臺灣博碩士論文加值系統

中文摘要
肝癌為國人最重要癌症之一。對於手術無法切除之肝癌，經肝動脈化學栓塞術為主要的治療方法之一，其中Daunorubicin為一重要的併用的化療藥物。Daunorubicin是一種anthracycline類抗生素，主要之作用機制為抑制topoisomerase II，但其詳細之分子機制尚待進一步研究。Activator protein-1(AP-1)是一種轉錄因子（transcription factor），研究顯示AP-1是造成tumor promotion及tumor progression的重要因子，AP-1活化會啟動許多基因轉錄，進行promotion。因此，AP-1可應用為一良好之腫瘤治療之分子標的。此外，Daunorubicin對於細胞週期之影響為現今daunorubicin作用之重要機制之一。因此在本研究中，我們希望藉助傳統之腫瘤實驗、AP-1活性分析及細胞週期分析，來探討daunorubicin對於肝細胞癌療效或細胞轉形之機制。本研究之方法包括有anchorage-independent transformation assay、Chang liver/AP-1及HepG2/AP-1重組細胞株的構築，測定luciferase的活性以評估AP-1的活性、細胞存活率試驗及細胞週期分析等實驗來評估daunorubicin對於肝細胞之作用機制。實驗結果顯示在anchorage-independent transformation assay實驗中daunorubicin確能有效抑制肝細胞的轉形，Daunorubicin可以抑制Chang liver/AP-1及HepG2/AP-1之AP-1活性，經由細胞週期分析可見Daunorubicin主要作用於抑制細胞週期之G2/M期。我們研究顯示在細胞實驗中，Daunorubicin確為良好之抗肝腫瘤藥物，其作用於肝細胞之主要機轉為對於G2/M細胞週期休止，對於其更詳細的機制值得進一步探討。

Abstract
Daunorubicin (DNR), an inhibitor of DNA topoisomerase II, was considered as the treatment of choice for hepatocellular carcinoma. Recent studies found that the therapeutic effect of DNR might be through mitogen-activated protein kinase pathway, apoptosis, and cell cycle arrest; however, the molecular basis of DNR on hepatocellular transformation is still unclear. To evaluate the anti-tumor mechanism of DNR on hepatocytes, we examined the effects of DNR on hepatocellular transformation by anchorage-independent transformation assay. Treatment of 0.01 μM DNR for 21 days inhibited colony formation, suggesting that DNR was able to suppress the hepatocellular transformation. Flow cytometry was further used to evaluate the effect of DNR on cell cycle distribution, and reporter assay was employed to examine the effect of DNR on activator protein 1 (AP-1) activity. The data showed that incubation of Chang liver and HepG2 cells with 0.01 μM DNR for 24 h resulted in the accumulation in G2/M phase but not the induction of apoptosis and AP-1 activity. However, the number of apoptotic cells would be slightly induced and AP-1 activity would be down-regulated by the treatment of 1 μM DNR for 24 h and 16 h, respectively. Therefore, the anti-hepatocellular transformation of DNR might be mediated by cell cycle arrest at G2/M checkpoint.

目錄
中文摘要 1
英文摘要 2
誌謝 3
目錄 4
圖目錄 5
第一章、前言 6
第二章、文獻探討 9
第三章、研究架構與研究設計 14
第四章、材料與方法 16
第五章、研究結果 20
第六章、討論 23
第七章、結論與建議 28
參考文獻 30
附圖 42
作者簡歷 48
著作權聲明 49

參考文獻
衛生署統計資訊網http://www.doh.gov.tw/statistic/index.htm
賴羿如。肝細胞轉型之分子機制及分子療程之探討。中國醫藥學院醫學研究所碩士論文。2001。
吳泰賢。以AP-1為標的開發抗肝細胞轉形藥物。中國醫藥學院醫學研究所碩士論文。2002。
Alani,R., Brown,P., Binetruy,B., Dosaka,H., Rosenberg,R.K., Angel,P., Karin,M., Birrer,M.J., 1991. The transactivating domain of the c-Jun proto-oncoprotein is required for cotransformation of rat embryo cells. Mol. Cell Biol. 11, 6286-6295.
Andrecht,S., Kolbus,A., Hartenstein,B., Angel,P., Schorpp-Kistner,M., 2002. Cell cycle promoting activity of JunB through cyclin A activation. J Biol. Chem. 277, 35961-35968.
Angel,P., Imagawa,M., Chiu,R., Stein,B., Imbra,R.J., Rahmsdorf,H.J., Jonat,C., Herrlich,P., Karin,M., 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49, 729-739.
Bachur,N.R., Gordon,S.L., Gee,M.V., 1978. A general mechanism for microsomal activation of quinone anticancer agents to free radicals. Cancer Res. 38, 1745-1750.
Befeler,A.S., Di Bisceglie,A.M., 2002. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology 122, 1609-1619.
Bernstein,L.R., Colburn,N.H., 1989. AP1/jun function is differentially induced in promotion-sensitive and resistant JB6 cells. Science 244, 566-569.
Bickers,D.R., Lowy,D.R., 1989. Carcinogenesis: a fifty-year historical perspective. J Invest Dermatol. 92, 121S-131S.
Boutwell,R.K., 1989. Model systems for defining initiation, promotion, and progression of skin neoplasms. Prog. Clin. Biol. Res. 298, 3-15.
Brechot,C., 1998. Molecular mechanisms of hepatitis B and C viruses related to liver carcinogenesis. Hepatogastroenterology 45 Suppl 3, 1189-1196.
Brown,K., Balmain,A., 1995. Transgenic mice and squamous multistage skin carcinogenesis. Cancer Metastasis Rev. 14, 113-124.
Castaneda,F., Kinne,R.K., 1999. Effects of doxorubicin, mitomycin C, and ethanol on Hep-G2 cells in vitro. J Cancer Res. Clin. Oncol. 125, 1-8.
Chang,B.D., Swift,M.E., Shen,M., Fang,J., Broude,E.V., Roninson,I.B., 2002. Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent. Proc. Natl. Acad. Sci. U. S. A 99, 389-394.
Chu,S.H., Kim,H., Seo,J.Y., Lim,J.W., Mukaida,N., Kim,K.H., 2003. Role of NF-kappaB and AP-1 on Helicobater pylori-induced IL-8 expression in AGS cells. Dig. Dis. Sci. 48, 257-265.
Colotta,F., Polentarutti,N., Sironi,M., Mantovani,A., 1992. Expression and involvement of c-fos and c-jun protooncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell lines. J Biol. Chem. 267, 18278-18283.
Dhar,A., Young,M.R., Colburn,N.H., 2002. The role of AP-1, NF-kappaB and ROS/NOS in skin carcinogenesis: the JB6 model is predictive. Mol. Cell Biochem. 234-235, 185-193.
Diao,J., Garces,R., Richardson,C.D., 2001. X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis. Cytokine Growth Factor Rev. 12, 189-205.
Domann,F.E., Levy,J.P., Birrer,M.J., Bowden,G.T., 1994. Stable expression of a c-JUN deletion mutant in two malignant mouse epidermal cell lines blocks tumor formation in nude mice. Cell Growth Differ. 5, 9-16.
Dong,Z., Birrer,M.J., Watts,R.G., Matrisian,L.M., Colburn,N.H., 1994. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc. Natl. Acad. Sci. U. S. A 91, 609-613.
Franklin,C.C., Kraft,A.S., 1995. Constitutively active MAP kinase kinase (MEK1) stimulates SAP kinase and c-Jun transcriptional activity in U937 human leukemic cells. Oncogene 11, 2365-2374.
Groupe d''Etude et de Traitement du Carcinome Hepatocellulaire 1995. A comparison of lipiodol chemoembolization and conservative treatment for unresectable hepatocellular carcinoma. Groupe d''Etude et de Traitement du Carcinome Hepatocellulaire. N Engl J Med 332, 1256-1261.
Hortobagyi,G.N., 1997. Anthracyclines in the treatment of cancer. An overview. Drugs 54 Suppl 4, 1-7.
Hsu,T.C., Young,M.R., Cmarik,J., Colburn,N.H., 2000. Activator protein 1 (AP-1)- and nuclear factor kappaB (NF-kappaB)-dependent transcriptional events in carcinogenesis. Free Radic. Biol. Med 28, 1338-1348.
Huang,C., Ma,W.Y., Dong,Z., 1996. Requirement for phosphatidylinositol 3-kinase in epidermal growth factor-induced AP-1 transactivation and transformation in JB6 P+ cells. Mol. Cell Biol. 16, 6427-6435.
Jochum,W., Passegue,E., Wagner,E.F., 2001. AP-1 in mouse development and tumorigenesis. Oncogene 20, 2401-2412.
Kamada,K., Nakanishi,T., Kitamoto,M., Aikata,H., Kawakami,Y., Ito,K., Asahara,T., Kajiyama,G., 2001. Long-term prognosis of patients undergoing transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma: comparison of cisplatin lipiodol suspension and doxorubicin hydrochloride emulsion. J Vasc. Interv. Radiol. 12, 847-854.
Kim,S.G., Sung,M., Kang,K.W., Kim,S.H., Son,M.H., Kim,W.B., 2001. DA-125, a novel anthracycline derivative showing high-affinity DNA binding and topoisomerase II inhibitory activities, exerts cytotoxicity via c-Jun N-terminal kinase pathway. Cancer Chemother. Pharmacol. 47, 511-518.
Kinoshita,I., Leaner,V., Katabami,M., Manzano,R.G., Dent,P., Sabichi,A., Birrer,M.J., 2003. Identification of cJun-responsive genes in Rat-1a cells using multiple techniques: increased expression of stathmin is necessary for cJun-mediated anchorage-independent growth. Oncogene 22, 2710-2722.
Kudoh,K., Ramanna,M., Ravatn,R., Elkahloun,A.G., Bittner,M.L., Meltzer,P.S., Trent,J.M., Dalton,W.S., Chin,K.V., 2000. Monitoring the expression profiles of doxorubicin-induced and doxorubicin-resistant cancer cells by cDNA microarray. Cancer Res. 60, 4161-4166.
Kuyvenhoven,J.P., Lamers,C.B., van Hoek,B., 2001. Practical management of hepatocellular carcinoma. Scand. J Gastroenterol. Suppl 82-87.
Lamb,R.F., Hennigan,R.F., Turnbull,K., Katsanakis,K.D., MacKenzie,E.D., Birnie,G.D., Ozanne,B.W., 1997. AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44. Mol. Cell Biol. 17, 963-976.
Laurent,G., Jaffrezou,J.P., 2001. Signaling pathways activated by daunorubicin. Blood 98, 913-924.
Lee,T.K., Lau,T.C., Ng,I.O., 2002. Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines. Cancer Chemother. Pharmacol. 49, 78-86.
Lee,W., Mitchell,P., Tjian,R., 1987. Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell 49, 741-752.
Li,J., Wang,W.L., Yang,X.K., Yu,X.X., Hou,Y.D., Zhang,J., 2000a. Inducible overexpression of Bak sensitizes HCC-9204 cells to apoptosis induced by doxorubicin. Acta Pharmacol. Sin. 21, 769-776.
Li,J., Wang,W.L., Yang,X.K., Yu,X.X., Hou,Y.D., Zhang,J., 2000b. Inducible overexpression of Bak sensitizes HCC-9204 cells to apoptosis induced by doxorubicin. Acta Pharmacol. Sin. 21, 769-776.
Li,J.J., Rhim,J.S., Schlegel,R., Vousden,K.H., Colburn,N.H., 1998. Expression of dominant negative Jun inhibits elevated AP-1 and NF-kappaB transactivation and suppresses anchorage independent growth of HPV immortalized human keratinocytes. Oncogene 16, 2711-2721.
Ling,Y.H., el Naggar,A.K., Priebe,W., Perez-Soler,R., 1996. Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34cdc2/cyclin B1 activity induced by doxorubicin in synchronized P388 cells. Mol. Pharmacol. 49, 832-841.
Llovet,J.M., Bruix,J., 2003. Systematic review of randomized trials for unresectable hepatocellular carcinoma: Chemoembolization improves survival. Hepatology 37, 429-442.
Lo,C.M., Ngan,H., Tso,W.K., Liu,C.L., Lam,C.M., Poon,R.T., Fan,S.T., Wong,J., 2002. Randomized controlled trial of transarterial lipiodol chemoembolization for unresectable hepatocellular carcinoma. Hepatology 35, 1164-1171.
Luo,D., Cheng,S.C., Xie,H., Xie,Y., 2000. Effects of Bcl-2 and Bcl-XL protein levels on chemoresistance of hepatoblastoma HepG2 cell line. Biochem. Cell Biol. 78, 119-126.
Luo,D., Cheng,S.C., Xie,Y., 1999. Expression of Bcl-2 family proteins during chemotherapeutic agents-induced apoptosis in the hepatoblastoma HepG2 cell line. Br. J Biomed. Sci. 56, 114-122.
Macdonald,A., Crowder,K., Street,A., McCormick,C., Saksela,K., Harris,M., 2003. The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing ras-ERK pathway signaling. J Biol. Chem. 278, 17775-17784.
Mansat-de Mas,V., Bezombes,C., Quillet-Mary,A., Bettaieb,A., D''orgeix,A.D., Laurent,G., Jaffrezou,J.P., 1999. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin. Mol. Pharmacol. 56, 867-874.
Matrisian,L.M., 1994. Matrix metalloproteinase gene expression. Ann. N Y. Acad. Sci. 732, 42-50.
Muller,M., Wilder,S., Bannasch,D., Israeli,D., Lehlbach,K., Li-Weber,M., Friedman,S.L., Galle,P.R., Stremmel,W., Oren,M., Krammer,P.H., 1998. p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp. Med 188, 2033-2045.
Osborn,M.T., Chambers,T.C., 1996. Role of the stress-activated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs. J Biol. Chem. 271, 30950-30955.
Painter,R.B., 1978. Inhibition of DNA replicon initiation by 4-nitroquinoline 1-oxide, adriamycin, and ethyleneimine. Cancer Res. 38, 4445-4449.
Park,K.J., Choi,S.H., Koh,M.S., Kim,D.J., Yie,S.W., Lee,S.Y., Hwang,S.B., 2001. Hepatitis C virus core protein potentiates c-Jun N-terminal kinase activation through a signaling complex involving TRADD and TRAF2. Virus Res. 74, 89-98.
Parkin,D.M., 2001. Global cancer statistics in the year 2000. Lancet Oncol. 2, 533-543.
Parkin,D.M., Bray,F., Ferlay,J., Pisani,P., 2001. Estimating the world cancer burden: Globocan 2000. Int. J Cancer 94, 153-156.
Passegue,E., Wagner,E.F., 2000. JunB suppresses cell proliferation by transcriptional activation of p16(INK4a) expression. EMBO J 19, 2969-2979.
Potter,A.J., Gollahon,K.A., Palanca,B.J., Harbert,M.J., Choi,Y.M., Moskovitz,A.H., Potter,J.D., Rabinovitch,P.S., 2002. Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin. Carcinogenesis 23, 389-401.
Puisieux,A., Ji,J., Guillot,C., Legros,Y., Soussi,T., Isselbacher,K., Ozturk,M., 1995. p53-mediated cellular response to DNA damage in cells with replicative hepatitis B virus. Proc. Natl. Acad. Sci. U. S. A 92, 1342-1346.
Sato,S., Iwaizumi,M., Handa,K., Tamura,Y., 1977. Electron spin resonance study on the mode of generation of free radicals of daunomycin, adriamycin, and carboquone in NAD(P)H-microsome system. Gann 68, 603-608.
Shaulian,E., Karin,M., 2001. AP-1 in cell proliferation and survival. Oncogene 20, 2390-2400.
Shaulian,E., Karin,M., 2002. AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4, E131-E136.
Sherr,C.J., 1993. Mammalian G1 cyclins. Cell 73, 1059-1065.
Tarn,C., Zou,L., Hullinger,R.L., Andrisani,O.M., 2002. Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes. J Virol. 76, 9763-9772.
Tritton,T.R., Murphree,S.A., Sartorelli,A.C., 1978. Adriamycin: a proposal on the specificity of drug action. Biochem. Biophys. Res. Commun. 84, 802-808.
Tyagi,A.K., Singh,R.P., Agarwal,C., Chan,D.C., Agarwal,R., 2002. Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth Inhibition, G2-M arrest, and apoptosis. Clin. Cancer Res. 8, 3512-3519.
Vogt,P.K., 2001. Jun, the oncoprotein. Oncogene 20, 2365-2377.
Vogt,P.K., Bos,T.J., 1990. jun: oncogene and transcription factor. Adv. Cancer Res. 55, 1-35.
Wagner,E.F., 2001. AP-1--Introductory remarks. Oncogene 20, 2334-2335.
Waldes,H., Center,M.S., 1981. The interaction of adriamycin with nuclear DNA: evidence for a drug induced compaction of isolated chromatin. Biochem. Biophys. Res. Commun. 98, 95-101.
Xiao,Z., Chen,Z., Gunasekera,A.H., Sowin,T.J., Rosenberg,S.H., Fesik,S., Zhang,H., 2003. Chk1 Mediates S and G2 Arrests through Cdc25A Degradation in Response to DNA-damaging Agents. J Biol. Chem. 278, 21767-21773.
Yamanaka,T., Shiraki,K., Sugimoto,K., Ito,T., Fujikawa,K., Ito,M., Takase,K., Moriyama,M., Nakano,T., Suzuki,A., 2000. Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines. Hepatology 32, 482-490.
Zhang,R.G., Guo,L.X., Wang,X.W., Xie,H., 2002. Telomerase inhibition and telomere loss in BEL-7404 human hepatoma cells treated with doxorubicin. World J Gastroenterol. 8, 827-831.
Zhao,Y., St Clair,D.K., 2003. Detection of the content and activity of the transcription factor AP-1 in a multistage skin carcinogenesis model. Methods Mol. Biol. 218, 177-184.