臺灣博碩士論文加值系統

癌症是國人十大死亡原因之一，而造成目前癌症治療上困難的最主要原因之一，即是癌細胞對許多抗癌藥物產生抗藥性，以致藥物無法累積達到足以殺死或抑制其生長的有效濃度。而主要原因之一，即是癌細胞中多重抗藥性蛋白過度表現所造成的。主要有P-glycoprotein,以及 multidrug resistance-associated protein (MRP) family兩大類。
對多重抗藥性蛋白而言，特別是multidrug resistance-associated protein 1 (MRP1) 在多數研究中顯示其細胞排除活性與臨床治療結果具有相關性。MRP1除參與一部分生理功能外，最主要是能減少抗癌藥物在細胞內的蓄積。因此，若能有效地調控MRP1的功能，不僅可以增加藥物在細胞內的蓄積量，增加抗癌藥物的療效，也可降低個體間藥物反應性的差異。發展具有調控MRP1功能的物質，以降低癌細胞對癌症藥物的多重抗藥性，可當作抗癌藥物治療的研究發展新方向之一。
本研究目的即在進行MRP1調控劑的開發與研究，主要是先利用HL-60/ADR細胞株為體外篩選模式輔以流式細胞儀分析，進行83種中藥藥引對MRP1作用影響之研究。本研究已完成各項工作，在體外篩選模式之建立與確效方面，包括模式藥物carboxyfluorescein適當細胞累積的時間及溫度、細胞穩定的排除活性時間決定點、藥物水解的完整度、MRP1活性的強弱的動力學特徵及陽性抑制劑不同濃度的抑制率等影因素評估。體外篩選結果顯示，具MRP1調控作用的中藥藥引以HUCHE 80、65、70、09、77、03、26、68、61可以增加細胞carboxyfluorescein蓄積為20～46倍 (P＜0.001)；HUCHE 81、10、71、79、34、02可以增加細胞carboxyfluorescein蓄積為11～19倍 (P＜0.001)；HUCHE 67、14、35、42、74、66、53、01、37、11、78皆可顯著地增加細胞carboxyfluorescein蓄積為4～8倍 (P＜0.05)。為進一步驗證上述體外有效成分是否同樣可在體內抑制MRP1功能，本研究先經由分離大白鼠血液中具有顯著表現rMRP1的紅血球進行離體模式建立與篩選。體內模式藥物為carboxyfluorescein diacetate (CFDA)，將上述仍具顯著調控效果的成分 (HUCHE 61、03、68) 合併模式藥物靜脈注射投與動物進行體內試驗。結果顯示HUCHE 61僅可使血球中CFDA吸收量增加為1.8倍 (P＜0.05)，但其他相關的藥動學參數皆無統計學上顯著的差異，表示抑制效果不佳，另兩種成分亦無任何顯著的抑制作用。究其原因可能為該藥引成份在體內所投予的劑量未能達到所需要的抑制濃度或親合度不佳等，但仍明顯地改善了動物個體間藥動學參數 (AUC, MRT等) 變異之程度，相關應用價值及其它具有調控潛力的藥引成份仍待進一步的探討。

Resistance against chemotherapeutic regimen has hampered the treatment of malignancies. Several mechanisms of cellular resistance have been incriminated. A major one results from reduced intracellular accumulation of structurally unrelated anticancer drugs and usually involves over expression of drug efflux pumps. P-glycoprotein (P-gp) is the first identified of these transporters. More recently, export pumps belonging to the multidrug resistance protein (MRP) subfamily, such as MRP1 and MRP2, have also been demonstrated to confer multidrug resistance on tumor.
The expression and function of the MRP subfamily might impact on the rate and extent of efflux of the substrates for the MRP subfamily. Elevated expression of P-gp, MRP have been reported in many cancers and has been shown to be associated with poor treatment response, indicating that P-gp and MRP are probably a predictive factor for the outcome of treatment of malignancies. The regulation of the activity of MRP may be a feasible direction to improve the treatment of malignancies with multidrug resistance.
The purpose of this study is to develop the MRP1 modulators. A simple and reliable in vitro screening method was setup and characterized using over expressed MRP1 HL60/ADR cell lines to study the effect of Chinese herbal enhancers (CHEs) on MRP1-mediated transport of a model substrate. A specific MRP1 substrate carboxyfluorescein (CF) was used as a fluorescent marker after hydrolysis from CF diacetate (CFDA) in the cell. The functional activity of MRP1 was evaluated by measuring CF retention/efflux in the presence of CHEs and MK517 used as a positive control. Intracellular accumulation of CF was measured by flow cytometry. The validation of the screening platform included accumulation kinetic under different temperatures, efflux kinetics, generation of the cell on efflux activity, and hydrolysis ability of CFDA in the cells. 83 pure constituents in CHEs has been completed up-to-now. The results indicated that the efflux of MRP1 was inhibited by some of the CHEs. Intracellular retention of CF increased 20-46 fold with No. 80, 65, 70, 09, 77, 03, 26, 68, and 61 CHEs at 100 M as compared with control (p＜0.001). As such as the same, increased 10~19 fold with No. 81, 10, 71, 79, 34, 02 (p＜0.001). Based on the in vitro screening studies has been done, top 20 CHEs would be further evaluated to study affinity. Function evaluation of rMRP1 on S.D. rat red blood cells in the present of CHEs have be conducted in vitro first. The effect of potent CHEs on the modulation of MRP1 function in vivo in rats have be further carried out by use IV administration of CHEs (HUCHE 61, 03, 68) combined with CFDA. The result show that only HUCHE 61 make the amount of CFDA uptake on RBCs increased 1.8 fold (p＜0.05), but not change with other pharmacokinetic parameters. Although it doesn’t successfully demonstrate the significant result, it may improve the individual variation of drug kinetic profile. It is still worth to develop these pure constituents in futher study.

正 文 目 錄

頁次
第一章、緒論…………….……………………………………………. 1
第一節、研究背景…….……………………………………..…. 1
第二節、多重抗藥性緣起…….…………………………….….. 4
第三節、MRP family的種類……..………………………….…. 8
第四節、MRP1的結構及其分佈…………….……………….... 13
第五節、MRP1的受質及其作用機轉………………….……… 17
第六節、MRP1的生理意義…………………….……………… 23
第七節、MRP1的臨床意義…...….…………………………… 23
第八節、MRP1抑制劑的介紹.………………………………… 26
第九節、Flavonoids與中藥藥引之簡介…...….……………… 30
第十節、MRP1模式藥物Carboxyfluorescein diacetate(CFDA) 38
第十一節、研究目的…………………………………………… 40

第二章、試劑與儀器…….……………………………………………. 42
第一節、藥品與試劑….……………………………………..…. 42
第二節、細胞株………….………………………………….….. 48
第三節、儀器…………………….………………………….…. 49
第三章、實驗方法…….………………………………………………. 51
體外篩選實驗方法
第一節、HL60/ADR及HL60細胞株…………………...…..…. 51
第二節、細胞培養基配製.………………………………….….. 51
第三節、細胞繼代培養…….………………………….……….. 53
第四節、細胞計數及存活測試………………………………… 53
第五節、Carboxyfluorescein diacetate (CFDA)於細胞內累積
相(Accumulation phase)動力學實驗…………........... 54
第六節、Carboxyfluorescein細胞排出實驗 (Efflux study)…... 56
第七節、HL60/ADR上p-gp活性的測試……………………... 59
第八節、HL60/ADR具有過量MRP1表現之試驗………..…... 60
第九節、體外篩選中藥藥引模式之建立……………………… 61
第十節、評估低濃度中草藥純成分調控效果………………… 62
第十一節、細胞毒性逆轉試驗 (Chemosensitize test by use
MTT assay)……..…………………………………. 65
離體動物實驗方法
第十二節、體外大白鼠紅血球上rMRP1功能測定………..…. 67
體內動物實驗方法
第十三節、動物……...……………………………..…….…….. 70
第十四節、S.D. rat手術…....…………………………….…….. 70
第十五節、體內模式藥物……………………………………… 71
第十六節、動物口服給藥實驗………………………………… 71
第十七節、Carboxyfluorescein的分析方法…………………… 72
1. 分析條件……………………………………….. 72
2. 檢品處理………………………………………. 72
3. 校正曲線製作………………………………….. 72
4. 數據處理及統計方法………………………….. 73
第四章、結果與討論…….……………………………………………. 75
第一節、體外篩選實驗 (體外MRP1調控劑篩選模式的建立) 75
第二節、中藥藥引體外篩選結果……………………………… 99
第三節、細胞毒性逆轉試驗…………………………………… 120
第四節、動物離体試驗………………………………………… 124
第五節、動物體內試驗………………………………………… 129
第五章、結論…….……………………………………………………. 143
第六章、參考文獻…….………………………………………………. 145
附錄一……………………………………………………………….... 154


表 目 錄

頁次
表1-1 MRP1家族各成員組成特性簡介…………………………….. 10
表1-2 MRP家族各成員功能特性簡介………………………………. 11
表1-3 MRP1運輸的受質種類………………...…...…...…......…..…. 20
表1-4 常見藥物或物質其運輸機制與GSH之關係……...…………. 22
表1-5 MRP1及相關MRP蛋白抑制劑種類…………………………. 29
表1-6 中藥藥引來源簡介…………………………………….............. 35
表4-1 體外模式篩選83種中藥藥引抑制MRP1功能之排序……… 104
表4-2 體外篩選方式不同濃度下之HUCHE 77對調控MRP1作用結果…………………………………………………………….. 107
表4-3 體外篩選方式不同濃度下之HUCHE 80對調控MRP1作用結果…………………………………………………………….. 108
表4-4 體外篩選方式不同濃度下之HUCHE 65 , 70對調控MRP1作用結果……………………………………………………….. 109
表4-5 體外篩選方式不同濃度下之HUCHE 3 , 9對調控MRP1作用結果………………………………………………………….. 110
表4-6 體外篩選方式不同濃度下之HUCHE 26 , 68對調控MRP1作用結果………………………………………….……………. 111
表4-7 體外篩選方式不同濃度下之HUCHE 61 , 81對調控MRP1作用結果………………………………………………………..
112
表4-8 體外篩選方式不同濃度下之HUCHE 10 , 71對調控MRP1作用結果……………………………………………………….. 113
表4-9 體外篩選方式不同濃度下之HUCHE 79 , 34對調控MRP1作用結果……………………………………………………….. 114
表4-10 體外篩選方式不同濃度下之HUCHE 2 , 14對調控MRP1作用結果……………………………………………………….. 115
表4-11 體外篩選方式不同濃度下之HUCHE 35 , 42對調控MRP1作用結果……………………………………………………….. 116
表4-12 體外篩選方式不同濃度下之HUCHE 74 , 11對調控MRP1作用結果……………………………………………………….. 117
表4-13 中藥藥引體外篩選模式中各項參數之變異程度…………….. 119
表4-14 抑制劑對老鼠紅血球排除carboxyfluorescein程度之影響….. 126
表4-15 中藥藥引 (高濃度) 對老鼠紅血球排除carboxyfluorescein程 度之影響……………………………………………………….. 127
表4-16 中藥藥引 (低濃度) 對老鼠紅血球排除carboxyfluorescein程 度之影響……………………………………………………….. 128
表4-17 Carboxyfluorescein於老鼠紅血球中同日間各標準濃度線性迴歸後之精密度及準確度試驗結果………………………….. 134
表4-18 Carboxyfluorescein於老鼠紅血球中同日間確效之精密度及
準確度試驗結果……………………………………………….. 135
表4-19 實驗組S.D. rat頸靜脈投予中藥藥引HUCHE 61 (~3.7 mg/rat)及CFDA 0.29 mg/kg，與控制組 (給PEG400 0.35 ml) 血球中藥動學參數之比較…………………………………….. 137
表4-20 實驗組S.D. rat頸靜脈投予中藥藥引HUCHE 03 (~3.6 mg/rat) 及CFDA 0.29 mg/kg，與控制組 (給PEG400 0.35 ml) 血球中之藥動學參數比較 (mean±SD, n=3)…………………. 139
表4-21 實驗組S.D. rat頸靜脈投予中藥藥引HUCHE 68 (~3.5 mg/rat) 及CFDA 0.29 mg/kg，與控制組(給PEG400 0.35 ml)血球中之藥動學參數作比較 (mean±SD, n=3)………………. 141
表4-22 上圖中各藥動學參數對照表 (mean±SD, n=3, 然陽性控制組n=2)…………………………………………………………. 142

圖 目 錄

頁次
圖1-1 腸道上皮細胞形成藥物進入障壁的示意圖..……………….... 3
圖1-2 癌細胞獲得後天性抗藥性的機制..…………………………… 7
圖1-3 肝臟細胞排除膽鹽及相關結合態藥物的示意圖…………….. 12
圖1-4ab MRP1之結構示意圖..………………………………………… 15
圖1-5 組織細胞上ATP Binding Cassette (ABC)運送物質的方向性…………………………………….......................................... 16
圖1-6 MRP1受質完整代謝途徑與GS-X pump示意圖……………. 21
圖1-7 GSSG細胞內氧化還原平衡示意圖…………………………... 21
圖1-8 Flavonoids 基本結構………………………………………….. 34
圖1-9 Carboxyfluorescein diacetate細胞內水解示意圖….…………. 39
圖3-1 體外篩選MRP1調控劑步驟….……………………………… 64
圖3-2 標準曲線之各濃度的配製方法……………………………….. 74
圖4-1 不同溫度下CFDA 0.5 M於細胞內累積之動力學趨勢……. 84
圖4-2 不同細胞代數間CFDA 0.5 M於冰浴溫度下累積相之趨勢 85
圖4-3
冰浴下累積CFDA後經流式細胞儀測定之細胞螢光強度分佈……………………………………………………………….. 86
圖4-4
常溫下累積CFDA後經流式細胞儀測定之細胞螢光強度分佈……………………………………………………………….
86
圖4-5 MK571於37℃下影響累積相細胞螢光強度分布………… 87
圖4-6 於不同實驗條件下細胞累積60分鐘後之螢光強度分佈…… 87
圖4-7 不同細胞代數下MRP1參與的carboxyfluorescein排除動力學趨勢………………………………………………………….. 88
圖4-8 細胞排除相經時變化螢光強度分佈圖 (control)…………….. 89
圖4-9 細胞排除相經時變化螢光強度分佈圖 (positive control)…… 89
圖4-10 冰浴累積CFDA 30 min飽合後，具或不具有抗藥性之細胞及抑制劑對carboxyfluorescein排除趨勢之影響……………
90
圖4-11 冰浴累積CFDA 30 min 飽合後，給予不同MRP1抑制劑下
細胞排除carboxyfluorescein動力學之趨勢…………………. 91
圖4-12 冰浴累積CFDA 30 min飽合後，不同低溫度下細胞排除carboxyfluorescein動力學之趨勢…………………………….. 92
圖4-13 冰浴累積CFDA 30 min飽合後，預先培養於冰浴20分鐘後 再進行排除相之細胞排出動力學趨勢 (含MK571 25 M)… 93
圖4-14 HL60/ADR細胞株上測定P-gp功能之試驗…………………. 94
圖4-15 細胞株上MRP1蛋白表現程度……………………………….. 95
圖4-16 MK571以所建立的篩選模式進行試驗，於不同濃度下對細胞內carboxyfluorescein滯留的影響…………………………. 96
圖4-17 Probenecid以所建立的篩選模式進行試驗，於不同濃度下對細胞內carboxyfluorescein滯留的影響………………………. 97
圖4-18 Indomethacin以所建立的篩選模式進行試驗，於不同濃度下對細胞內carboxyfluorescein滯留的影響…………………….
98
圖4-19 體外篩選方式不同濃度下之HUCHE 77對調控MRP1作用之曲線圖………………………………………………………. 107
圖4-20 體外篩選方式不同濃度下之HUCHE 80對調控MRP1作用之曲線圖………………………………………………………. 108
圖4-21 體外篩選方式不同濃度下之HUCHE 65 , 70對調控MRP1作用之曲線圖…………………………………………………. 109
圖4-22 體外篩選方式不同濃度下之HUCHE 3 , 9對調控MRP1作用之曲線圖……………………………………………………. 110
圖4-23 體外篩選方式不同濃度下之HUCHE 26 , 68對調控MRP1作用之曲線圖…………………………………………………. 111
圖4-24 體外篩選方式不同濃度下之HUCHE 61 , 81對調控MRP1作用之曲線圖…………………………………………………. 112
圖4-25 體外篩選方式不同濃度下之HUCHE 10 , 71對調控MRP1作用之曲線圖…………………………….……………………. 113
圖4-26 體外篩選方式不同濃度下之HUCHE 79 , 34對調控MRP1作用之曲線圖…………………………………………………. 114
圖4-27 體外篩選方式不同濃度下之HUCHE 2 , 14對調控MRP1作用之曲線圖……………………………………………………. 115
圖4-28 體外篩選方式不同濃度下之HUCHE 35 , 42對調控MRP1作用之曲線圖…………………………………………………. 116
圖4-29 體外篩選方式不同濃度下之HUCHE 74 , 11對調控MRP1作用之曲線圖…………………………………………………. 117
圖4-30 本實驗室所篩選出P-gp的抑制劑和MRP1功能抑制的關係 118
圖4-31 細胞濃度和formazan生成量的標準曲線 (反應1小時)........ 122
圖4-32 細胞濃度和formazan生成量的標準曲線 (反應四小時)........ 122
圖4-33 中藥藥引對癌細胞株逆轉抗藥性的試驗.................................. 123
圖4-34 抑制劑對老鼠紅血球排除carboxyfluorescein趨勢之影響…. 126
圖4-35 中藥藥引(高濃度)對老鼠紅血球排除carboxyfluorescein趨勢之影響………………………………………………………….. 127
圖4-36 中藥藥引(低濃度)對老鼠紅血球排除carboxyfluorescein趨勢
之影響………………………………………………………….. 128
圖4-37 Carboxyfluorescein於老鼠紅血球中同日間之標準曲線……. 134
圖4-38 實驗組S.D. rat頸靜脈投予中藥藥引HUCHE 61 (~3.7 mg/rat) 及CFDA 0.29 mg/kg，與控制組 (給PEG400 0.35 ml) 之血球中濃度比較 (mean±SD, n=3)………………………… 136
圖4-39 實驗組S.D. rat頸靜脈投予中藥藥引HUCHE 3 (~3.6 mg/rat) 及CFDA 0.29 mg/kg，與控制組 (給PEG400 0.35 ml) 之血球中濃度比較 (mean±SD, n=3)……………………………… 138
圖4-40 實驗組S.D. rat頸靜脈投予中藥藥引HUCHE 68 (~3.5 mg/rat) 及CFDA 0.29 mg/kg，與控制組 (給PEG400 0.35 ml) 之血球中濃度比較 (mean±SD, n=3)………………………… 140
圖4-41 實驗組S.D. rat腹腔注射投予probenecid (400 mg/kg) 及頸靜脈注射CFDA 0.29 mg/kg，與控制組 (0.5 M HEPES buffer 0.9 ml ) 之血球中濃度比較 (mean±SD, n=3, 然陽性控制組n=2)…………………………………………………………….. 142
圖附錄一~一 體外篩選方式100μM HUCHE 01～05對調控MRP1作用之比較圖…………………………………………………………. 154
圖附錄
一~二 體外篩選方式100μM HUCHE 06～10對調控MRP1作用之比較圖…………………………………………………………. 155
圖附錄
一~三 體外篩選方式100μM HUCHE 11～15對調控MRP1作用之比較圖…………………………………………………………. 156
圖附錄
一~四 體外篩選方式100μM HUCHE 16～20對調控MRP1作用之比較圖…………………………………………………………. 157
圖附錄
一~五 體外篩選方式100μM HUCHE21～25對調控MRP1作用之比較圖…………………………………………………………. 158
圖附錄
一~六 體外篩選方式100μM HUCHE26～30對調控MRP1作用之比較圖…………………………………………………………. 159
圖附錄
一~七 體外篩選方式100μM HUCHE 31～35對調控MRP1作用之比較圖…………………………………………………………. 160
圖附錄
一~八 體外篩選方式100μM HUCHE 36～40對調控MRP1作用之比較圖…………………………………………………………. 161
圖附錄
一~九 體外篩選方式100μM HUCHE 41～45對調控MRP1作用之比較圖…………………………………………………………. 162
圖附錄
一~十 體外篩選方式100μM HUCHE 46～50對調控MRP1作用之比較圖…………………………………………………………. 163
圖附錄
一~十一 體外篩選方式100μM HUCHE 51～55對調控MRP1作用之比較圖…………………………………………………………. 164
圖附錄
一~十二 體外篩選方式100μM HUCHE56～60對調控MRP1作用之比較圖…………………………………………………………. 165
圖附錄
一~十三 體外篩選方式100μM HUCHE 61～65對調控MRP1作用之比較圖…………………………………………………………. 166
圖附錄
一~十四 體外篩選方式100μM HUCHE 66～70對調控MRP1作用之比較圖…………………………………………………………. 167
圖附錄
一~十五 體外篩選方式100μM HUCHE 71～75對調控MRP1作用之比較圖…………………………………………………………. 168
圖附錄
一~十六 體外篩選方式100μM HUCHE 76～80對調控MRP1作用之比較圖…………………………………………………………. 169
圖附錄
一~十七 體外篩選方式100μM HUCHE 81～83對調控MRP1作用之比較圖…………………………………………………………. 170

Alqawi O, Georges E. The multidrug resistance protein ABCC1 drug- binding domains show selective sensitivity to mild detergents Biochem Biophys Res Commun. 303: 1135–1141, 2003.

Ballatori N, Hammond CL, Cunningham JB, Krance SM, Marchan R. Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins. Toxicol Appl Pharmacol. 204: 238– 255, 2005.

Bhalla K, Hindenburg A, Taub RN, Grant S. Isolation and characteri- zation of an anthracycline-resistance human leukemic cell line. Can Res. 45: 3657-3662, 1985.

Bogman K, Erne-Brand F, Alsenz J, Drewe J. The role of surfactants in the reversal of active transport mediated by multidrug resistance proteins. J Pharm Sci. 92: 1250-1261, 2003.

Borst P, Evers R, Kool M, Wijnholds J. The multidrug resistance protein family. Biochim Biophys Acta. 1461: 347–357, 1999.

Borst P, Evers R, Kool M, Wijnholds J. A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst. 92: 1295 –1302, 2000.

Boumendjel A, Pietro AD, Dumontet C, and Barron D. Recent advances in the discovery of flavonoids and analogs with high-affinity binding to P-glycoprotein responsible for cancer cell multidrug resistance. Med Res Rev. 22: 512-529, 2002.

Chan LMS, Lowes S, Hirst BH. The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. Eur J Pharm Sci. 21: 25–51, 2004.

Chuman Y, Chen ZS, Seto K, Sumizawa T, Furukawa T, Tani A, Haraguchi M, Niwa K, Yamada K, Aikou T, Akiyama S. Reversal of MRP-mediated vincristine resistance in KB cells by buthionine sulfoximine in combination with PAK-104P. Cancer Lett. 129: 69–76, 1998.

Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV, Deeley RG. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Sci. 258: 1650–1654, 1992.

Cole SPC. Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother Pharmacol. 17: 259-263, 1986.

Cole SPC, Deelay RG Multidrug resistance-associated protein: sequence correction. Sci. 260: 879, 1993.

Cui Y, Konig J, Buchholz JK, Spring H, Leier I, and Keppler D. Drug-resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol. 55: 929–937, 1999.
David FVL, 57 varieties: the human cytochromes P450. Pharmacogenomics. 5:305–318, 2004.
David RH, Roger GD, Cole SPC. Structural, mechanistic and clinical aspects of MRP1. Biochimica et Biophysica Acta 1461: 359-376, 1999.

Dogan L, Legrand O, Faussat AM, Perrot JY, Marie JP. Evaluation and comparison of MRP1 activity with three fluorescent dyes and three modulators in leukemic cell lines, Leu Res. 28: 619–622, 2004.

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 USA. 95: 15665-15670, 1998.

Draper MP, Martell RL, Levy SB. Indomethacin-mediated reversal of multidrug resistance and drug efflux in human and murine cell lines overexpressing MRP, but not P-glycoprotein. Br J Cancer. 75: 810–815, 1997.

Efferth T, Davey M, Olbrich A, Rucker G, Gebhart E, and Davey R. Activity of Drugs from Traditional Chinese Medicine toward Sensitive and MDR1- or MRP1- Overexpressing Multidrug-Resistant Human CCRF-CEM Leukemia Cells. Blood Cells Mol Dis. 28: 160–168, 2002.

Gekeler V, Ise W, Sanders KH, Ulrich WR, Beck J. The leukotriene LTD4 recept- or antagonist MK571 specifically modulates MRP associated multidrug resistance. Biochem Biophys Res Commun. 208: 345–352, 1995.

Gollapudi S, Kim CH, Tran BN, Sangha S, Gupta S. Probenecid reverses multidrug resistance in resistance-associated protein overexpressing HL60/AR and H69/AR cells but not in P-glycoprotein-overexpressing HL60/Tax and P388/ADR cells. Cancer Chemother Pharmacol. 40: 150–155, 1997.

Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer. 2: 48-58, 2002.

Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole SPC, Deeley RG. Overexpression of multidrug resistance-associated protein (MRP) increase resistance to natural product drugs. Cancer Res. 54: 357–361, 1994.

Hart SM, Ganeshaguru K, Hoffbrand AV, Prentice HG, Mehta AB. Expression of the multidrug resistance-associated protein (MRP) in acute leukaemia. Leukemia 8: 2163–2168, 1994.

Haimeur A, Conseil G, Deeley RG, Cole SPC. The MRP-Related and BCRP/ABCG2 Multidrug Resistance Proteins: Biology, Substrate Specificity and Regulation. Curr Drug Metab. 5: 21-53, 2004.

Hipfner DR, Deeley RG, Cole SPC. Structural, mechanistic and clinical aspects of MRP1. Biochim Biophys Acta. 1461: 359–376, 1999.

Hugger ED, Audus KL, Borchardt RT. Effects of polyethylene glycol on efflux transporter activity in Caco-2 cell monolayers. J Pharm Sci. 91: 1980–1990, 2002.

Hu O.Y., Acyclovir Transdermal Drug Delivery Systems, U.S. Patent: 6,162,459, 2000.

Jedlitschky G, Burchell B, and Keppler D. The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. J Biol Chem. 275: 30069–30074, 2000.

Jedlitschky G, Leier I, Buchholz U, Barnouin K, Kurz G, Keppler D. Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump. Cancer Res. 56: 988–994, 1996.

Kartenbeck J., Leuschner U, Mayer R, Keppler D. Absence of the canalicul- ar isoform of the MRP geneencoded conjugate export pump from the hepatocytes in Dubin–Johnson syndrome. Hepatology. 23: 1061–1066, 1996.

Faber KN, Muller M, Jansen PL. Drug transport proteins in the liver. Adv Drug Deliv Rev. 55: 107–124, 2003.

Klokouzas A, Barrand MA, Hladky SB. Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells. Eur J Biochem. 268: 6569–6577, 2001.

Klokouzas A, Wu CP, van Veen HW, Barrand MA, Hladky SB. cGMP and glutathione-conjugate transport in human erythrocytes. The roles of the multidrug resistance-associated proteins, MRP1, MRP4 and MRP5. Eur J Biochem. 270: 3696–3708, 2003.

Konig J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta. 1461: 377– 394, 1999.

Kool M, de Haas M, Scheffer GL, Scheper RJ, van Eijk MJ, Juijn JA, Baas F, Borst P. Analysis of expression of cMOAT (MRP2), MRP3,MRP4, and MRP5, homologues of the multidrug resistance- associated protein gene (MRP1), in human cancer cell lines. Cancer Res. 57: 3537–3547, 1997.

Kool M, van der Linden M, de Haas M, Scheffer GL, de Vree JM, Smith AJ, Jansen G, Peters GJ, Ponne N, Scheper RJ, Elferink RP, Baas F, Borst P. MRP3, an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci USA. 96: 6914–6919, 1999.

Kruh GD, Belinsky1 MG. The MRP family of drug efflux pumps. Oncogene. 22: 7537–7552, 2003.

Lai L, and Tan TM. Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues. Biochem J. 361: 497–503, 2002.

Laupeze B, Amiot L, Payen L, Drenou B, Grosset JM, Lehne G, Fauchet R, Fardel O. Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood. Life Sci. 68:1323–1331, 2001.

Laupeze B, Amiot L, Drenou B, Bernard M, Branger B, Grosset JM, Lamy T, Fauchet R. High multidrug resistance protein activity in acute myeloid leukaemia is associated with poor response to chemotherapy and reduced patient survival. Br J Haematol. 116: 834–838, 2002.

Legrand O, Simonin G, Beauchamp-Nicoud A, Zittoun R, Marie JP. Simultaneous activity of MRP1 and Pgp is correlated with in vitro resistance to daunorubicin and with in vivo resistance in adult acute myeloid leukemia. Blood 94: 1046–1056, 1999.

Legrand O, Simonin G, Perrot JY, Zittoun R, Marie JP. Pgp and MRP activities using calcein-AM are prognostic factors in adult acute myeloid leukemia patients. Blood 91: 4480–4488, 1998.

Legrand O, Zittoun R, Marie JP. Role of MRP1 in multidrug resistance in acute myeloid leukemia. Leukemia 13: 578–584, 1999.

Leier I, Jedlitschky G, Buchholz U, Cole SPC, Deeley RG, Keppler D. The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 25: 27807–27810, 1994.

Leslie EM, Roger GD, Cole SPC. Toxicological relevance of the multidrug resistance protein1, MRP1 (ABCC1) and related transporters. Toxicol. 167: 3–23, 2002.

Loe DW, Deeley RG, Cole SPC. Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Res. 58: 5130–5136, 1998.

Marbeuf-Gueye C, Salerno M, Quidu P, Garnier-Suillerot A. Inhibition of P-glycoprotein and multidrug resistance protein mediated efflux of anthracyclines and calceinacetoxymethyl ester by PAK-104P. Eur J Pharmacol. 391: 207- 216, 2000.

Marie JP, Zhou DC, Gurbuxani S, Legrand O, Zittoun R. MDR1 /P-glycoprotein in haematological neoplasms. Eur J Cancer. 32: 1034– 1038, 1996.

Massou S, Albigot R, Prats M. Carboxyfluorescein fluorescence experiments, Biochem Educ. 28: 171-173, 2000.

McGrath T, Center MS. Adriamycin resistance in HL60 cells in the absence of detectable p-glycoprotein. Biochem Biophys Res Commun. 145: 1171- 1176, 1987.

McGrath T, Latoud C, Arnold ST, Safa AR, Felsted RL, Center MS. Mechanisms of multidrug resistance in HL60 cells. Biochem Pharmacol. 38: 3611- 3619, 1989.

Meaden ER, Hoggard PG, Khoo SH, Back DJ. Determination of P-gp and MRP1 expression and function in peripheral blood mononuclear cells in vivo. J Immunol Methods. 262: 159– 165, 2002.

Muller M, Meijer C, Zaman GJR, Borst P, Scheper RJ, Mulder NH, de Vries EG, Jansen PL. Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP dependent glutathione S-conjugate transport. Proc Natl Acad Sci USA. 91: 13033– 13037, 1994.

Nguyen H, Zhang S, Morris ME. Effect of Flavonoids on MRP1- Mediated Transport in Panc-1 Cells. J pharm sci. 92: 250-257, 2003.

Pastan I, Gottesman MM. Multiple-drug resistance in human cancer. N Eng J Med. 316: 1388-1393, 1987.

Paulusma CC. A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin–Johnson syndrome. Hepatology. 25: 1539–1542, 1997.

Pietro AD, Conseil G, Perez-Victoria JM, Dayan G, Baubichon-Cortay H, Trompier D, Steinfels E, Jault JM, de Wet H, Maitrejean M, Comte G, Boumendjel A, Mariotte AM, Dumontet C, McIntosh DB, Goffeau A, Castanys S, Gamarro F, and Barron D. Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters. Cell Mol Life Sci. 59: 307-322, 2002.

Rao VV, Dahlheimer JL, Bardgett ME, Snyder AZ, Finch RA, Sartorelli AC, Piwnica-Worms D. Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood–cerebrospinal-fluid drug-permeability barrier. Proc Natl Acad Sci USA. 96: 3900–3905, 1999.

Ross DD. Novel mechanisms of drug resistance in leukemia. Leukemia 14: 467–473, 2000.

Rudas M, Filipits M, Taucher S, Stranzl T, Steger GG, Jakesz R, Pirker R, Pohl G. Expression of MRP1, LRP and Pgp in breast carcinoma patients treated with preoperative chemotherapy. Breast Cancer Res Treatment. 81: 149–157, 2003.

Salerno M, Garnier-Suillerot A. Kinetics of glutathione and daunorubicin efflux from multidrug resistance protein overexpressing small-cell lung cancer cells. Eur J Pharmacol. 421: 1–9, 2001.

Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev. 55: 3–29, 2003.

Schultz MJ, Wijnholds J, Peppelenbosch MP, Vervoordeldonk MJBM, Speelman P, van Deventer SJH, Borst P, van der Poll T. J Immunol. 166: 4059-4064, 2001.

Sikic BI, Fisher GA, Lum BL, Halsey J, Beketic-Oreskovic L, Chen G. Modulation and prevention of multidrug resistance by inhibitors of P-glycoprotein. Cancer Chemother Pharmacol. 40(suppl): S13-9, 1997.

Soroka CJ, Lee JM, Azzaroli F, Boyer JL. Cellular localization and upregulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver. Hepatology 33: 783–791, 2001.

Scheffer GL, Kool M, Heijn M, de Haas M, Pijnenborg AC, Wijnholds J, van Helvoort A, de Jong MC, Hooijberg JH, Mol CA, van der Linden M, de Vree JM, van der Valk P, Elferink RP, Borst P, Scheper RJ. Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies: Cancer Res. 60: 5269-5277, 2000.

van Brussel JP, Oomen MA, Vossebeld PJ, Wiemer EA, Sonneveld P, Mickisch GH. Identification of multidrug resistance-associated protein 1 and glutathione as multidrug resistance mechanisms in human prostate cancer cells: chemosensitization with leukotriene D4 antagonists and buthionine sulfoximine. BJU Int. 9:1333-1338, 2004.

van der Kolk DM, de Vries EGE, Koning JA, van den Berg E, Muller M, Vellenga E. Activity and expression of the multidrug resistance proteins MRP1and MRP2 in acute myeloid leukemia cells, tumor cell lines, and normal hematopoietic CD34+ peripheral blood cells. Clin Cancer Res. 4: 1727–1736, 1998.

van der Kolk DM, de Vries EGE, van Putten WLJ, Verdonck LF, Ossenkoppele G., Verhoef GEG, and Vellenga E. P-glycoprotein and Multidrug Resistance Protein Activities in Relation to Treatment Outcome in Acute Myeloid Leukemia. Clin Cancer Res. 6: 3205–3214, 2000.

van Zandena JJ, Wortelboerb HM, Bijlsma S, Punt A, Usta M, van Bladeren JP, Rietjens IMCM, Cnubbenb NHP. Quantitative structure activity relationship studies on the flavonoid mediated inhibition of multidrug resistance proteins 1 and 2. Biochem Pharmacol. 69: 699–708, 2005.

Versantvoort CH, Broxterman HJ, Bagrij T, Scheper RJ, Twentyman PR. Regulation by glutathione of drug transport in multidrug resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein. Br J Cancer. 72: 82–89, 1995.

Vezmar M, Georges E. Reversal of MRP-mediated doxorubicin resistance with quinoline-based drugs. Biochem Pharmacol. 59: 1245–1252, 2000.

Westlake CJ, Cole SPC, Deeley RG. Role of the NH2-terminal membrane spanning domain of multidrug resistance protein 1/ABCC1 in protein processing and trafficking.Mol. Biol Cell 16: 2483–2492, 2005.

Wijnholds J, Lange ECM, Scheffer GL, van den Berg DJ, Mol CAAM, van der Valk M, Schinkel AH, Scheper RJ, Breimer DD, Borst P. Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood–cerebrospinal fluid barrier. J Clin Invest. 105: 279–285, 2000.

Xania-Pietrzak B, Michalak K, Hendricha AB, Mosiadz D, Grynkiewiczb G, Motohashic N, Shiratakid Y. Modulation of MRP1 protein transport by plant, and synthetically modified flavonoids. Life Sci. 77: 1879–1891, 2005.

Yeh JJ, Hsu NY, Hsu WH, Tsai CH, Lin CC, Liang JA. Comparison of chemotherapy response with P-glycoprotein, multidrug resistance- related protein-1, and lung resistance related protein expression in untreated small cell lung cancer. Lung. 183: 177–183, 2005.

Zhang DW, Nunoya K, Vasa M, Gu HM, Cole SPC, Deeley RG. Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity. Drug Metab Dispo. 34: 539-546, 2006.

Zijlstra JG, De Vries EGE. Mulder NH. Multifactorial drug resistance in an adriamycin-resistant human small cell lung carcinoma cell line.Cancer Res. 47: 1780–4, 1987.

Ziping Y, Cheryl SWL, Danny DS, and Rodney JYH. Cloning and Characterization of the Rat Multidrug Resistance-Associated Protein 1. AAPS PharmSci. 4 (3): E15, 2002.