本研究探討三種抗腫瘤藥物cisplatin、fluorouracil及temozolomide對於人類口腔癌細胞株OC2在細胞存活能力及細胞內鈣濃度([Ca2+]i) 的影響，同時也檢驗cisplatin相關的增生因子活化型激酶(mitogen-activated protein kinase；MAPKs) 磷酸化的現象。口腔癌細胞在投藥後，細胞存活能力在cisplatin藥物濃度25-150 μM之間隨著濃度增加而下降，同樣的情形也可以在藥物濃度50-1000 μM之間的fluorouracil及藥物濃度50-600 μM之間的temozolomide實驗組觀察到。在進行細胞內鈣傳訊的研究，以上三種抗腫瘤藥物都未能引發細胞內鈣濃度([Ca2+]i) 的上升，因此這三種抗腫瘤藥物引發細胞死亡之方式應該為非鈣離子依賴的途徑。在西方墨點分析實驗中，發現口腔癌細胞背景狀態中，ERK、JNK及p38皆有磷酸化之表現，當以100 μM cisplatin投藥後，在不同的時間點會影響ERK、 JNK及p38增生因子活化型激酶。

The present study explored the effect of three anti-tumor drugs (cisplatin, fluorouracil, and temozolomide) on viability and cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells. The effect of cisplatin related mitogen-activated protein kinases (MAPKs) phosphorylation was also examined. Cisplatin at concentration of 25-150 μM decreased viability in a concentration-dependent manner, and so did fluorouracil (50-1000 μM) and temozolomide (50-600 μM). The three anti-tumor drugs all failed to induce a [Ca2+]i increase; thus it seemed that these drugs induced cell death via Ca2+-independent pathways. Immunoblotting showed that OC2 cells have background phospho-ERK, phospho-JNK and phospho-p38 MAPKs. It was found that cisplatin influenced the phosphorylation of ERK, JNK and p38 MAPKs at different time points.

致謝與感言 ……………………………………………….. 2
中文摘要 …………………………………………………… 7
Abstract ……………………………………………………… 8
I. Introduction ………………………………………………... 9
II. Materials and Methods…………………………………… 15
1. General aims……………………………………………………… 15
2. Procedures …...…………………………………………………… 15
3. Material ...……………………..…………………………………. 15
(1) Cell culture ……………………………………...……………. 16
(2) Chemicals …………………………………………………….. 16
(3) [Ca2+]i measurements ………………………………..……….. 16
(4) Cell viability assays …………………………………………... 18
(5) Assessments of MAPKs by Western Immunoblotting ……….. 19
4. Statistics ………………………………………………………….. 20
III. Results …...………………………………………………. 22
1. Cisplatin on OC2 cells …………………………………………… 22
(1) Effect of cisplatin on the viability of OC2 cells ……………… 22
(2) Effect of cisplatin on [Ca2+]i in Ca2+-containing medium ……. 22
(3) Effect of cisplatin on MAPKs phosphorylation ……………… 23
2. Fluorouracil on OC2 cells ………………………………………... 24
(1) Effect of fluorouracil on the viability of OC2 cells ………….. 24
(2) Effect of fluorouracil on [Ca2+]i in Ca2+-containing medium … 25
3. Temozolomide on OC2 cells …………………………………...… 25
(1) Effect of temozolomide on the viability of OC2 cells ……….. 25
(2) Effect of temozolomide on [Ca2+]i in Ca2+-containing medium ...…………………………………………………….26
IV. Discussion ……………………………………………….. 27
V. Conclusion ………………………………………………... 30
VI. References ………………………………………………. 31
VII. Figures ………………………………………………….. 39
1. Cisplatin on OC2 cells …………………………………………… 39
Figure 1. Effect of cisplatin on the viability of OC2 cells ……….. 39
Figure 2. Effect of cisplatin on [Ca2+]i in Ca2+-containing medium ….……………………………………………... 40
Figure 3. Effect of cisplatin on ERK phosphorylation …………... 41
Figure 4. Effect of cisplatin on JNK phosphorylation …………… 43
Figure 5. Effect of cisplatin on p38 phosphorylation ……………. 45
2. Fluorouracil on OC2 cells ………………………………………... 47
Figure 6. Effect of fluorouracil on the viability of OC2 cells ……. 47
3. Temozolomide on OC2 cells ……………………………………... 48
Figure 7. Effect of temozolomide on the viability of OC2 cells …. 48

Akaboshi M, Kawai K, Ujeno Y, Takada S, Miyahara T. 1994. Binding characteristics of (-)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutane dicarboxylato)-2-platinum(II) to DNA, RNA and protein molecules in HeLa cells and its lethal effect: comparison with cis- and trans-diamminedichloroplatinums(II), Jpn. J Cancer Res 85:106-111.
Basu A, Tu H. 2005. Activation of ERK during DNA damage-induced apoptosis involves protein kinase Cdelta. Biochem Biophys Res Commun 334:1068-1073.
Brini M, Carafoli E. 2000. Calcium signalling: a historical account, recent developments and future perspectives. Cell Mol Life Sci 57:354-370.
Bootman MD, Berridge MJ, Lipp P. 1997. Cooking with calcium: the recipes for composing global signals from elementary events. Cell 91:367-373.
Brozovic A, Osmak M. 2007. Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin resistance. Cancer letters 251:1-16.
Chu ST, Huang CC, Huang CJ, Cehng JS, Chai KL, Cheng HH, Fang YC, Chi CC, Su HH, Chou CT, Jan CR. 2007. Tamoxifen-induced [Ca2+]i rises and Ca2+-independent cell death in human oral cancer cells. J Recept Signal Transduct Res 27:1-15.
Das S, Bhattacharyya S, Ghosh S, Majumdar S. 1999. TNF-alpha induced altered signaling mechanism in human neutrophils. Mol Cell Biochem 197:97-108.
Dehdashti AR, Hegi ME, Regli L, Pica A, Stupp R. 2006. New trends in the medical management of glioblastoma multiforme: the role of temozolomide chemotherapy. Neurosurg Focus 20:E6.
Del Bello B, Moretti D, Gamberucci A, Maellaro E. 2007. Cross-talk between calpain and caspase-3/-7 in cisplatin-induced apoptosis of melanoma cells: a major role of calpain inhibition in cell death protection and p53 status. Oncogene 26:2717-2726.
Department of Health, Executive Yuan, R.O.C.(TAIWAN). 2008. Statistics of causes of death. 中華民國行政院衛生署. 95年死因統計結果分析. (2008年7月8日取自http://www.doh.gov.tw/statistic/ data/衛生統計叢書2/95/上冊/表5.xls)
Fang F, Hoskins J, Butler JS. 2004. 5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs. Mol Cell Biol 24:10766-76.
Fuertes MA, Alonso C, Perez JM. 2003. Biochemical modulation of cisplatin mechanisms of action: enhancement of antitumor activity and circumvention of drug resistance. Chem Rev 103:645-662.
Gebauer G, Mirakhur B, Nguyen Q, Shore SK, Simpins H, Dhanasekaran N. 2000. Cisplatin-resistance involves the defective processing of MEKK1 in human ovarian adenocarcinoma 2008/C13 cells. Int J Oncol 16:321-325.
Gonzalez VM, Fuertes MA, Alonso C, Perez JM. 2001. Is cisplatin-induced cell death always produced by apoptosis? Mol Pharmacol 59:657-663.
Grynkiewicz G, Poenie M, Tsien RY. 1985. A new generation of [Ca2+]i indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450.
Halestrap AP. 2006. Calcium, mitochondria and reperfusion injury a pore way to die. Biochem Soc Trans 34:232-237.
Hegi ME, Diserens AC, Gorlia T, Hamou MF, Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JEC, Hau P, Mirimanoff RO, Cairncross JG, Janzer RC, Stupp R. 2005. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352:997-1003.
Ho CC, Yang XW, Lee TL, Liao PH, Yang SH, Tsai CH, Chou MY. 2003. Activation of p53 signalling in acetylsalicylic acid-induced apoptosis in OC2 human oral cancer cells. Eur J Clin Invest 33:875-882.
Huang TH, Ding SJ, Hsu TZ, Lee ZD, Kao CT. 2004. Root cancal sealers induce cytotoxicity and necrosis. J Master Sci Mater Med 15:767-771.
Huang JK, Huang CJ, Chen WC, Liu SI, Hsu SS, Chang HT, Tseng LL, Chou CT, Chang CH, Jan CR. 2005. Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells. Naunyn Schmiedebergs Arch Pharmacol 372:88-94.
Huang TH, Tsai CY, Chen S, Kao CT. 2002. An evaluation of the cytotoxic effects of orthodontic bonding adhesives upon a primary human oral gingival fibroblast culture and a permanent, human oral cancer-cell line. J Biomed Mater Res 63:814-821.
Jordan P, Carmo-Fonseca M. 2000. Molecular mechanisms involved in cisplatin cytotoxicity. Cell Mol Life Sci 57:1229-1235.
Khateeb ME, Appleton Tg, Gahan LR, Charles BG, Berners-Price SJ, Bolton AM. 1999. Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques. J Inorg Biochem 77:13-21.
Kim S, Shim J. 2008. A forward genetic approach for analyzing the mechanism of resistance to the anti-cancer drug, 5-fluorouracil, using Caenorhabditis elegans. Mol Cells 25:119-123.
Kingsley K, O'Malley S, Ditmyer M, Chino M. 2008. Analysis of oral cancer epidemiology in the US reveals state-specific trends: implications for oral cancer prevention. BMC Public Health 8:87.
Koo MS, Kwo YG, Park JH, CHoi WJ, Biliar TR, Kim YM. 2002. Signaling and function of Caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis. Mol Cells 13:194-201.
Levresse V, Marek L, Blumberg D, Heasley LE. 2002. Regulation of platinum-compound cytotoxicity by th c-Jun N-terminal kinase and c-Jun signaling pathway in small-cell lung cancer cells. Mol Pharmacol 62:689-697.
Lu YC, Huang CC, Huang CJ, Chu ST, Chi CC, Su HH, Hsu SS, Wang JL, Chen IS, Liu SI, Huang JK, Ho CM, Kuo SJ, Jan CR. 2007. Effects of antrodia camphorate on viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation in MG63 human osteosarcoma cells. Drug Dev Res 68:71-78.
Mandic A, Viktorsson K, Heiden T, Hansson J, Shoshan MC. 2001. The MEK1 inhibitor PD98059 sensitizes C8161 melanoma cells to cisplatin –induced apoptosis. Melanoma Res 11:11-19.
Mansouri A, Ridgway LD, Korapati AL, Zhang Q, Tian L, Wang Y, Siddik ZH, Mills GB, Claret FX. 2003. Sustained activation of JNK/p38 MAPK pathways in response to cisplatin leads to Fas ligand induction and cell death in ovarian carcinoma cells. J Biol Chem 278:19245-19256.
Matuszyk J, Kobzdej M, Ziolo E, Kalas W, Kisielow P, Strzadala L. 1998. Thymic lymphomas are resistant to Nur77-mediated apoptosis. Biochem Biophys Res Commun 249:279-282.
Pérez RP. 1998. Cellular and molecular determinants of cisplatin resistance. Eur J Cancer 34: 1535-1544.
Persons DL, Yazlovitskaya EM, Pelling JC. 2000. Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin. J Biol Chem 275:35778-35785.
Posner MR. 2005. Paradigm shift in the treatment of head and neck cancer: the role of neoadjuvant chemotherapy. Oncologist 10 Suppl 3:11-9.
Schweyer S, Soruri S, Heintze A, Radzun HJ, Fayyazi A. 2004. The role of reactive oxygen species in cisplatin-induced apoptosis in human malignant testicular germ cell lines. Int J Oncol 25:1671-1676.
Wei SQ, Sui LH, Zheng JH, Zhang GM, Kao YL. 2004. Role of ERK1/2 kinase in cisplatin-induced apoptosis in human ovarian carcinoma cells. Chin Med Sci J 19:125-129.
Wong DY, Chang KW, Chen CF, Chang RC. 1990. Characterization of two new cell lines derived from oral cavity human squamous cell carcinomas – OC1 and OC2. J Oral Maxillofac Surg 48:385-390.
Quirt I, Verma S, Petrella T, Bak K, Charette M. 2007. Temozolomide for the treatment of metastatic melanoma: a systematic review. Oncologist. 12:1114-23.
Yoshida I, Monji A, Tashiro K, Nakamura K, Inoue R, Kanba S. 2006. Depletion of intracellular Ca2+ store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells. Neurochem Int 48:696-702.
Zhang QX, Feng R, Zhang W, Ding Y, Yang JY, Liu GH. 2005. Role of stress-activated MAP kinase P38 in cisplatin- and DTT-induced apoptosis of the esophageal carcinoma cell line Eca109. World J Gastroenterol 11:4451-4456.