Chou TH, Ding HY, Chen PR, Chiang FY, Kuo PL, Liang CH. Apigenin induces apoptosis via tumor necrosis factor receptor- and Bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatin. Biochim Biophys Acta. 1820(7): 1081-1091, 2012.
Chen YC, Huang YL, Huang BM. Cordyceps sinensis mycelium activates PKA and PKC signal pathways to stimulate steroidogenesis in MA-10 mouse Leydig tumor cells. Int J Biochem Cell Biol. 37(1): 214-223, 2005.
Choi S, Lim MH, Kim KM, Jeon BH, Song WO, Kim TW. Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor. Toxicology and Appl Pharmacol. 257(2): 165–173, 2011.
Cory S. Cell death throes. Proc Natl Acad Sci USA. 95(21): 12077-12079, 1998.
Cryns V, Yuan J. Proteases to die for. Genes Dev.12(11): 1551-1570, 1998.
Cunningham KG, Hutchinson SA, Manson W, Spring FS. Cordycepin, a metabolic product isolated from cultures of Cordyceps militaris. Nature. 166(4231): 949, 1950.
Department of Health, Executive Yuan, Taiwan, R.O.C. 2011
Forastiere AA. Is there a new role for induction chemotherapy in the treatment of head and neck cancer? J Natl Cancer Inst. 96(22): 1647–1649, 2004.
Forastiere AA, Goepfert H, Maor M, Pajak TF, Weber R et al. Concurrent chemotherapy and radiotherapy for organ preservation in advanced laryngeal cancer. N Eng J Med. 349(22): 2091–2098, 2003.
Green DR, Reed JC. Mitochondria and apoptosis. Science. 281(5381): 1309-1312, 1998.
Gupta PC, Warnakulasuriya S. Global epidemiology of areca nut usage. Addict Biol. 7(1): 77–83, 2002.
Gupta S. Molecular steps of death receptor and mitochondrial pathways of apoptosis. Life Sci. 69(25-26): 2957–2964, 2001.
Ho PS, Ko YC, Yang YH, Shieh TY, Tsai CC. The incidence of oropharyngeal cancer in Taiwan: an endemic betel quid chewing area. J Oral Pathol Med. 31(4): 213–219, 2002.
Hung SH, Lee FP, Su CH, Tseng H. Effect of all-trans retinoic acid on the growth of two nasopharyngeal cancer cell lines and its treatment potential in combination with cisplatin. Eur Arch Otorhinolaryngol. (Epub ahead of print), 2012.
Jen CY, Lin CY, Leu SF, Huang BM. Cordycepin Induced MA-10 Mouse Leydig Tumor Cell Apoptosis through Caspase-9 Pathway. Evid Based Complement Alternat Med; in press.
Jeng JH, Chang MC, Hahn LJ. Role of areca nut in betel quid-associated chemical carcinogenesis: current awareness and future perspectives. Oral Oncol. 37(6): 477–492, 2001.
Juvekar AS, Adwankar MK, Tongaonkar HB. Effect of cisplatin-based chemotherapy on emergence of cisplatin resistance, and its correlation with intracellular glutathione levels and accumulation of p53 protein in human ovarian cancer. Cancer Biother Radiopharm.15(3): 295-300, 2000.
Kaczirek K, Schindl M, Weinhausel A, Scheuba C, Passler C, Prager G, Raderer M, Hamilton G, Mittlbock M, Siegl V, Pfragner R and Niederle B. Cytotoxic activity of camptothecin and paclitaxel in newly established continuous human medullary thyroid carcinoma cell lines. J Clin Endocrinol Metab. 89(5): 2397-2401, 2004.
Kim GY, Mercer SE, Ewton DZ, Yan Z, Jin K, Friedman E. The stress-activated protein kinases p38 alpha and JNK1 stabilize p21(Cip1) by phosphorylation. J Biol Chem. 277(33): 29792-29802, 2002.
Koo MS, Kwo YG, Park JH, Choi WJ, Billiar TR, Kim YM. Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis. Mol Cells. 13(2):194-201, 2002.
Lallas GC, Courtis N, Havredaki M. K562 cell sensitization to 5-fluorouracil- or interferon-alpha-induced apoptosis via cordycepin (3-deoxyadenosine): fine control of cell apoptosis via poly(A) polymerase upregulation. Int J Biol Markers. 19(1): 58–66, 2004.
Lee JH, Hong SM, Yun JY, Myoung H, Kim MJ. Anti-Cancer Effects of Cordycepin on Oral Squamous Cell Carcinoma Proliferation and Apoptosis in Vitro. Journal of Cancer Therapy. 2: 224-234, 2011.
Levresse V, Marek L, Blumberg D, Heasley LE. Regulation of platinum-compound cytotoxicity by the c-Jun N-terminal kinase and c-Jun signaling pathway in small-cell lung cancer cells. Mol Pharmacol. 62(3): 689-697, 2002.
Li R, Zang Y, Li C, Patel NS, Grandis JR, Johnson DE. ABT-737 synergizes with chemotherapy to kill head and neck squamous cell carcinoma cells via a Noxa-mediated pathway. Mol Pharmacol. 75(5): 1231-1239, 2009.
Lieu CH, Chang YN and Lai YK. Dual cytotoxic mechanisms of submicromolar tacol on human leukemia HL-60 cells. Biochem Pharmacol. 53(11): 1587-1596, 1997.
Lin SC, Liu CJ, Chiu CP, Chang SM, Lu SY, Chen YJ. Establishment of OC3 oral carcinoma cell line and identification of NF-kappa B activation responses to areca nut extract. J Oral Pathol Med. 33(2): 79-86, 2004.
Lowry OH, Rosenborough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol regent. J Biol Chem. 193(1): 265-275, 1951.
Mandic A, Viktorsson K, Heiden T, Hansson J, Shoshan MC. The MEK1 inhibitor PD98059 sensitizes C8161 melanoma cells to cisplatin-induced apoptosis. Melanoma Research.11(1): 11-19, 2001.
Mandic R, Rodgarkia-Dara CJ, Krohn V, Wiegand S, Grénman R, Werner JA. Cisplatin resistance of the HNSCC cell line UT-SCC-26A can be overcome by stimulation of the EGF-receptor. Anticancer Res. 29(4): 1181-1187, 2009.
Meng CL, Yang CY, Shen KL, Wong PY, Lee HK. Inhibition of the synthesis of eicosanoid-like substances in a human oral cancercell line by interferon-gamma and eicosapentaenoic acid. Arch Oral Biol. 43(12): 979-986, 1998.
Nakamura K, Yoshikawa N, Yamaguchi Y, Kagota S, Shinozuka K, Kunitomo M. Antitumor effect of cordycepin (3- deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation. Anticancer Res. 26(1A): 43-47, 2006.
Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6(11): 1028-1042, 1999.
Park SA, Choi KS, Bang JH, Huh K, Kim SU. Cisplatin-Induced Apoptotic Cell Death in Mouse Hybrid Neurons Is Blocked by Antioxidants Through Suppression of Cisplatin-Mediated Accumulation of p53 but Not of Fas/Fas Ligand. Journal of Neurochemistry. 75(3): 946–953, 2000.
Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat. 7(2): 97-110, 2004.
Psyrri A, Kwong M, DiStasio S, Lekakis L, Kassar M, Sasaki C et al. Cisplatin, fluorouracil, and leucovorin induction chemotherapy followed by concurrent cisplatin chemoradiotherapy for organ preservation and cure in patients with advanced head and neck cancer: long-term follow-up. J Clin Oncol. 22(15): 3061–3069, 2004.
Pushkarev VM, Starenki DV, Saenko Va, Namba H, Kurebayashi J, Tronko MD and Yamashita S. Molecular mechanisms of the effects of low concentrations of taxol in anaplastic thyroid canacer cells. Endocrinology. 145(7): 3143-3152, 2004.
Thomadaki H, Tsiapalis CM, Scorilas A. Polyadenylate polymerase modulations in human epithelioid cervix and breast cancer cell lines, treated with etoposide or cordycepin, follow cell cycle rather than apoptosis induction. Biol Chem. 386(5): 471–480, 2005.
Schweyer S, Soruri A, Meschter O, Heintze A, Zschunke F, Miosge N, Thelen P, Schlott T, Radzun HJ and Fayyazi A. Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation. Br J Cancer. 91(3): 589–598, 2004.
Smith KS, Folz BA, Adams EG, Bhuyan BK. Synergistic and additive combinations of several antitumor drugs and other agents with the potent alkylating agent adozelesin. Cancer Chemother Pharmacol. 35(6): 471-482, 1995.
Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 281(5381): 1312–1316, 1998.
Toh WH, Siddique MM, Boominathan L, Lin KW, Sabapathy K. C-Jun regulates the stability and activity of the p53 homologue, p73. J Biol Chem. 279(43): 44713-44722, 2004.
van den Broek GB, Wildeman M, Rasch CR, Armstrong N, Schuuring E, Begg AC et al. Molecular markers predict outcome in squamous cell carcinoma of the head and neck after concomitant cisplatin-based chemoradiation. Int J Cancer. 124(11): 2643-2650, 2009.
Wada T and Penninger Jm. Mitogen-activated protein kinases in apoptosis regulation. Oncogene. 23(16):2838-2849, 2004.
Wang G, Reed E, Li QQ. Molecular basis of cellular response to cisplatin chemotherapy in non-small cell lung cancer (Review). Oncol Rep. 12(5): 955-965, 2004.
Wang LG, Liu XM, Kreis W and Budman DR. The effect of antimicrotubule agents on signal transduction pathways of apoptosis: a review. Cancer Chemother Pharmacol. 44(5): 355-361, 1999
Wu WC, Hsiao JR, Lian YY, Lin CY, Huang BM. The apoptotic effect of cordycepin on human OEC-M1 oral cancer cell line. Cancer Chemother Pharmacol. 60(1): 103-111, 2007.
Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 284(5756): 555–556, 1980.
Yang X, Chang HY, Baltimore D. Autoproteolytic activation of pro-caspases by oligomerization. Mol Cell. 1(2): 319–325, 1998.
Yan X, Fraser M, Qiu Q, Tsang BK. Over-expression of PTEN sensitizes human ovarian cancer cells to cisplatin-induced apoptosis in a p53-dependent manner. Gynecol Oncol. 102(2): 348-355, 2006.
Yoon CY, Park MJ, Lee JS, Lee SC, Oh JJ, Park H et al. The histone deacetylase inhibitor trichostatin A synergistically resensitizes a cisplatin resistant human bladder cancer cell line. J Urol. 185(3): 1102-1111, 2011.
Yuan J, Horvitz HR. A first insight into the molecular mechanisms of apoptosis. Cell. 116(2 Suppl): S53-6, 2004.
Zahid H Siddik. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene. 22(47): 7265–7279, 2003.
Zhang QX, Feng R, Zhang W, Ding Y, Yang JY, Liu GH. Role of stress-activated MAP kinase P38 in cisplatin- and DTT-induced apoptosis of the esophageal carcinoma cell line Eca109. World J Gastroenterol. 11(29): 4451-4456, 2005.
Zieve GW, Roemer EJ. Cordycepin rapidly collapses the intermediate Wlament networks into juxtanuclear caps in Wbroblasts and epidermal cells. Exp Cell Res. 177(1):19–26, 1988.
Zieve GW, Feeney RJ, Roemer EJ. Cordycepin disrupts the microtubule networks and arrests Nil 8 hamster Wbroblasts at the onset of mitosis. Cell Motil Cytoskeleton. 7(4):337–446, 1987.