癌症連續十年位居國人十大死因之首，其中肝癌是造成男性死亡人數最多而女性次之的疾病。原發性肝癌主要屬於肝細胞腫瘤 (hepatocellular carcinoma, HCC)。肝癌的治療除了肝臟移植、手術切除外還有局部摘除治療與肝動脈血管栓塞。肝動脈栓塞化療術是一種局部性的治療，將化療藥物導入肝臟腫瘤。但是癌症病人在接受化學治療後，常常對藥物具有抗藥性 (drug resistance)。癌細胞對藥物抗藥性的研究顯示，ABC運輸蛋白扮演重要的角色，尤其是 ABCB1、ABCC1、ABCG2 等這些主要與抗藥性有關的ABC運輸蛋白，這些ABC運輸蛋白也分布於肝臟細胞中。臨床上以 5FU、Cisplatin、Mitomycin-C 等化療藥物運用在治療肝癌上。本研究假設以ABC運輸蛋白抑制劑 Verapamil 配合化療藥物可以加強治療的效能。並且我們觀察化療藥物、抑制劑對於肝癌細胞株 HepG2 與 Hep3B 細胞ABC運輸蛋白的影響。MTT assay 的結果顯示化療藥物配合 Verapamil 能有效殺死癌細胞，若合併三種化療藥物配合抑制劑更能有效降低肝癌細胞存活率。RT-PCR 的結果顯示 Verapamil 對於肝癌細胞ABC運輸蛋白 mRNA 的表現量沒有顯著性的影響。流式細胞儀的結果顯示處理藥物 1 或 24 小時會誘導細胞膜的 ABCB1 及 ABCG2 增加。由 P-glycoprotein 功能測試發現，Verapamil 能抑制 P-glycoprotein，使細胞內 Rhodamine 123 滯留量增加。綜合以上實驗結果發現，經由化療藥物處理後 HepG2 及 Hep3B細胞在ABC運輸蛋白的反應不同。雖然 Verapamil 對於ABC運輸蛋白 mRNA 的表現量沒有影響，但是會促使細胞膜的 ABCB1 與 ABCG2 增加。因此化療藥物由 Verapamil 本身毒殺效果與抑制藥物運送出細胞外的能力增加癌細胞對於化療藥物的敏感性。希望藉由此研究結果提供更有效治療肝癌的方法。

Cancer remains the most cause death disease in Taiwan at least ten years. Liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), is the most common cause of cancer mortality in men and the second most in women. Not only liver section and liver transplantation are used in HCC therapy but also local ablation therapy and transarterial therapy. Transarterial chemoembolization (TACE) is one of the local therapies that inject chemotherapeutic drugs directly into liver tumor. However, drug resistance is the mainly restriction in patient after chemotherapy. Moreover, it is known that drug resistance was associated to over-expression of certain ABC transporter genes, especially ABCB1, ABCG2, and ABCC family in cancer cell and those ABC transporters were also expressed in liver. Base on clinical study, they use 5-fluororuacil, cisplatin and mitomycin-C for liver cancer treatment. In this study, we hypothesized that cancer therapies may be augmented through blocked the drug efflux ABC channels with the ABC transporter inhibitors such as verapamil. The associations among drug treatments, inhibitor incorporation and the expression of ABC transporters were evaluated in HepG2 and Hep3B cells. MTT assay demonstrated that the cell viability was considerable decreased by treating triple drugs with verapamil. RT-PCR data showed that ABC transporters mRNA expression has no significantly change. However, membrane ABCB1 and ABCG2 were induced after drugs and inhibitors treatment either 1 or 24 hours by flow cytometry analysis. P-glycoprotein functional assay also showed p-glycoprotein was inhibited by verapamil, and hence Rhodamine 123 retention was increased. Taken together, there are different response of ABC transporters in HepG2 and Hep3B after drugs and inhibitors treatment. Membrane ABCB1 and ABCG2 were induced by drugs and inhibitors treatment. However, p-glycoprotein’s function was restrained simultaneously by inhibitors treatment. Therefore, verapamil can enhance cell death by inhibiting ABC transporters and its cytotoxic effect rather than the increased expression of ABC transporters. This finding might provide a better way in liver cancer therapy.

目錄
中文摘要 1
英文摘要 2
縮寫表 3
壹、緒論 6
一. 肝細胞腫瘤與肝癌的治療 6
1-1 肝細胞腫瘤 (Hepatocellular carcinoma, HCC) 6
1-2 肝癌的治療 9
二. 多重抗藥性與ABC運輸蛋白 13
2-1 多重抗藥性 (Multidrug Resistance) 13
2-2 ABC運輸蛋白 (ABC transporter) 14
2-2-1 ABCB1 16
2-2-2 ABCG2 17
2-2-3 ABCC family 19
2-3 化學致敏藥物 (Chemosensitiser) 21
2-3-1 維拉帕米 (Verapamil) 22
2-3-2 苯溴馬隆 (Benzbromarone) 23
三. 癌症幹細胞 (Cancer Stem Cells) 24
3-1 癌症幹細胞 (Cancer Stem Cells, CSC) 24
3-2 癌症幹細胞標記 (Cancer Stem Cells Marker) 27
3-2-1 CD133 27
3-2-2 周邊細胞 (Side population, SP) 30
3-3 癌症幹細胞與多重抗藥性 31
貳、研究目的 33
叁、實驗材料與方法 34
一. 細胞培養 (Cell culture) 34
二. CD133+/- 細胞的分析與分選 (Sorting) 37
三. 細胞存活率試驗 (MTT assay) 40
四. 反轉錄聚合酶連鎖反應 (RT-PCR) 43
五. ABCB1 蛋白功能試驗 49
六. 免疫細胞化學法 (Immonocytochemistry, ICC)) 52
七. 間接免疫螢光法 (Indirect Immunofluorescence
assay) 54
肆、結果 57
一. ABC運輸蛋白抑制劑及化療用藥對於肝癌細胞的影
響 57
(一) ABC運輸蛋白抑制劑之肝細胞毒性測試 57
(二) 單一藥物或合併抑制劑對細胞存活性的影響 60
(三) 多重藥物 (3D) 合併抑制劑對細胞存活性的影響
63
二. ABC運輸蛋白抑制劑及化療用藥對於ABC運輸蛋
mRNA 表現的影響 66
(一) 單一藥物或抑制劑對細胞ABC運輸蛋白 mRNA 表
現的影響 66
(二) 多重藥物 (3D) 及ABC運輸蛋白抑制劑對mRNA
表現的影響 70
三. ABC運輸蛋白抑制劑或/及化療用藥 (3D) 對於ABC
運輸蛋白在細胞膜分佈的影響 74
(一) ABCB1 蛋白與 ABCG2 蛋白在肝癌細胞中的分佈
情形 74
(二) 抑制劑 Verapamil或/及化療用藥 (3D) 可增加肝癌
細胞膜上 ABCB1 蛋白的表現 77
(三) ABC運輸蛋白抑制劑或/及化療用藥 (3D) 對於
ABCB1 及ABCG2表現於細胞膜上的影響 80
四. ABC運輸蛋白抑制劑或/及化療用藥 (3D) 對於肝癌
細胞排藥功能的影響 86
(一) ABC運輸蛋白抑制劑對肝癌細胞排藥功能之影響
86
(二) ABC運輸蛋白抑制劑合併化療用藥 (3D) 對於肝癌
細胞排藥功能的影響 91
伍、討論 95
陸、未來工作 102
柒、參考文獻 103
捌、附錄 108
附錄一、蛋白質電泳與西方點墨法 (Western Blots,
WB 108
(一) 實驗材料與方法 108
(二) 實驗結果 114
1. HepG2 及 Hep3B細胞中 ABCB1、pan-
cadherin、 GAPDH 的表現 114
附錄二、肝癌細胞 CD133 的分選與藥物測試 118
(一) 實驗結果 118
1. CD133 在肝癌細胞株中的分佈與分選 118
(1) CD133 在肝癌細胞株的分佈 118
(2) CD133+/- 細胞分選 (Sorting) 120
(3) 分選後在 CD133+/- 與 Un-sorted 細胞中
CD133 的含量 125
2. 化療用藥及ABC運輸蛋白抑制劑對於 CD133+ 與
CD133- 細胞的影響 128
(1) CD133+/- 細胞在分選後的生長 (Proliferation)
128
(2) CD133+/- 細胞對於化療用藥及ABC運輸蛋白抑
制劑的影響 131
(二) 討論 135
(三) 未來工作 137

圖表目錄
圖一 ABC運輸蛋白抑制劑在肝癌細胞之細胞毒性 59
圖二 單一藥物或合併抑制劑對細胞存活性的影響 62
圖三 3D合併抑制劑對細胞存活率的影響 65
圖四 單一藥物或抑制劑對ABC運輸蛋白mRNA表現的影
響 68
圖五 多重藥物(3D)及抑制劑對ABC運輸蛋白mRNA表現
的影響 72
圖六 ABCB1蛋白與ABCG2蛋白在肝癌細胞中的分佈情形
76
圖七 抑制劑 Verapamil或/及化療用藥 (3D) 可增加肝癌
細胞膜上 ABCB1 蛋白的表現 79
圖八 ABC運輸蛋白抑制劑或/及化療用藥 (3D) 對於
ABCB1 及 ABCG2表現於細胞膜上的影響 82
圖九 ABC運輸蛋白抑制劑對肝癌細胞排藥功能之影響 89
圖十 ABC運輸蛋白抑制劑合併化療用藥 (3D) 對於肝癌細
胞排藥功能的影響 93
圖十一 HepG2 及 Hep3B細胞中的 ABCB1、pan-
cadherin、GAPDH 117
圖十二 分選 HepG2細胞 與 Hep3B細胞中 CD133+ 與
CD133- 族群 123
圖十三 CD133+ 與 CD133- 細胞在分選後的生長情形
130
圖十四 化療藥物及ABC運輸蛋白抑制劑對於 CD133 +/-
細胞的影響 133
表一 CD133在各肝癌細胞株中的含量 119
表二 CD133 族群 (%) 在 CD133+/- 及 Un-sorted 細胞中
的含量 126
表三 CD133+ 與 CD133- 細胞或 Un-sorted 細胞的比值
127

Al-Hajj M, Becker MW, Wicha M, Weissman I, Clarke MF. Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 2004;14:43-7.
Bakos E, Evers R, Sinko E, Varadi A, Borst P, Sarkadi B. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol 2000;57:760-8.
Bakos E, Homolya L. Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1). Pflugers Arch 2007;453:621-41.
Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006;444:756-60.
Belpomme D, Gauthier S, Pujade-Lauraine E, Facchini T, Goudier MJ, Krakowski I, Netter-Pinon G, Frenay M, Gousset C, Marie FN, Benmiloud M, Sturtz F. Verapamil increases the survival of patients with anthracycline-resistant metastatic breast carcinoma. Ann Oncol 2000;11:1471-6.
Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730-7.
Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004;127:S5-S16.
Brabec V, Kasparkova J. Molecular aspects of resistance to antitumor platinum drugs. Drug Resist Updat 2002;5:147-61.
Challen GA, Little MH. A side order of stem cells: the SP phenotype. Stem Cells 2006;24:3-12.
Chaudhary PM, Roninson IB. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 1991;66:85-94.
Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A, Nakauchi H, Taniguchi H. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 2006;44:240-51.
Choi CH. ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal. Cancer Cell Int 2005;5:30.
Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AM, Deeley RG. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992;258:1650-4.
Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer 2005;5:275-84.
Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 2001;11:1156-66.
Deeley RG, Cole SP. Substrate recognition and transport by multidrug resistance protein 1 (ABCC1). FEBS Lett 2006a;580:1103-11.
Deeley RG, Westlake C, Cole SP. Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol Rev 2006b;86:849-99.
Doyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 2003;22:7340-58.
Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, Ross DD. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 1998;95:15665-70.
Eilers M, Roy U, Mondal D. MRP (ABCC) transporters-mediated efflux of anti-HIV drugs, saquinavir and zidovudine, from human endothelial cells. Exp Biol Med (Maywood) 2008;233:1149-60.
El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557-76.
Ford JM. Experimental reversal of P-glycoprotein-mediated multidrug resistance by pharmacological chemosensitisers. Eur J Cancer 1996;32A:991-1001.
Gillet JP, Efferth T, Remacle J. Chemotherapy-induced resistance by ATP-binding cassette transporter genes. Biochim Biophys Acta 2007;1775:237-62.
Gomes CM, van Paassen H, Romeo S, Welling MM, Feitsma RI, Abrunhosa AJ, Botelho MF, Hogendoorn PC, Pauwels E, Cleton-Jansen AM. Multidrug resistance mediated by ABC transporters in osteosarcoma cell lines: mRNA analysis and functional radiotracer studies. Nucl Med Biol 2006;33:831-40.
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797-806.
Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2:48-58.
Gottesman MM, Ling V. The molecular basis of multidrug resistance in cancer: the early years of P-glycoprotein research. FEBS Lett 2006;580:998-1009.
Hadnagy A, Gaboury L, Beaulieu R, Balicki D. SP analysis may be used to identify cancer stem cell populations. Exp Cell Res 2006;312:3701-10.
Hendrikse NH, Franssen EJ, van der Graaf WT, Vaalburg W, de Vries EG. Visualization of multidrug resistance in vivo. Eur J Nucl Med 1999;26:283-93.
Heppner GH. Tumor heterogeneity. Cancer Res 1984;44:2259-65.
Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415-28.
Lai EC, Choi TK, Cheng CH, Mok FP, Fan ST, Tan ES, Wong J. Doxorubicin for unresectable hepatocellular carcinoma. A prospective study on the addition of verapamil. Cancer 1990;66:1685-7.
Lau WY, Lai EC. Hepatocellular carcinoma: current management and recent advances. Hepatobiliary Pancreat Dis Int 2008;7:237-57.
Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, Lu L, Irvin D, Black KL, Yu JS. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 2006;5:67.
Liu ZL, Onda K, Tanaka S, Toma T, Hirano T, Oka K. Induction of multidrug resistance in MOLT-4 cells by anticancer agents is closely related to increased expression of functional P-glycoprotein and MDR1 mRNA. Cancer Chemother Pharmacol 2002;49:391-7.
Livraghi T. Guidelines for treatment of liver cancer. Eur J Ultrasound 2001;13:167-76.
Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3:330-8.
Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 2007;132:2542-56.
Ma S, Chan KW, Lee TK, Tang KH, Wo JY, Zheng BJ, Guan XY. Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol Cancer Res 2008a;6:1146-53.
Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY. CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene 2008b;27:1749-58.
Mansilla S, Rojas M, Bataller M, Priebe W, Portugal J. Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells. Biochem Pharmacol 2007;73:934-42.
Mao Q, Unadkat JD. Role of the breast cancer resistance protein (ABCG2) in drug transport. Aaps J 2005;7:E118-33.
Merino V, Jimenez-Torres NV, Merino-Sanjuan M. Relevance of multidrug resistance proteins on the clinical efficacy of cancer therapy. Curr Drug Deliv 2004;1:203-12.
Mizrak D, Brittan M, Alison MR. CD133: molecule of the moment. J Pathol 2008;214:3-9.
Oguri T, Bessho Y, Achiwa H, Ozasa H, Maeno K, Maeda H, Sato S, Ueda R. MRP8/ABCC11 directly confers resistance to 5-fluorouracil. Mol Cancer Ther 2007;6:122-7.
Pennock GD, Dalton WS, Roeske WR, Appleton CP, Mosley K, Plezia P, Miller TP, Salmon SE. Systemic toxic effects associated with high-dose verapamil infusion and chemotherapy administration. J Natl Cancer Inst 1991;83:105-10.
Petriz J, Garcia-Lopez J. Flow cytometric analysis of P-glycoprotein function using rhodamine 123. Leukemia 1997;11:1124-30.
Pine MB, Citron PD, Bailly DJ, Butman S, Plasencia GO, Landa DW, Wong RK. Verapamil versus placebo in relieving stable angina pectoris. Circulation 1982;65:17-22.
Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105-11.
Robey RW, Polgar O, Deeken J, To KW, Bates SE. ABCG2: determining its relevance in clinical drug resistance. Cancer Metastasis Rev 2007;26:39-57.
Sarkadi B, Ozvegy-Laczka C, Nemet K, Varadi A. ABCG2 -- a transporter for all seasons. FEBS Lett 2004;567:116-20.
Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507-12.
Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 2003;55:3-29.
Shmelkov SV, St Clair R, Lyden D, Rafii S. AC133/CD133/Prominin-1. Int J Biochem Cell Biol 2005;37:715-9.
Staud F, Pavek P. Breast cancer resistance protein (BCRP/ABCG2). Int J Biochem Cell Biol 2005;37:720-5.
Suetsugu A, Nagaki M, Aoki H, Motohashi T, Kunisada T, Moriwaki H. Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells. Biochem Biophys Res Commun 2006;351:820-4.
Takeda M, Mizokami A, Mamiya K, Li YQ, Zhang J, Keller ET, Namiki M. The establishment of two paclitaxel-resistant prostate cancer cell lines and the mechanisms of paclitaxel resistance with two cell lines. Prostate 2007;67:955-67.
Tomasz M. Mitomycin C: small, fast and deadly (but very selective). Chem Biol 1995;2:575-9.
Tsuruo T, Iida H, Tsukagoshi S, Sakurai Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res 1981;41:1967-72.
van de Water FM, Masereeuw R, Russel FG. Function and regulation of multidrug resistance proteins (MRPs) in the renal elimination of organic anions. Drug Metab Rev 2005;37:443-71.
Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008;8:755-68.
Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PW, Lam CT, Poon RT, Fan ST. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008;13:153-66.
Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J, Buck DW. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002-12.
Yin S, Li J, Hu C, Chen X, Yao M, Yan M, Jiang G, Ge C, Xie H, Wan D, Yang S, Zheng S, Gu J. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer 2007;120:1444-50.
Zen Y, Fujii T, Yoshikawa S, Takamura H, Tani T, Ohta T, Nakanuma Y. Histological and culture studies with respect to ABCG2 expression support the existence of a cancer cell hierarchy in human hepatocellular carcinoma. Am J Pathol 2007;170:1750-62.
Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028-34.