Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells

STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo . However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P-glycopro-tein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [¹²⁵l]azidoagosterol A-photolabeling of P-gp, but not that of MRP1. K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.     

Reversal of P-glycoprotein mediated multidrug resistance by a newly synthesized 1,4-benzothiazipine derivative,JTV-519

A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in K562/MDR and KB/MRP cells, respectively. JTV-519 at 3 microM reversed the resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16), adriamycin (ADM) and actinomycin D and at 0.5 or 1 microM reversed their resistance to STI571. JTV-519 at 10 microM enhanced the accumulation of ADM in K562/MDR cells to the level in parental K562 cells and inhibited the efflux of ADM from K562/MDR cells. Photoaffinity labeling of P-gp with 3H-azidopine was almost completely inhibited by 500 microM JTV-519. JTV-519 at 3 microM also partially reversed the resistance of KB/MRP cells to VCR and at 500 microM partially inhibited the photoaffinity labeling of MRP1 with (125)I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1 overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1, and competitively inhibiting transport of the anti-cancer agents.     

干扰素和环孢霉素A 协同逆转K562/ ADM细胞对阿霉素的耐药性

  目的 观察干扰素(α-Interleron，α-IFN)和环孢霉素A(Cyclosporine A,CsA)对白血病K562/ADM细胞耐药性的协同逆转效应。方法 以多药耐药基因／P-糖蛋白(Muhidrug resistance gene／P-glycoprotein，mdrl／P-gp)超表达的K562／ADM细胞为靶细胞，MTT比色法检测药物的细胞毒效应；流式细胞仪检测细胞P-糖蛋白(P-glycoprotein，P-gp)表达水平；激光共聚焦显微镜观察细胞内阿霉素含量变化。结果 K562／ADM细胞对阿霉素呈高度耐药性，并与柔红霉素和鬼臼乙叉甙交叉耐药，但与CsA无交叉耐药。CsA和α-IFN单独或联合应用均对K562／ADM细胞的耐药性有较强的抑制效应。流式细胞仪和激光共聚焦显微镜分析发现α-IFN和CsA单独或联合均不能下调细胞mdrl/P-gp的表达，反而应激性地刺激耐药细胞P-gp的合成增加，但可抑制P-gp的功能、增加K562／ADM细胞内阿霉素的积聚。结论 α-IFN和CsA联合可协同逆转耐药白血病细胞的耐药性，其作用机制为抑制P-gp的功能而非下调mdrl／P-gp的表达水平。     

Circumvention of multidrug resistance by a newly synthesized quinoline derivative, MS-073.

Newly synthesized quinoline derivatives were investigated for their efficacy to reverse multidrug resistance (MDR). In this study, one of the most effective quinoline derivatives, MS-073, was compared with verapamil with regard to its ability to overcome MDR in vitro and in vivo. MS-073 at 0.1 microM almost completely reversed in vitro resistance to vincristine (VCR) in VCR-resistant P388 cells. The compound also reversed in vitro VCR, adriamycin (ADM), etoposide, and actinomycin D resistance in ADM-resistant human myelogenous leukemia K562 (K562/ADM) cells, ADM-resistant human ovarian carcinoma A2780 cells, and colchicine-resistant human KB cells. MS-073 administered i.p. daily for 5 days with VCR enhanced the chemotherapeutic effect of VCR in VCR-resistant P388-bearing mice. Increases in life span of 19-50% were obtained by the combination of 100 micrograms/kg of VCR with 3-100 mg/kg of MS-073, as compared to the control. The ability of MS-073 to reverse MDR was remarkably higher, especially at low MS-073 doses, than that of verapamil, both in vitro and in vivo. MS-073 enhanced accumulation of [3H]VCR in K562/ADM cells. Photolabeling of P-glycoprotein with 200 nM [3H]azidopine in K562/ADM plasma membranes was completely inhibited by 10 microM MS-073, indicating that MS-073 reverses MDR by competitively inhibiting drug binding to P-glycoprotein.     

RNAi对白血病细胞mdr-1基因和多药耐药表型的影响

目的:探讨RNA干扰技术(RNAi)对慢性粒细胞白血病急变细胞系K562/AO2细胞mdr1基因的抑制和耐药表型的逆转作用。方法:选择合成封闭mdr-1基因的小干扰序列(si-MDR1),以1个碱基突变的si-MDR1-mut为对照序列,在脂质体介导下转染至K562/AO2细胞系。RT-PCR和Western blot检测mdr1 mRNA及P-gp蛋白水平,流式细胞术分析细胞内柔红霉素(daunorubicin,DNR)积累量,并以四甲基唑蓝快速比色法(MTT)反映K562/AO2对阿霉素、长春新碱、足叶乙甙药物敏感性的变化。结果:实验证实该序列能高效封闭K562/AO2细胞内mdr-1基因表达,增加细胞内化疗药物DNR积累量,增强K562/AO2细胞对阿霉素、长春新碱、足叶乙甙的敏感性。结论:RNAi可以通过抑制mdr1基因表达,逆转K562/AO2细胞耐药表型。     

Circumvention of multidrug resistance by a quinoline derivative, MS-209, in multidrug-resistant human small-cell lung cancer cells and its synergistic interaction with cyclosporin A or verapamil

Purpose and methods: To develop a clinically useful approach to circumvent P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MDR human small-cell lung cancer (SCLC), we examined the ability of a novel quinoline compound, MS-209, to reverse MDR by inhibition of P-gp function in combination with other MDR-reversing drugs using a cytotoxicity assay. Results: We established MDR human SCLC cells by culture in medium with gradually increasing concentrations of adriamycin (ADM). Compared with the parental human SCLC cells, SBC-3, the MDR variant SBC-3 cells obtained (SBC-3/ADM) were highly resistant to various chemotherapeutic agents due to P-gp expression. MS-209 reversed the resistance to ADM and vincristine (VCR) of SBC-3/ADM and H69/VP cells in a dose-dependent manner. Moreover, MS-209 in combination with cyclosporin A (CsA) or verapamil (VER) synergistically enhanced the antitumor effects of ADM and VCR on SBC-3/ADM cells. MS-209 restored ADM incorporation and this effect was enhanced by CsA and VER, suggesting that these synergistic effects were due to competitive inhibition of P-gp function. Conclusion: MS-209 in combination with CsA or VER might increase the efficacy of these chemotherapeutic agents against MDR human SCLC cells. Received: 10 December 1997 / Accepted: 16 April 1998     

Reversal of multidrug resistance by a novel quinoline derivative,MS-209

MS-209, a novel quinoline derivative, was examined for its reversing effect on multidrug-resistant tumor cells. MS-209 at 1–10 M completely reversed resistance against vincristine (VCR) in vitro in multidrug-resistant variants of mouse leukemia P388 cells (VCR-resistant P388/VCR and Adriamycin (ADM)-resistant P388/ADM) and human leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM). MS-209 at 1–10 M also completely reversed resistance against ADM in vitro in P388/VCR cells, K562/VCR cells, and K562/ADM cells. In ADM-resistant P388 (P388/ADM) cells, however, ADM resistance was only partially reversed at the MS-209 concentrations tested. MS-209 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When MS-209 was given p.o. at 80 mg/kg twice a day (total dose, 160 mg/kg per day) with 100 g/kg VCR, a treated/control (T/C) value of 155% was obtained. MS-209 also enhanced the chemotherapeutic effect of ADM in P388/ADM-bearing mice. The most prominent effects were obtained when MS-209 was given with 2 mg/kg ADM, yielding T/C values of 150%–194% for the combined treatment at an MS-209 dose of 200–450 mg/kg. MS-209 inhibited [³H]-azidopine photolabeling of P-glycoprotein efficiently. Furthermore, the accumulation of ADM in K562/ADM cells was increased more eficiently by MS-209 than by verapamil. These results indicate that MS-209, like verapamil, directly interacts with P-glycoprotein and inhibits the active efflux of antitumor agents, thus overcoming multidrug resistance in vitro and in vivo.This work was supported by grants-in-aid from the Ministry of Education Science and Culture, Japan     

Competitive inhibition by verapamil of ATP-dependent high affinity vincristine binding to the plasma membrane of multidrug-resistant K562 cells without calcium ion involvement

Verapamil, a calcium channel blocker, can inhibit the efflux of antitumor agents from multidrug-resistant cells and reverse drug resistance. We have recently reported that the plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells showed ATP/Mg2+-dependent high affinity binding of vincristine (VCR), which is closely related to the drug transport mechanism in this cell line. To clarify how calcium channel blockers inhibit the transport of antitumor agents from the resistant cells, we analyzed the effect of calcium channel blockers and Ca2+ ion on the VCR binding to K562/ADM plasma membrane. The ATP-dependent VCR binding was inhibited by calcium channel blockers (verapamil, nicardipine, and diltiazem), which are known to inhibit drug efflux from the resistant cells. Addition of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid or high concentration of Ca2+ decreased the amount of VCR binding to some extent; however, a substantial amount of VCR still could bind to K562/ADM plasma membrane. The inhibitory effect of verapamil on the VCR binding was observed regardless of the Ca2+ concentration. Klotz plot analysis revealed that the inhibition of the VCR binding to K562/ADM plasma membrane by verapamil was competitive. Dissociation constant (Kd) of VCR and apparent inhibitory constant (Kiapp) of verapamil were calculated to be 0.1 +/- 0.1 microM (SD) and 1 +/- 1 microM, respectively. These results indicate that Ca2+ ion is not required for the VCR binding and that verapamil competitively inhibits the VCR binding without concerning Ca2+ ion. Antitumor agents (vinblastine, actinomycin D, ADM, and colchicine) and other agents known to reverse multidrug resistance (nicardipine, diltiazem, cyclosporin A, quinidine, and trifluoperazine) also inhibited the VCR binding competitively.     

鲎素抑制多药耐药细胞HL-60/VCR生长的研究

目的：探讨鲎血细胞来源的多肽鲎素是否能够抑制白血病多药耐药细胞HL-60／VCR细胞的生长。方法：HL-60细胞采用持续低浓度和逐渐增加长春新碱（VCR）浓度间歇诱导，建立多药耐药细胞HL-60／VCR；MTT法检测细胞耐药性及鲎素对HL-60／VCR细胞和HL-60细胞生长的影响；流式细胞仪检测HL-60／VCR细胞P-糖蛋白（P-glycoprotein，P-gP）和多药耐药相关蛋白（multidrugresistance-associatedprotein，MRP）的表达水平。结果：HL~0／VCR细胞对多种化疗药物产生耐药；HL-60／VCR细胞表面P-gP、MRP表达上调，分别为78．3％和58．3％（P〈0．01）。鲎素无论对HL-60／VCR细胞还是对HL-60细胞均有抑制作用，作用呈剂量依赖性。HL-60／VCR细胞和HL-60细胞对鲎素具有相似的敏感性。鲎素能增强HL-60／VCR细胞对VCR的敏感性，但不影响HL-60／VCR细胞表达P-gP和MRP。结论：鲎素具有抗HL-60／VCR细胞耐药性并增加其对VCR敏感性的作用。     

Comparative Study on Reversal Efficacy of SDZ PSC 833, Cyclosporin a and Verapamil on Multidrug Resistance in vitro and in vivo

A non-immunosuppressive cyclosporin, SDZ PSC 833 (PSC833), shows a reversal effect on multidrug resistance (MDR) by functional modulation of MDR1 gene product, P-glycoprotein. The objective of the present study was to compare the reversal efficacy of three multidrug resistance modulators, PSC833, cyclosporin A (CsA) and verapamil (Vp). PSC833 has approximately 3-10-fold greater potency than CsA and Vp with respect to the restoring effect on reduced accumulation of doxorubicin (ADM) and vincristine (VCR) in ADM-resistant K562 myelogenous leukemia cells (K562/ADM) in vitro and also on the sensitivity of K562/ADM to ADM and VCR in in vitro growth inhibition. The in vivo efficacy of a combination of modifiers (PSC833 and CsA: 50 mg/kg, Vp 100 mg/kg administered p.o. 4 h before the administration of anticancer drugs) with anticancer drugs (ADM 2.5 mg/kg i.p., Q4D days 1, 5 and 9, VCR 0.05 mg/kg i.p., QD days 1-5) was tested in ADM-resistant P388-bearing mice. PSC833 significantly enhanced the increase in life span by more than 80%, whereas CsA and Vp enhanced by less than 50%. This reversal potency, which exceeded that of CsA and Vp, was confirmed by therapeutic experiments using colon adenocarcinoma 26-bearing mice. These results demonstrated that PSC833 has signficant potency to reverse MDR in vitro and in vivo, suggesting that PSC833 is a good candidate for reversing multidrug resistance in clinical situations.     

环孢素A对白血病耐药细胞化疗药物敏感性的研究

目的 研究环孢素A对K562/DOX细胞多药耐药的逆转作用.方法 用流式细胞仪和MTT法观察了CsA对K562/DOX细胞P-糖蛋白的抑制作用及对阿霉素(doxorubicin,DOX)、长春新碱(vincristine,VCR)耐药的逆转作用.结果 CsA能剂量相关性地增加K562/DOX细胞内罗丹明123(rhodamine123,Rh123)的累积,明显抑制P-gp介导的Rh123外排,显著增强DOX、VCR的细胞毒作用,增加VCR诱导的细胞凋亡和G2/M期细胞百分率.结论 CsA能显著抑制P-gp的外排功能,逆转K562/DOX细胞的多药耐药性.     

Anti-proliferative effect of the abl tyrosine kinase inhibitor STI571 on the P-glycoprotein positive K562/ADM cell line

STI571, an abl tyrosine kinase inhibitor, is less effective in chronic myelogenous leukemia (CML) patients in the accelerated phase and in blastic crisis. We addressed whether STI571 is effective for the CML blastic crisis cell line K562 and the P-glycoprotein (P-gp) positive, multidrug resistance cell line K562/ADM. The present results demonstrate that P-gp positive K562/ADM cells were more resistant than K562 cells to the anti-proliferative and apoptotic effect of STI571, but the co-addition of a P-gp modulator augmented the sensitivity of K562/ADM cells to STI571. For patients in CML blastic crisis, simultaneous use of a P-gp modulator may increase the efficacy of STI571.     

MDR1基因短发卡样RNA表达载体逆转K562/A02人白血病细胞多药耐药的研究

目的构建MDR1基因短发卡样RNA(shRNA)真核表达载体,观察对K562/A02人白血病细胞株MDR1基因的沉默作用以及对P-糖蛋白(P-gp)表达及功能的影响。方法以基因重组技术构建表达质粒,转染重组质粒pEGFP-C1/U6/MDR1-A和pEGFP-C1/U6/MDR1-B至K562/A02细胞株,通过半定量RT-PCR和蛋白质印迹法,检测MDR1基因表达及P-gp表达水平的变化;以MTT法检测阿霉素(ADM)对K562/A02细胞的半数抑制浓度(IC_(50));高效液相色谱(HPLC)法检测细胞内ADM含量。结果构建的2种重组质粒pEGFP-C1/U6/MDR1-A和pEGFP-C1/U6/MDR1-B均明显抑制K562/A02细胞株MDR1基因表达,抑制率最高为48．2%±2．5%;同时抑制P-gp蛋白的表达,抑制率最高为50．67%。对ADM药物敏感性的相对逆转效率分别为40．8%和62．4%;同时使K562/ A02细胞内ADM含量增加。结论shRNA表达载体可明显抑制K562/A02细胞MDR1 mRNA的转录和P-gp蛋白的表达,增加K562/A02细胞内ADM含量,恢复K562/A02细胞对化疗药物的敏感性,逆转MDR1基因编码蛋白P-gp介导的多药耐药。     

雷公藤红素逆转K562/A02细胞多药耐药的实验研究

目的探讨雷公藤红素逆转人慢性粒细胞白血病红白血病急变细胞株K562/A02多药耐药的效果。方法采用CCK-8法测定细胞的药敏性及耐药逆转性,应用流式细胞术检测细胞内ADM浓度、P-gp蛋白表达。结果雷公藤红素对K562/A02、K562的半数抑制率浓度（IC50）分别为（295.58±23.288）μmol/L、（411.59±26.551）μmol/L。K562/A02细胞对ADM的耐药性是K562细胞的79.78倍。细胞毒剂量的雷公藤红素作用后,ADM对K562/A02细胞的IC50显著下降（P〈0.05）,逆转倍数为117.860倍。细胞毒剂量（IC50）和非细胞毒剂量（IC10）的雷公藤红素处理后的K562/A02细胞内的ADM浓度显著增加（P〈0.05）,增加倍数分别为1.537倍和1.102倍。雷公藤红素能明显下调K562/A02细胞的P-gp表达。结论雷公藤红素对逆转K562/A02细胞的耐药性有一定的作用,其机制可能与下调P-gp表达有关。     

沉默 UVRAG 对白血病K562/ADM 细胞自噬及耐药性的影响

目的：探讨沉默紫外线抵抗相关基因（UVRAG）对人白血病 K562/ ADM 细胞自噬及耐药性的影响。方法 Western Blotting 检测 UVRAG 蛋白在 K562及 K562/ ADM 细胞中表达差异。特异性干扰 UVRAG 基因的 UVRAG siRNA 及 Scramble siRNA 在 LipofectamineTM2000介导下转染 K562/ ADM 细胞,CCK-8法、MDC荧光染色及 Western Blotting 分别检测 UVRAG siRNA 转染前后 K562/ ADM 细胞耐药性、自噬水平以及 P-糖蛋白（P-gp）表达的变化。结果 Western Blotting 检测显示 K562/ ADM 细胞中 UVRAG 蛋白表达明显高于K562细胞（P ﹤0.05）;与 K562/ ADM 组及 Scramble siRNA 转染组相比,UVRAG siRNA 转染组 UVRAG 蛋白表达显著下降（P ﹤0.05）,以48 h 效果最佳,提示 UVRAG siRNA 能高效沉默 K562/ ADM 细胞 UVRAG;CCK-8法显示与 K562/ ADM 组及 Scramble siRNA 转染组相比,UVRAGsiRNA 组对阿霉素敏感性显著增高,IC50值明显下降（P ﹤0.05）;MDC 染色荧光显微镜观察到 UVRAG siRNA 转染后 K562/ ADM 细胞胞浆中自噬泡明显减少;Western Blotting 显示 K562/ ADM 细胞中 Beclin-1、P-gp 表达及 P62降解明显高于 K562细胞,与K562/ ADM 细胞及 Scramble siRNA 转染组相比,UVRAG siRNA 转染组 Beclin-1、P-gp 表达及 P62降解显著降低（P 均﹤0.05）。结论 UVRAG 蛋白在 K562/ ADM 细胞中高表达,与白血病 MDR 密切相关;UVRAG siR-NA 下调 UVRAG 表达可降低 K562/ ADM 细胞耐药性,其机制可能与降低自噬水平及下调 P-gp 表达有关。     

Mechanism of cytotoxic effects of taxotere in the K562 human premyelocytic leukemia line

Background Taxotere, a semisynthetic derivative of Taxol, is known to possess cytotoxic effects on various animal cells. Methods To better understand the precise mechanism of this drug action, the human promyelocytic leukemia cell line, K562, and its adriamycin and vincristine resistant sublines, respectively termed K562/ADM and K562/VCR, were used as targets. Results The IC₅₀ for taxotere was almost equal to that for VCR. Due to cross-resistance in the K562/ADM cells, the IC₅₀ value was 42.3 times greater with taxotere, although it was still lower than with ADM and VCR. A much lower cross-resistance was noted with the K562/VCR cells. Assessment of MDR-1 mRNA indicated that expression of the multidrug resistance gene product p-glycoprotein in the cell membrane was partly responsible for the resistance. K562 cells treated with taxotere accumulated in G₂M of the cell cycle, and morphologically, cells in metaphase were found to be remarkably increased. This indicates inhibition of mitosis. Unlike vincristine or vinblastine, taxotere enhanced the assembly of tubulin into microtubules in the absence of guanosine triphosphate (GTP). Moreover, microtubule disassembly was inhibited even in the presence of calcium ions. Conclusion These results suggest that the tubulin equilibrium was shifted towards formation and away from degradation of microtubules that lead to metaphase arrest and eventual cell death. Syntheses of DNA, RNA and protein were not inhibited, and topoisomerases I and II were unaffected. Thus taxotere is an analogue of Taxol, showing a similar mechanism of cytotoxic effect to Taxol on the human K562 leukemia cell line as well as on rodent tumor cell lines.     

Effect of bisbenzylisoquinoline (biscoclaurine) alkaloids on multidrug resistance in KB human cancer cells

Cepharanthine, a bisbenzylisoquinoline (biscoclaurine) alkaloid, completely overcomes resistance of a multidrug-resistant subline, ChR-24, derived from human KB carcinoma cells, to vincristine, actinomycin D, and daunomycin, and partially overcomes resistance to Adriamycin. Another biscoclaurine alkaloid, berbamine, partially overcomes resistance to these anticancer agents. Accumulation of [3H]daunomycin in ChR-24 cells is about 10% of that in both the parental KB and revertant cell line (Rev-2) which is derived from ChR-24. Cepharanthine prominently increases the accumulation of daunomycin in resistant ChR-24 cells, but not in parental KB and Rev-2 cells. Enhanced efflux of daunomycin from the resistant cells is completely inhibited by cepharanthine. Cellular uptake of [3H]daunomycin is not significantly affected in the resistant cells by cepharanthine. Accumulation of [3H]cepharanthine is observed at similar levels in both KB and ChR-24. Phosphatidylserine specifically inhibited the accumulation of [3H]cepharanthine in KB and ChR-24 cells when tested by adding various phospholipids such as phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin to culture medium. The enhanced accumulation of [3H]daunomycin in cepharanthine-treated ChR-24 cells is inhibited in the presence of 20 micrograms/ml phosphatidylserine. Cepharanthine may overcome multidrug resistance by binding to phosphatidylserine in the plasma membrane and perturbing membrane function.     

5-溴汉防己甲素与汉防己甲素对K562/A02细胞多药耐药性逆转作用的比较

背景与目的:5-溴汉防己甲素(5-bromotetrandrine,BrTet)是汉防己甲素(tetrandrine,Tet)的溴化产物,具有逆转P-糖蛋白(P-glyeoprotein,P-gp)介导的肿瘤多药耐药(muhidrug resistance,MDR)的作用。本研究旨在比较BrTet与Tet对人白血病细胞K562/A02多药耐药的逆转作用。方法:采用四甲基偶氮唑蓝法(MTT)法检测不同浓度BrTet对K562细胞和K562/A02细胞的增殖抑制效应;检测阿霉素(adfiamycin,ADM)对K562细胞和K562/A02细胞增殖的抑制作用,以及加用BrTet、Tet时上述抑制作用的变化,并计算半数抑制浓度(IC50)及逆转倍数。Westernblot法检测各组细胞P-gp的表达,流式细胞仪检测各组细胞内ADM的蓄积。结果:K562/A02细胞对ADM的耐药倍数为49.51倍。2.0μmol/L及更低浓度的BrTet和1.5μmol/L及更低浓度的Tet对K562细胞和K562/A02细胞抑制率均小于10%,无明显细胞毒性作用。加入1.0μmol/L的Tet后,K562/A02细胞对ADM的耐药倍数为12.17倍。加入0.25、0.5和1.0μmol/L的BrTet后,K562/A02细胞对ADM的耐药倍数分别为17.88、9.97和4.24倍。1.0"mol/L的BrTet和Tet分别使K562/A02细胞内ADM浓度提高了69.0%和51.6%,使P-gp表达分别下调了51.1%和43.73%,其差异具有统计学意义(P<0.05)。结论:BrTet及Tet均可逆转K562/A02细胞耐药,且前者较后者逆转作用更强,逆转机制与抑制P-gp的表达、增加细胞内抗肿瘤药物浓度有关。     

Reversal of multidrug resistance by an immunosuppressive agent FK-506

Summary FK-506, a novel immunosuppressive agent, was examined for its reversing effect on multidrug-resistant tumor cells. FK-506 at 3 M completely reversed the resistance against vincristine (VCR) in vitro in VCR-resistant mouse leukemia P388 cells (P388/VCR). FK-506 also enhanced the cytotoxicity of VCR in Adriamycin(ADM)-resistant human ovarian cancer A2780 cells (AD₁₀) and ADM-resistant human myelogenous leukemia K562 cells (K562/ADM) in vitro. FK-506 was also effective in modulating sensitivity to ADM in AD₁₀ cells in vitro. FK-506 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When 20 mg/kg FK-506 was combined with 200 g/kg VCR, a T/C value of 151% was obtained. Under the protocol used in this study, FK-506 was more potent than cyclosporin A (CsA) and verapamil. FK-506 inhibited [³H]azidopine binding to P-glycoprotein efficiently. The binding of VCR to K562/ADM plasma membrane was inhibited by FK-506 as effectively as by CsA. Moreover, the accumulation of VCR in AD₁₀ cells was increased by FK-506 as efficiently as that of CsA and verapamil. These results indicate that FK-506 directly interacts with P-glycoprotein like CsA and verapamil, inhibits the active efflux of vincristine from resistant cells, increases the vincristine accumulation in resistant cells, and thus overcomes multidrug resistance in vitro and in vivo.Abbreviations VCR vincristine - ADM Adriamycin - CsA cyclosporin A - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue) - T/C mean survival time of treated group of mice divided by mean survival time of control group - T/V mean survival time of treated group of mice divided by mean survival time of the group of mice treated with vincristine alone This work was supported by grants-in-aid for cancer Research from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare, Japan     

Inhibition of P-glycoprotein by flavonoid derivatives in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells.

We investigated the effects of natural flavones, quercetin and morin, and their pentamethyl, pentaethyl, pentapropyl, pentabutyl and pentaallyl ethers, on the function of P-glycoprotein (P-gp) assessed by an increase in the uptake of [3H]vincristine by human myelogenous leukemia (K562) cells and adriamycin-resistant human myelogenous leukemia (K562/ADM) cells. Pentamethyl, pentaethyl, pentapropyl and pentaallyl ethers of morin and quercetin (20 microM) all increased the uptake of [3H]vincristine by K562/ADM cells, while quercetin, morin and their pentabutyl ethers had no effect. Pentamethylquercetin, pentaallylquercetin and pentaethylmorin remarkably increased the uptake of [3H]vincristine by K562/ADM cells by 10.6, 10.8 and 14.4-fold, respectively. These inhibitory potencies for P-gp were more potent than typical P-gp inhibitors, cyclosporine A and verapamil. Taking into consideration that these flavonoid derivatives possess antitumor promoter activity, they may become candidates of effective multidrug resistance-reversing agents in cancer chemotherapy.