中文摘要
抗癌藥物之多重抗藥性(MDR)，是癌症化學治療上最主要失敗的主因。癌細胞會大量表現出P-glycoprotein (P-gp)，及多重相關性蛋白(MRPs)，而P-gp它主要是MDR1基因產物。這些蛋白通常會造成降低腫瘤細胞內藥物的累積以及增加腫瘤細胞內，藥物的排出。 P-gp及MRP是存在於細胞膜上的醣蛋白，他們是由ATP提供能量的藥物運輸器，能給與腫瘤細胞對於多種藥物之抗藥性。
在這次研究中，我們挑選了天然物純成份，他們廣泛存在蔬菜、水果、大豆、茶葉中。這次研究中我們試著評估這些天然純成份當作拮抗劑之可能性。實驗中，我們選用了epirubicin為當作模式抗癌藥物，它已經廣泛運用於治療多種不同的癌症，例如乳癌、卵巢癌、胃癌及腸癌。
細胞毒性分析已證實所使用的天然物純成份，在1~25 mM濃度範圍間是較無毒性的。 我們也將epirubicin與天然物純成份合用，並且利用flow cytometer，發現天然物純成份能顯著地增加epirubicin積存在Caco-2細胞內。而這些多重抗劑(modulators)可以顯著地抑制Caco-2細胞上之, MDR1、MRP1、MRP2、MRP3以及MRP4 mRNA的表現。此外，我們近一步評估天然物純成份對hMDR1基因序列上DNA element之效應，此基因能轉錄出MDR1。我們利用transient transfetion去分析，natural compounds是否能調控Caco-2 細胞上hMDR1之 promoter 區域。利用luciferase assay分析，這些天然物純成份發現可以顯著地抑制hMDR1 promoter的表現。
我們的結果顯示出這些調節劑，已被證實能增加epirubicin進入Caco-2細胞內，並且經由拮抗抗癌藥物之多重抗藥性，抑制MDR1、MRP1、MRP2、MRP3以及MRP4蛋白表現之方式。我們將抗癌藥物與天然物純成份合用，能加強癌症化學法之療效。這些不具有毒性的天然物純成份，應用於MDR之調控機轉上，可降低全身之傷害並且提供更有效的對同屬於多重藥型範圍之藥物之生體可用率。

Abstract

Multidrug resistance (MDR) is the major cause of failure of cancer chemotherapy. Over expression of P-glycoprotein (P-gp), the product of multidrug resistance gene (MDR1), and multidrug-resistance associated protein (MRPs) is often associated with enhanced drug efflux, resulting in decreased intracellular drug accumulation in the tumor cells. P-gp and MRPs are plasma membrane glycoproteins that confer multidrug resistance on cancer cells, by virtue of the be ability to these proteins to act ATP-driven drug-export pumps for excluding cytotoxic drugs.
In this study, we selected natural compounds, which are extracted from fruit, vegetables, soybeans and herbs, to test their possibility as multidrug resistance (MDR) antagonists. Epirubicin was selected as a model anticancer drug. It is used extensively in the treatment of a variety of cancers including breast, ovarian, stomach and bowel cancers.
The cytotoxicity assay demonstrated that these selected natural compounds were non-toxic in the test concentration range from 1 to 25 mM. Co-incubation of epirubicin with natural compounds resulted in an increase in the cytotoxicity of epirubicin. These compounds were shown to significantly enhance the intracellular accumulation of epirubicin in Caco-2 cells, as demonstrated by the flow cytometric study. These modulators were found to markedly inhibit MDR1, MRP1, MRP2, MRP3, and MRP4 mRNA levels in Caco-2 cells. Furthermore, regulation of the human multidrug resistance gene (hMDR1) was studied by mapping DNA element in the proximal promoter necessary for efficient transcription . The effect of natural compounds on the transient transfection analysis in the regulatory promoter region of Caco-2 cells was then examined. These compounds were found to suppress hMDR1 promoter expression using the luciferase assay in Caco-2 cells.
In conclusion, these modulators were demonstrated to increase the intracellular uptake of epirubicin and antagonize MDR in Caco-2 cells, through their inhibitory effects on intestinal MDR1, MRP1, MRP2, MRP3, and MRP4. The combined used of anticancer drugs with these natural compounds may have significant implications in circumventing MDR of cancer chemotherapy. As MDR modulators in drug formulations, such non-toxic natural compounds may reduce systemic side-effects and improve oral bioavailability of drugs in the MDR spectrum.

目錄
中文摘要……………………………………………………………………Ⅰ-Ⅱ
英文摘要……………………………………………………………………Ⅲ-Ⅳ
目錄…………………………………………………………………………….Ⅴ
圖目錄……………………………………………………………………..IX-XI
縮寫表………………………………………………………………….XII-XIII
第一章 緒論…………………………………………………………………0-1
第二章 抗癌藥物簡介……………………………………………………….2-5
◎ Epirubicin………………………………………………………………….2
◎ Anthracyclines……………………………………………………………..2
◎ Vinblastine………………………………………………………………….3
◎ Epipodophyllotoxins………………………………………………………..3
◎ Cispatin……………………………………………………………………...3
◎ Methotrexate………………………………………………………………..3
◎ Paclitaxel……………………………………………………………………4
◎ Cyclophospamide…………………………………………………………..4
◎ Antibotics……………………………………………………………………4
◎ Cyclosporine A……………………………………………………………...5
◎ Antineoplastic agents………………………………………………………5
第三章 Flavonoids各論………………………………………………………6
第四章 細胞抗藥性機轉………………………………………………………7
第五章 各種藥物運輸蛋白文獻之整理……………………………………8-12
1. MDR1 (multi-drug resistance)……………………………………………...8
2. MRP1 (multidrug -resistance associated protein 1)……………………9-10
3. MRP2 (multidrug -resistance associated protein 2)……………………...10
4. MRP3 (multidrug -resistance associated protein 3)……………………...11
5. MRP4 (multidrug -resistance associated protein 2)……………………...12
第四章 研究目的……………………………………………………………...13
第五章 實驗材料與方法………………………………………………….14-29
1-1. 儀器………………………………………………………………………14
1-2. 化學藥品及試劑……………………………………………………...15-16
1-3. 套組試劑………………………………………………………………….17
2-1. 細胞株…………………………………………………………………….18
2-2. 細胞培養………………………………………………………………….19
2-3. 細胞存活率試驗………………………………………………………….20
2-4. 流式細胞技術儀………………………………………………………….21
2-5. RNA抽取………………………………………………………………….22
2-6. RNA電泳………………………………………………………………….22
2-7. RT-PCR………………………………………………………………22-23
2-8. Cloning……………………………………………………………….24-29
2-8-1. Genomic DNA Extraction……………………………………………24
2-8-2. PCR…………………………………………………………………….25
2-8-3. DNA purification………………………………………………………26
2-8-4. Purification restriction of DNA and vector…………………………..27
2-8-5. Ligation…………………………………………………………………27
2-8-6. E coli處理………………………………………………………………27
2-8-7. Transformation………………………………………………………...28
2-8-8. Mini-plate of plasmid抽取…………………………………………….28
2-8-9. Maxi-plate of plasmid抽取………………………………………...28-29
2-8-10. Transfection…………………………………………………………..29
第六章 結果……………………………………………………………….30-37
1. Luteolin, Myricetin, Modulator C, Modulator D, Modulator E, Modulator F, Modulator G, Modulator H, Modulator I, Modulator J細胞毒性之評估………………………………………………………………………30
2. Luteolin, Myricetin, Modulator C, Modulator D, Modulator E, Modulator F, Modulator G, Modulator H, Modulator I, Modulator J與抗癌藥物Epirubicin合用能否增加毒殺癌細胞能力……………………………31
3. Luteolin, Myricetin, Modulator C, Modulator D, Modulator E, Modulator F, Modulator G, Modulator H, Modulator I, Modulator J與抗癌藥物Epirubicin合用能否增加抗癌藥物進入癌細胞的能力……………32-33
4. Luteolin, Myricetin, Modulator C, Modulator D, Modulator E, Modulator F, Modulator G, Modulator H, Modulator I, Modulator J對多種藥物運輸蛋白之mRNA表現的影響…………………………………………34
5. Luteolin, Myricetin, Modulator C, Modulator D, Modulator E, Modulator F, Modulator G, Modulator H, Modulator I, Modulator J與Epirubicin合用是否能加強抑制多種藥物運輸蛋白基因的表現……….35-36
6. Luteolin, Myricetin, Modulator C, Modulator D, Modulator E, Modulator F, Modulator G, Modulator H, Modulator I, Modulator J是否能影響MDR1 promoter的表現………………………………………………...37
第七章 討論………………………………………………………………..38-40
參考文獻……………………………………………………………………75-79





圖目錄
Fig 1. Structure of flavonoids………………………………………………...41
Fig 2A. Effect of Luteolin on the cell viability of Caco-2 cells ……………42
Fig 2B.Effect of Myricetin on the cell viability of Caco-2 cells ……………43
Fig 2C. Effect of Modulator C on the cell viability of Caco-2 cells ……….44
Fig 2D. Effect of Modulator D on the cell viability of Caco-2 cells ……….45
Fig 3A. Effect of Luteolin on Epirubicin –induced Cytotoxicity…………..46
Fig 3B. Effect of Myricetin on Epirubicin –induced Cytotoxicity…………47
Fig 3C. Effect of Modulator C on Epirubicin –induced Cytotoxicity……..48
Fig 3D. Effect of Modulator D on Epirubicin –induced Cytotoxicity……..49
Fig 4A. Intracellular Accumulation of Epirubicin…………………………50
Fig 4B. Intracellular Accumulation of Epirubicin…………………………51
Fig 4C. Intracellular Accumulation of Epirubicin…………………………52
Fig 5A. RT-PCR assay of Modulators on mRNA expression of MDR1……53
Fig 5B. RT-PCR assay of Modulators on mRNA expression of MDR1……54
Fig 5C. RT-PCR assay of Modulators on mRNA expression of MDR1……55
Fig 5D. RT-PCR assay of Modulators on mRNA expression of MRP1……56
Fig 5E. RT-PCR assay of Modulators on mRNA expression of MRP1……57
Fig 5F. RT-PCR assay of Modulators on mRNA expression of MRP2……58
Fig 5I. RT-PCR assay of Modulators on mRNA expression of MRP2……59
Fig 5J. RT-PCR assay of Modulators on mRNA expression of MRP3……60
Fig 5K. RT-PCR assay of Modulators on mRNA expression of MRP3……61
Fig 5L. RT-PCR assay of Modulators on mRNA expression of MRP4……62
Fig 5M. RT-PCR assay of Modulators on mRNA expression of MRP4…...63
Fig 6A. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MDR1………………………………………………………….64
Fig 6B. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MDR1………………………………………………………….65
Fig 6C. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP1………………………………………………………….66
Fig 6D. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP1………………………………………………………….67
Fig 6E. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP2………………………………………………………….68
Fig 6F. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP2………………………………………………………….69
Fig 6G. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP3………………………………………………………….70
Fig 6G. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP3………………………………………………………….71
Fig 6I. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP4………………………………………………………….72
Fig 6J. RT-PCR assay of Modulators combine Epirubicin on mRNA expression of MRP4………………………………………………………….73
Fig 7 Effect of Modulators on hMDR1 promoter activity in Caco-2 cells…74

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