臺灣博碩士論文加值系統

中文摘要
本研究的目的是測試雷公藤內酯醇是否可抑制口腔癌。雷公藤內酯醇是從中草藥雷公藤中提鍊出三氧二萜內酯化合物。相關研究顯示其對一些腫瘤有抑制作用。但對口腔癌並無相關報導。首先，雷公藤內酯醇可顯著抑制口腔癌細胞生長及促進細胞凋亡，在奈體積莫耳濃度範圍內雷公藤內酯醇可促進細胞凋亡，可在annexin V staining下確認磷脂醯絲氨酸(phosphatidylserine，PS) 於口腔癌細胞膜外翻，並活化凋亡蛋白酵素Caspase 3、8、9。在腫瘤的活體實驗，雷公藤內酯醇內亦有顯著的抗癌效果及促進細胞凋亡。其次，多抗藥性是癌細胞所表現出之特性，常見的原因是化療藥產生抗藥性P-糖蛋白及多抗藥性蛋白。雷公藤內酯醇可顯著抑制P-糖蛋白及多抗藥性蛋白，並促進細胞凋亡藉由降低Mcl-1和XIAP 這類抗細胞凋亡蛋白的表現。在腫瘤活體實驗，抗藥性口腔癌細胞亦有顯著的抗癌效果藉由促進腫瘤細胞凋亡。再者，雷公藤內酯醇與傳統治療口腔癌化療藥物5-Fu合併使用有協同作用，即便是在多抗藥性口腔癌細胞亦也有相同的效果。實驗結果顯示雷公藤內酯醇提供口腔癌細胞之化學治療及5-氟尿嘧啶合併使用，提供未來臨床之應用。

Abstract
The aim of this study was to test whether Triptolide possess inhibitory effect of oral cancer. Triptolide (TPL), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F. Recently, TPL has been reported to exhibit potential on anti-tumor proliferation in many cancer cells. However, the treatment effect of TPL on oral cancer or cancer cell lines are still undetermined. Herein we reported that TPL induced prominent growth inhibition and apoptosis in three oral cancer cell lines (KB, SCC25 and OEC-M1 cell line). First, at nM range TPL induced apoptosis which was also confirmed by annexin V staining to detect externalization of phosphatidylserine on the cell membrane. TPL also induced caspase 8, caspase 9 and caspase 3 activaton in these cancer cells. In vivo, TPL had anticancer effect in tumor bearing model via inducing cancer cells apoptosis by Tunel assay. Second, we also demonstrate that triptolide possesses an anti-cancer effect on drug-sensitive parental KB cells and multidrug-resistant KB-7D and KB-tax cells that overexpress MRP and MDR, respectively. Our data revealed that triptolide decreases the expression of MRP and MDR in both KB-7D and KB-tax cells. It also induces apoptosis in these multidrug-resistant cancer cells by activating caspase-3, and decreasing Mcl-1 and XIAP. Triptolide not only inhibits tumor growth but also induces apoptosis of these drug-resistant cancer cells in xenograft mouse models. Third, we also reveal that triptolide combined with 5-fluorouracil could be an alternative strategy for chemotherapy enhancement. These results may lead the usage of TPL on chemotherapy and chemoprevention in oral cancer, and when combined with 5-fluorouracil for the enhancement of cancer therapy in multidrug-resistant cells.

CONTENTS

CHINESE ABSRACT………………………………………………………………1
ABSRACT……………………………………………………………………………2
INTRODUCTION……………………………………………………………… 3-6
MATERIALS AND METHODS………………………………………………… 7
Cells and chemicals……………………………………………………………… 7
Growth inhibition assay ………………………………………………………… 7
Annexin V staining……………………………………………………………… 8
Determination of caspase 3、8、9 activity……………………………………… 8
Quantitative real-time PCR ……………………………………………………… 8
Protein extraction and western blot analysis…………………………………… 9
Xenograft tumor model ………………………………………………………… 10
TUNEL assay …………………………………………………………………… 11
Statistical analysis……………………………………………………………… 11
RESULTS………………………………………………………………………… 13
Growth inhibitory effect ……………………………………………………… 13
Induction of apoptosis………………………………………………………… 13
Enhancement of caspase activities…………………………………………… 13
Anti-tumor effect in vivo…………………………………………………… 14
Overexpression of MRP and MDR in drug-resistant cells……………………… 15
Growth inhibitory effect of TPL on drug-resistant cells………………………… 15
In vitro downregulation of MRP and MDR genes by TPLtreatment…………… 16
Induction of apoptosis by activation of caspase-3 and inhibition of Mcl-1 and XIAP
…………………………………………………………………………………… 17
The effects of TPL on body weight in mice …………………………………… 17
Anti-tumor effect on drug-resistant cells in vivo………………………………… 18
Induction of tumor cell apoptosis in vivo……………………………………… 18
Synergistic effect of TPL combined with 5-FU on drug-resistant cells in vivo… 19
DISCUSSION…………………………………………………………………21-26
REFERENCES…………………………………………………………………27-33
FIGURES AND FIGURE LEGENDS…………………………………………34-50

REFERENCES
1.Chen YJ, Lin SC, Kao T, Chang CS, Hong PS, Shieh TM, Chang KW. 2004. Genome-wide profiling of oral squamous cell carcinoma. J Pathol 204: 326-32
2.Pentenero M, Gandolfo S, Carrozzo M. 2005. Importance of tumor thickness and depth of invasion in nodal involvement and prognosis of oral squamous cell carcinoma: a review of the literature. Head Neck 27: 1080-91
3.Lo WL, Kao SY, Chi LY, Wong YK, Chang RC. 2003. Outcomes of oral squamous cell carcinoma in Taiwan after surgical therapy: factors affecting survival. J Oral Maxillofac Surg 61: 751-8
4.McCarty MF. 2001. Current prospects for controlling cancer growth with non-cytotoxic agents--nutrients, phytochemicals, herbal extracts, and available drugs. Med Hypotheses 56: 137-54
5.Kupchan SM, Court WA, Dailey RG, Jr., Gilmore CJ, Bryan RF. 1972. Triptolide and tripdiolide, novel antileukemic diterpenoid triepoxides from Tripterygium wilfordii. J Am Chem Soc 94: 7194-5
6.Qiu D, Kao PN. 2003. Immunosuppressive and anti-inflammatory mechanisms of triptolide, the principal active diterpenoid from the Chinese medicinal herb Tripterygium wilfordii Hook. f. Drugs R D 4: 1-18
7.Qiu D, Zhao G, Aoki Y, Shi L, Uyei A, Nazarian S, Ng JC, Kao PN. 1999. Immunosuppressant PG490 (triptolide) inhibits T-cell interleukin-2 expression at the level of purine-box/nuclear factor of activated T-cells and NF-kappaB transcriptional activation. J Biol Chem 274: 13443-50
8.Qin WZ, Zhu GD, Yang SM, Han KY, Wang J. 1983. Clinical observations on Tripterygium wilfordii in treatment of 26 cases of discoid lupus erythematosus. J Tradit Chin Med 3: 131-2
9.Tao XL, Sun Y, Dong Y, Xiao YL, Hu DW, Shi YP, Zhu QL, Dai H, Zhang NZ. 1989. A prospective, controlled, double-blind, cross-over study of tripterygium wilfodii hook F in treatment of rheumatoid arthritis. Chin Med J (Engl) 102: 327-32
10.Kiviharju TM, Lecane PS, Sellers RG, Peehl DM. 2002. Antiproliferative and proapoptotic activities of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells. Clin Cancer Res 8: 2666-74
11.Krishna G, Liu K, Shigemitsu H, Gao M, Raffin TA, Rosen GD. 2001. PG490-88, a derivative of triptolide, blocks bleomycin-induced lung fibrosis. Am J Pathol 158: 997-1004
12.Lee HH, Dadgostar H, Cheng Q, Shu J, Cheng G. 1999. NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci U S A 96: 9136-41
13.Chang WT, Kang JJ, Lee KY, Wei K, Anderson E, Gotmare S, Ross JA, Rosen GD. 2001. Triptolide and chemotherapy cooperate in tumor cell apoptosis. A role for the p53 pathway. J Biol Chem 276: 2221-7
14.Carter BZ, Mak DH, Schober WD, McQueen T, Harris D, Estrov Z, Evans RL, Andreeff M. 2006. Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells. Blood 108: 630-7
15.Yang S, Chen J, Guo Z, Xu XM, Wang L, Pei XF, Yang J, Underhill CB, Zhang L. 2003. Triptolide inhibits the growth and metastasis of solid tumors. Mol Cancer Ther 2: 65-72
16.Young RC. 1990. Mechanisms to improve chemotherapy effectiveness. Cancer 65: 815-22
17.Deng L, Tatebe S, Lin-Lee YC, Ishikawa T, Kuo MT. 2002. MDR and MRP gene families as cellular determinant factors for resistance to clinical anticancer agents. Cancer Treat Res 112: 49-66
18.Larkin A, O'Driscoll L, Kennedy S, Purcell R, Moran E, Crown J, Parkinson M, Clynes M. 2004. Investigation of MRP-1 protein and MDR-1 P-glycoprotein expression in invasive breast cancer: a prognostic study. Int J Cancer 112: 286-94
19.Fillpits M, Suchomel RW, Dekan G, Stiglbauer W, Haider K, Depisch D, Pirker R. 1997. Expression of the multidrug resistance-associated protein (MRP) gene in colorectal carcinomas. Br J Cancer 75: 208-12
20.van den Heuvel-Eibrink MM, Wiemer EA, Prins A, Meijerink JP, Vossebeld PJ, van der Holt B, Pieters R, Sonneveld P. 2002. Increased expression of the breast cancer resistance protein (BCRP) in relapsed or refractory acute myeloid leukemia (AML). Leukemia 16: 833-9
21.van den Heuvel-Eibrink MM, Wiemer EA, de Boevere MJ, van der Holt B, Vossebeld PJ, Pieters R, Sonneveld P. 2001. MDR1 gene-related clonal selection and P-glycoprotein function and expression in relapsed or refractory acute myeloid leukemia. Blood 97: 3605-11
22.van der Kolk DM, de Vries EG, van Putten WJ, Verdonck LF, Ossenkoppele GJ, Verhoef GE, Vellenga E. 2000. P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia. Clin Cancer Res 6: 3205-14
23.Brinkhuis M, Izquierdo MA, Baak JP, van Diest PJ, Kenemans P, Scheffer GL, Scheper RJ. 2002. Expression of multidrug resistance-associated markers, their relation to quantitative pathologic tumour characteristics and prognosis in advanced ovarian cancer. Anal Cell Pathol 24: 17-23
24.Diestra JE, Condom E, Del Muro XG, Scheffer GL, Perez J, Zurita AJ, Munoz-Segui J, Vigues F, Scheper RJ, Capella G, Germa-Lluch JR, Izquierdo MA. 2003. Expression of multidrug resistance proteins P-glycoprotein, multidrug resistance protein 1, breast cancer resistance protein and lung resistance related protein in locally advanced bladder cancer treated with neoadjuvant chemotherapy: biological and clinical implications. J Urol 170: 1383-7
25.de Figueiredo-Pontes LL, Pintao MC, Oliveira LC, Dalmazzo LF, Jacomo RH, Garcia AB, Falcao RP, Rego EM. 2008. Determination of P-glycoprotein, MDR-related protein 1, breast cancer resistance protein, and lung-resistance protein expression in leukemic stem cells of acute myeloid leukemia. Cytometry B Clin Cytom 74: 163-8
26.Ng IO, Lam KY, Ng M, Kwong DL, Sham JS. 1998. Expression of P-glycoprotein, a multidrug-resistance gene product, is induced by radiotherapy in patients with oral squamous cell carcinoma. Cancer 83: 851-7
27.Guo X, Ma N, Wang J, Song J, Bu X, Cheng Y, Sun K, Xiong H, Jiang G, Zhang B, Wu M, Wei L. 2008. Increased p38-MAPK is responsible for chemotherapy resistance in human gastric cancer cells. BMC Cancer 8: 375
28.Liu Q, Chen T, Chen H, Zhang M, Li N, Lu Z, Ma P, Cao X. 2004. Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation. Biochem Biophys Res Commun 319: 980-6
29.Kim YH, Lee SH, Lee JY, Choi SW, Park JW, Kwon TK. 2004. Triptolide inhibits murine-inducible nitric oxide synthase expression by down-regulating lipopolysaccharide-induced activity of nuclear factor-kappa B and c-Jun NH2-terminal kinase. Eur J Pharmacol 494: 1-9
30.Wartenberg M, Ling FC, Schallenberg M, Baumer AT, Petrat K, Hescheler J, Sauer H. 2001. Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by reactive oxygen species. J Biol Chem 276: 17420-8
31.Bao X, Cui J, Wu Y, Han X, Gao C, Hua Z, Shen P. 2007. The roles of endogenous reactive oxygen species and nitric oxide in triptolide-induced apoptotic cell death in macrophages. J Mol Med 85: 85-98
32.Shi X, Jin Y, Cheng C, Zhang H, Zou W, Zheng Q, Lu Z, Chen Q, Lai Y, Pan J. 2009. Triptolide inhibits Bcr-Abl transcription and induces apoptosis in STI571-resistant chronic myelogenous leukemia cells harboring T315I mutation. Clin Cancer Res 15: 1686-97
33.Longley DB, Harkin DP, Johnston PG. 2003. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 3: 330-8
34.Jacobs C, Lyman G, Velez-Garcia E, Sridhar KS, Knight W, Hochster H, Goodnough LT, Mortimer JE, Einhorn LH, Schacter L, et al. 1992. A phase III randomized study comparing cisplatin and fluorouracil as single agents and in combination for advanced squamous cell carcinoma of the head and neck. J Clin Oncol 10: 257-63
35.Lin JC, Jan JS, Hsu CY, Wong DY. 1999. High rate of clinical complete response to weekly outpatient neoadjuvant chemotherapy in oral carcinoma patients using a new regimen of cisplatin, 5-fluorouracil, and bleomycin alternating with methotrexate and epirubicin. Cancer 85: 1430-8
36.Wendt TG, Grabenbauer GG, Rodel CM, Thiel HJ, Aydin H, Rohloff R, Wustrow TP, Iro H, Popella C, Schalhorn A. 1998. Simultaneous radiochemotherapy versus radiotherapy alone in advanced head and neck cancer: a randomized multicenter study. J Clin Oncol 16: 1318-24
37.Forastiere AA, Metch B, Schuller DE, Ensley JF, Hutchins LF, Triozzi P, Kish JA, McClure S, VonFeldt E, Williamson SK, et al. 1992. Randomized comparison of cisplatin plus fluorouracil and carboplatin plus fluorouracil versus methotrexate in advanced squamous-cell carcinoma of the head and neck: a Southwest Oncology Group study. J Clin Oncol 10: 1245-51
38.Clavel M, Vermorken JB, Cognetti F, Cappelaere P, de Mulder PH, Schornagel JH, Tueni EA, Verweij J, Wildiers J, Clerico M, et al. 1994. Randomized comparison of cisplatin, methotrexate, bleomycin and vincristine (CABO) versus cisplatin and 5-fluorouracil (CF) versus cisplatin (C) in recurrent or metastatic squamous cell carcinoma of the head and neck. A phase III study of the EORTC Head and Neck Cancer Cooperative Group. Ann Oncol 5: 521-6
39.Kuo CC, Hsieh HP, Pan WY, Chen CP, Liou JP, Lee SJ, Chang YL, Chen LT, Chen CT, Chang JY. 2004. BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res 64: 4621-8
40.Bensadoun RJ, Benezery K, Dassonville O, Magne N, Poissonnet G, Ramaioli A, Lemanski C, Bourdin S, Tortochaux J, Peyrade F, Marcy PY, Chamorey E, Vallicioni J, Seng H, Alzieu C, Gery B, Chauvel P, Schneider M, Santini J, Demard F, Calais G. 2006. French multicenter phase III randomized study testing concurrent twice-a-day radiotherapy and cisplatin/5-fluorouracil chemotherapy (BiRCF) in unresectable pharyngeal carcinoma: Results at 2 years (FNCLCC-GORTEC). Int J Radiat Oncol Biol Phys 64: 983-94
41.Lin J, Chen LY, Lin ZX, Zhao ML. 2007. The effect of triptolide on apoptosis of glioblastoma multiforme (GBM) cells. J Int Med Res 35: 637-43
42.Tengchaisri T, Chawengkirttikul R, Rachaphaew N, Reutrakul V, Sangsuwan R, Sirisinha S. 1998. Antitumor activity of triptolide against cholangiocarcinoma growth in vitro and in hamsters. Cancer Lett 133: 169-75
43.Krysko DV, Vanden Berghe T, D'Herde K, Vandenabeele P. 2008. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 44: 205-21
44.Krysko DV, D'Herde K, Vandenabeele P. 2006. Clearance of apoptotic and necrotic cells and its immunological consequences. Apoptosis 11: 1709-26
45.Engels IH, Stepczynska A, Stroh C, Lauber K, Berg C, Schwenzer R, Wajant H, Janicke RU, Porter AG, Belka C, Gregor M, Schulze-Osthoff K, Wesselborg S. 2000. Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis. Oncogene 19: 4563-73
46.von Haefen C, Wieder T, Essmann F, Schulze-Osthoff K, Dorken B, Daniel PT. 2003. Paclitaxel-induced apoptosis in BJAB cells proceeds via a death receptor-independent, caspases-3/-8-driven mitochondrial amplification loop. Oncogene 22: 2236-47
47.Juang SH, Pan WY, Kuo CC, Liou JP, Hung YM, Chen LT, Hsieh HP, Chang JY. 2004. A novel bis-benzylidenecyclopentanone derivative, BPR0Y007, inducing a rapid caspase activation involving upregulation of Fas (CD95/APO-1) and wild-type p53 in human oral epidermoid carcinoma cells. Biochem Pharmacol 68: 293-303
48.Liu X, Zhou B, Xue L, Shih J, Tye K, Lin W, Qi C, Chu P, Un F, Wen W, Yen Y. 2006. Metastasis-suppressing potential of ribonucleotide reductase small subunit p53R2 in human cancer cells. Clin Cancer Res 12: 6337-44
49.Kim MS, Li SL, Bertolami CN, Cherrick HM, Park NH. 1993. State of p53, Rb and DCC tumor suppressor genes in human oral cancer cell lines. Anticancer Res 13: 1405-13
50.Kamiya Y, Ohshima T. 2005. The Individual Cell Properties of Oral Squamous Cell Carcinoma and p53 Tumor Suppressor Gene Mutation. Oral Science International 2: 104-17
51.Wang X, Matta R, Shen G, Nelin LD, Pei D, Liu Y. 2006. Mechanism of triptolide-induced apoptosis: Effect on caspase activation and Bid cleavage and essentiality of the hydroxyl group of triptolide. J Mol Med 84: 405-15
52.Wan CK, Wang C, Cheung HY, Yang M, Fong WF. 2006. Triptolide induces Bcl-2 cleavage and mitochondria dependent apoptosis in p53-deficient HL-60 cells. Cancer Lett 241: 31-41
53.Kuzuya K. 2004. Chemoradiotherapy for uterine cancer: current status and perspectives. Int J Clin Oncol 9: 458-70
54.Psyrri A, Fountzilas G. 2006. Advances in the treatment of locally advanced non-nasopharyngeal squamous cell carcinoma of the head and neck region. Med Oncol 23: 1-15
55.Tang XY, Zhu YQ, Tao WH, Wei B, Lin XL. 2007. Synergistic effect of triptolide combined with 5-fluorouracil on colon carcinoma. Postgrad Med J 83: 338-43
56.Wang W, Yang S, Su Y, Xiao Z, Wang C, Li X, Lin L, Fenton BM, Paoni SF, Ding I, Keng P, Okunieff P, Zhang L. 2007. Enhanced antitumor effect of combined triptolide and ionizing radiation. Clin Cancer Res 13: 4891-9
57.Leonessa F, Green D, Licht T, Wright A, Wingate-Legette K, Lippman J, Gottesman MM, Clarke R. 1996. MDA435/LCC6 and MDA435/LCC6MDR1: ascites models of human breast cancer. Br J Cancer 73: 154-61
58.O'Connor R. 2007. The pharmacology of cancer resistance. Anticancer Res 27: 1267-72
59.Feng G, Wang DZ, Chen HQ, Hu J, He J. 2008. A new method to induce multi-drug resistance to carboplatin in a mouse model of human tongue squamous cell carcinoma. Int J Oral Maxillofac Surg
60.Yoshida N, Takagi A, Kitazawa H, Kawakami J, Adachi I. 2005. Inhibition of P-glycoprotein-mediated transport by extracts of and monoterpenoids contained in Zanthoxyli fructus. Toxicol Appl Pharmacol 209: 167-73
61.Zhang B, Gojo I, Fenton RG. 2002. Myeloid cell factor-1 is a critical survival factor for multiple myeloma. Blood 99: 1885-93
62.Nakagawa Y, Abe S, Kurata M, Hasegawa M, Yamamoto K, Inoue M, Takemura T, Suzuki K, Kitagawa M. 2006. IAP family protein expression correlates with poor outcome of multiple myeloma patients in association with chemotherapy-induced overexpression of multidrug resistance genes. Am J Hematol 81: 824-31
63.Chen YW, Lin GJ, Chia WT, Lin CK, Chuang YP, Sytwu HK. 2009. Triptolide exerts anti-tumor effect on oral cancer cells in vitro and in vivo. Oral Oncol In press
64.Chen YW, Lin GJ, Chia WT, Lin CK, Chuang YP, Sytwu HK. 2009. Triptolide exerts anti-tumor effect on oral cancer and KB cells in vitro and in vivo. Oral Oncol 45: 562-8
65.He MF, Liu L, Ge W, Shaw PC, Jiang R, Wu LW, But PP. 2009. Antiangiogenic activity of Tripterygium wilfordii and its terpenoids. J Ethnopharmacol 121: 61-8
66.Zhu W, Ou Y, Li Y, Xiao R, Shu M, Zhou Y, Xie J, He S, Qiu P, Yan G. 2009. A small-molecule triptolide suppresses angiogenesis and invasion of human anaplastic thyroid carcinoma cells via down-regulation of the nuclear factor-kappa B pathway. Mol Pharmacol 75: 812-9
67.Lee KY, Chang W, Qiu D, Kao PN, Rosen GD. 1999. PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells. J Biol Chem 274: 13451-5
68.Jiang XH, Wong BC, Lin MC, Zhu GH, Kung HF, Jiang SH, Yang D, Lam SK. 2001. Functional p53 is required for triptolide-induced apoptosis and AP-1 and nuclear factor-kappaB activation in gastric cancer cells. Oncogene 20: 8009-18
69.Lee KY, Park JS, Jee YK, Rosen GD. 2002. Triptolide sensitizes lung cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inhibition of NF-kappaB activation. Exp Mol Med 34: 462-8
70.Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC. 1998. Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 91: 991-1000
71.Moshynska O, Sankaran K, Pahwa P, Saxena A. 2004. Prognostic significance of a short sequence insertion in the MCL-1 promoter in chronic lymphocytic leukemia. J Natl Cancer Inst 96: 673-82