环胞菌素A 逆转数种人类胶质瘤细胞多药耐药性的体外实验研究

 目的　探讨环胞菌素A体外逆转人类胶质瘤细多药耐药性的作用及其机理。方法　采用有MDR1和MRP表达的人类胶质瘤细系 ,通过放射自显影观察应用环胞菌素A前后抗癌药物在细胞内的积累与外排量。结果　在有MDR1和MRP表达的人类胶质瘤细胞系中 ,加入环胞菌素A后细胞内抗癌药物积累量显著增加 (P <0 .0 5 )。结论　环胞菌素A可增加耐药细胞对抗癌药的敏感性 ,在体外能有效地逆转入类胶质瘤细胞的多药耐药性。其作用机制除了与细胞膜上的P -糖蛋白竞争性结合、抑制药物外排 ,对MRP过度表达的胶质瘤细胞系也有逆转作用 ,这可能与细胞类型有关。     

Multidrug Resistance in Cultured Human Leukemia and Lymphoma Cell Lines Detected by a Monoclonal Antibody, MRK16

Forty cultured human leukemia and lymphoma cell lines never exposed to anticancer agents in culture, apart from doxorubicin (ADM)-resistant K562/ADM, were examined for reactivity with a monoclonal antibody, MRK16 in F(ab')₂ form [MRK16-F(ab')₂], which recognizes P-glycoprotein (P-gp). The relative resistance index to various drugs was calculated by dividing the 50% growth inhibitory concentration (IC₅₀) of the test cell line by IC₅₀ of K562, which was the negative control in the antibody experiment. MRK16-F(ab')₂ reacted with four cell lines, K562/ADM, KYO-1, HEL and CMK, which had relative resistance index values of 2 or more to vincristine (VCR), vindesine, vinblastine, ADM, daunorubicin, mitoxantrone (MIT), etoposide (VP-16) and actinomycin-D (ACT-D). The level of resistance to VCR and ADM in these cell lines decreased significantly in the presence of 10 μ M verapamil in vitro . Significant expression of mRNA of P-gp gene was also detected in K562/ADM, KYO-1 and HEL. MRK16-F(ab')₂ did not react with 36 other cell lines. Among them, three cell lines, PL-21, P31/FUJ and KOPM-28, had relative resistance index values of 2 or more to anthracyclines, MIT and VP-16, but not to vinca alkaloids or ACT-D. The level of ADM-resistance in these cell lines did not decrease significantly in the presence of 10 μ M verapamil. Five cell lines, ATL-1K, HL-60, KMOE-2, ML-1 and U266, had relative resistance index values of 2 or more to some of the drugs, but not to the others, and 19 other cell lines did not. These results indicate that the reactivity of MRK16-F(ab')₂ correlates with a relative resistance index of 2 or more to all these drugs in cultured human leukemia and lymphoma cell lines.     

A Novel Multidrug Resistance in Cultured Leukemia and Lymphoma Cells Detected by a Monoclonal Antibody to 85-kDa Protein, MRK20

A monoclonal antibody, MRK20, in F(ab')₂ form [MRK20-F(ab')₂], which reacts with 85-kDa membrane protein in a doxorubicin (ADM)-resistant subline (K562/ADM) of human myelogenous leukemia cell line, K562, was examined for reactivity with 41 cultured human leukemia and lymphoma cell lines. None of these cell lines had ever been exposed to any anticancer agent in vitro except K562/ ADM. The relative resistance index to various drugs was calculated by dividing the 50% growthinhibitory concentration (IC₅₀) of the test cell line by IC₅₀ of K562 (the negative control in the antibody experiment). MRK20-F(ab')₂ reacted with seven cell lines, KYO-1 derived from chronic myelogenous leukemia in blastic crisis (CMLbc), CMK from acute megakaryoblastic leukemia, HEL from erythroleukemia, P31/FUJ from acute monocytic leukemia, KOPM-28 from CMLbc, PL-21 from acute promyelocytic leukemia and K562/ADM, MRK20-F(ab')₂ did not react with 34 other cell lines. All seven MRK20-F(ab')₂-positive cell lines had relative resistance index values of 2 or more to anthracyclines (ADM, pyrarubicin, daunorubicin), mitoxantrone, etoposide, bleomycin, and pepleomycin. There was no distinct correlation between the reactivity to MRK20-F(ab')₂ and a higher relative resistance index than 2 to vinca alkaloids, actinomycin-D, cisplatin, 4-hydroperoxycyclophos-phamide, nimustine hydrochloride, methotrexate or cytarabine. These results indicate that MRK20-F(ab')₂ detects a novel multidrug resistance to anthracyclines, mitoxantrone, etoposide, bleomycin and pepleomycin in cultured human leukemia and lymphoma cells.     

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。     

Enhancement of Reversing Effect of Cyclosporin A on Vincristine Resistance by Anti-P-glycoprotein Monoclonal Antibody MRK-16

The synergistic effect of MRK-16, a monoclonal antibody against P-glycoprotein, and cyclosporin A (CsA) on the modulation of vincristine resistance was studied by isobologram analysis in three different, highly multidrug-rcsistant tumor cells. In all cell lines, the synergistic effect was demonstrated at various concentrations of MRK-16 and CsA. While MRK-16 alone did not enhance the sensitivity of the moderately resistant KB-8–5 cells to vincristine, it increased two-fold the reversing effect of cyclosporin A at 1 μM , an achievable blood concentration. Since MRK-16 alone showed therapeutic effects against multidrug-resistant tumors, the combined use of MRK-16, CsA and antitumor agents should provide therapeutic benefits for the treatment of resistant tumors.     

非霍奇金淋巴瘤多药耐药逆转的临床研究

目的 :探讨环胞霉素 A(Cs A)联合他莫昔芬 (TAM)逆转非霍奇金淋巴瘤 (NHL )多药耐药的临床疗效。方法 :采用免疫组织化学标记链亲和素生物素法检测 NHL的 P-糖蛋白表达。将 P-糖蛋白表达阳性者分层随机分为两组 ,比较 Cs A联合 TAM加化疗组与单用化疗组的疗效。结果 :P-糖蛋白的阳性表达率在初治 NHL 为 9.6 % ,在复发难治 NHL 为 79.2 % (P<0 .0 0 1)。Cs A联合 TAM对逆转 NHL 的多药耐药有一定作用 ,加逆转剂组的化疗疗效高于不加逆转剂组 (P<0 .0 5 )。结论 :Cs A联合 TAM逆转 NHL 的多药耐药是安全和具有一定疗效的。     

Enhancement by Verapamil of Neocarzinostatin Action on Multidrug-resistant Chinese Hamster Ovary Cells: Possible Release of Nonprotein Chromophore in Cells

Multidrug-resistant CH^RC5 cells were about 10-fold more resistant to the proteinaceous anticancer drug neocarzinostatin (NCS) and its nonprotein chromophore (NPC) than the parental AUXB1 cells. There was little difference in cell growth, glutathione content, or activities of several antioxidant enzymes between the two cell lines. The degree of intracellular incorporation and extracellular excretion of fiuorescein isothiocyanate-labeled NCS by CH^RC5 cells was similar to that of AUXB1 cells. On the other hand, 20 μM verapamil or 27 μM cepharanthine restored the susceptibility of CH^RC5 cells to NCS and NPC to the level of AUXB1 cells. In addition, NPC was found to suppress the photolabeling of [³H]azidopine (a known P-glycoprotein-binding ligand) to plasma membranes of CH^HC5 cells. All these findings favor the possibility that NPC was excreted via P-glycoprotein, which may contribute to the resistance of CH^RC5 cells to NCS.     

环孢霉素A逆转白血病耐药细胞系K562／VCR耐药性的研究

为寻找克服Ｐ－糖蛋白介导肿瘤多药耐药的途径。选择环孢霉素Ａ（ＣｓＡ）作为耐药逆转剂,分别采用台盼蓝拒染法、ＭＴＴ法和免疫组化法分析ＣｓＡ对人白血病细胞株的毒性及逆转效果,观察ＣｓＡ及长春新碱（ＶＣＲ）对Ｋ－５６２／ＶＣＲ生长的抑制作用及检测Ｋ５６２／ＶＣＲ细胞膜Ｐ－ｇｐ的表达情况,结果显示,ＣｓＡ在非毒性剂量浓度４μｇ／ｍｌ以下能逆转细胞耐药,与ＶＣＲ合用能抑制Ｋ５６２／ＶＣＲ生长;Ｋ５６２／ＶＣ     

异搏定对耐阿霉素人乳腺癌细胞系多药耐受的逆转作用

多药耐受是肿瘤化疗失败的主要原因之一.耐阿霉素的人乳腺癌细胞系MCF_7~(adr)具有多药耐受表型,对阿霉素和罗丹明123表现交叉耐受.本文应用MTT法证实;钙通道阻滞剂异搏定能够逆转 MCF_7~(adr)细胞对阿霉素或罗丹明 123的耐受;荧光显微镜技术和 FCM进一步证实了无毒浓度异搏定(10μm)可增加MCF_(adr)~7细胞内阿霉素或罗丹明123的荧光强度.该作用系由于异搏定使耐药细胞表达的大量P一糖蛋白功能失活,细胞摄取药物量增加或延长药物在细胞内滞留时间所致.     

Intracellular Levels of Two Cyclosporin Derivatives Valspodar (PSC 833) and Cyclosporin A Closely Associated with Multidrug Resistance-modulating Activity in Sublines of Human Colorectal Adenocarcinoma HCT-15

P-Glycoprotein, which mediates multidrug resistance (MDR) in cancer chemotherapy, is a principal target of Cyclosporin A and [3'-keto-Bmt¹]-[Val²]-cyclosporin (valspodar; PSC 833). To clarify mechanisms contributing to the different MDR-modulating activities of valspodar and Cyclosporin A, we investigated the relation of the intracellular levels of the two Cyclosporin derivatives to their modulating effect on MDR in different P-glycoprotein-expressing human colorectal carcinoma HCT-15 cells (parental HCT-15 and adriamycin-resistant sublines). In this study, valspodar was found to be much more potent than Cyclosporin A in both sensitizing resistant cells to MDR-related anticancer drugs (e.g., adriamycin, vincristine and paclitaxel (taxol)) and increasing 2-[6-amino-3-imino-3H-xanthen-9-yl]benzoic acid methyl ester (rhodamine 123) retention and [G-³H]vincristine sulfate ([³H]vincristine) accumulation in these cells. Furthermore, a good correlation was detected between P-glycoprotein levels and the MDR-reversing effect of valspodar. In contrast, the effects of Cyclosporin A could not be linked to P-glycoprotein levels in the MDR cells. In addition, the intracellular accumulation of valspodar was found to be 3–6 fold higher than that of Cyclosporin A in four sublines and verapamil, an inhibitor of P-glycoprotein-mediated transport, enhanced the accumulation of Cyclosporin A, but not valspodar. These results suggested that valspodar accumulation is not actively regulated by the P-glycoprotein-mediated efflux system     

Inhibitory Effects of a Cyclosporin Derivative, SDZ PSC 833, on Transport of Doxorubicin and Vinblastine via Human P-Glycoprotein

The inhibitory effects of SDZ PSC 833 (PSC833), a non-immunosuppressive cyclosporin derivative, on the P-glycoprotein (P-gp)-mediated transport of doxorubicin and vinblastine were compared with those of cyclosporin A (Cs-A). The transcellular transport of the anticancer drugs and PSC833 across a monolayer of LLC-GA5-COL150 cells, which overexpress human P-gp, was measured. Both PSC833 and Cs-A inhibited P-gp-mediated transport of doxorubicin and vinblastine in a concentration-dependent manner and increased the intracellular accumulation of doxorubicin and vinblastine in LLC-GA5-COL150 cells. The values of the 50%-inhibitory concentration (IC₅₀) of PSC833 and Cs-A for doxorubicin transport were 0.29 and 3.66 μ M , respectively, and those for vinblastine transport were 1.06 and 5.10 μ M , respectively. The IC₅₀ of PSC833 for doxorubicin transport was about 4-fold less than that for vinblastine transport, suggesting that the combination of PSC833 and doxorubicin might be effective. PSC833 itself was not transported by P-gp and had higher lipophilicity than Cs-A. These results indicated that the inhibitory effect of PSC833 on P-gp-mediated transport was 5- to 10-fold more potent than that of Cs-A, and this higher inhibitory effect of PSC833 may be related to the absence of PSC833 transport by P-gp and to the higher lipophilicity of PSC833.     

Modulation of Adriamycin Resistance in Human Breast Carcinoma MCF-7 Cells in vitro and in vivo by Medroxyprogesterone Acetate

The combination effect of adriamycin (ADM) and medroxyprogesterone acetate (MPA) was examined in vitro against human breast carcinoma MCF-7 and its ADM-resistant line (MCF-7/ADM). MCF-7 cells, which are positive for estrogen receptors, progesterone receptors and high-affinity MPA-binding activity, were more susceptible to the growth-inhibitory activity of ADM or MPA than MCF-7/ADM cells. A combination effect of ADM and MPA was observed against MCF-7/ADM cells, which are negative for steroid receptors, and furthermore against human nasopharynx carcinoma KB and its ADM-resistant line KB-A1. This combination effect of ADM and MPA against MCF-7/ADM cells was demonstrated to be synergistic by using the median effect plot method. The activity of MPA was almost equivalent to that of chlormadinone acetate or tamoxifen, greater than that of progesterone, and less than that of verapamil. The accumulation of ADM in MCF-7/ADM cells was enhanced by treatment with 10 μ M MPA as well as 10 μ M verapamil. The efflux of accumulated ADM from MCF-7/ADM cells was also partially inhibited by treatment with MPA or verapamil. MPA augmented the growth-inhibitory activity of ADM against MCF-7/ADM tumors inoculated into nude mice, although statistical significance was not observed. It is suggested that the clinical advantage of the combination of MPA with ADM against advanced breast cancers may be partly explained by the modulation of ADM resistance by MPA.     

Reversing Effect of Agosterol A, a Spongean Sterol Acetate, on Multidrug Resistance in Human Carcinoma Cells

The effect of agosterol A, a novel polyhydroxylated sterol acetate isolated from a marine sponge, on P-glycoprotein (P-gp)-mediated multidrug-resistant cells (KB-C2) and the multidrug resistance associated protein (MRPl)-mediated multidrug-resistant cells (KB-CV60) was examined. Agosterol A reversed the resistance to colchicine in KB-C2 cells and also the resistance to vincristine in KB-CV60 cells at 3 to 10 μM concentration. Agosterol A at 3 μM increased the vincristine concentration in both KB-C2 cells and KB-CV60 cells to the level in parental KB-3-1 cells. Agosterol A also decreased the efflux of vincristine from both KB-C2 cells and KB-CV60 cells to the level seen in KB-3-1 cells. Agosterol A inhibited the [³H]azidopine-photolabeling of P-gp and also inhibited the uptake of [³H]S-(2,4-dinitrophenyl)glutathione (DNP-SG) in inside-out membrane vesicles prepared from KB-CV60 cells. We conclude that agosterol A directly inhibited drug efflux through P-gp and/or MRP1.     

Cyclosporin A Enhances Susceptibility of Multi-drug Resistant Human Cancer Cells to Anti-P-glycoprotein Antibody-dependent Cytotoxicity of Monocytes, but Not of Lymphocytes

Cyclosporin A (CsA) was previously found to bind to P-glycoprotein expressed on multidrug-resistant (MDR) cancer cells. In the present study, the effect of CsA on anti-P-glycoprotein monoclonal antibody (mAb)-dependent cell-mediated cytotoxicity (ADCC) against human MDR cells was examined. The ADCC reaction was assessed by 4-h ⁵¹Cr-release assay. Highly purified lymphocytes (> 99%) and monocytes (>99%) obtained from blood mononuclear cells (MNC) of healthy donors were used as effector cells. CsA decreased the cytotoxic activity of MNC against MDR cells, but enhanced their ADCC activity in the presence of anti-P-glycoprotein mAb MRK16. Lymphocyte-mediated ADCC and natural killer activity against MDR cells were also suppressed by addition of CsA. CsA induced a significant dose-dependent increase in monocyte-mediated ADCC activity. Interestingly, pretreatment of MDR cancer cells, but not of monocytes, with CsA significantly enhanced ADCC activity mediated by monocytes, but not by lymphocytes. A CsA analog (PSC833) and FK-506, but not verapamil also increased the sensitivity of MDR cells to ADCC by monocytes. CsA did not affect the binding of monocytes to MDR cells in the presence of MRK16 mAb. These results indicate that CsA may directly enhance the susceptibility of MDR cancer cells to the monocyte-mediated ADCC reaction.     

Monoclonal Anti-P-glycoprotein Antibody-dependent Killing of Multidrug-resistant Tumor Cells by Human Mononuclear Cells

Mouse monoclonal antibodies (MRK16 and MRK17) against human multidrug-resistant cancer cell lines were tested for antibody-dependent cytotoxicity mediated by human blood mononuclear cells, using a 4-h ⁵¹Cr release assay. MRK16 (IgG₂₈ isotype) was shown to be more effective than MRK17 (IgG₁ isotype). Moreover, when four pairs of drug-resistant and their parent sensitive human cancer cells were tested for antibody-dependent cell-mediated cytolysis (ADCC) using MRK16, only the drug-resistant cell lines were susceptible to ADCC reaction. When highly purified lymphocytes (>99%) and monocytes (>97%) were isolated from blood mononuclear cells by centrifugal elutriation and adherence, MRK16 promoted both lymphocyte- and monocyte-mediated tumor cell killing, whereas MRK17 induced only a lymphocyte-mediated ADCC reaction. These results suggest that MRK16 of IgG₂₈ subtype may be a useful therapeutic agent in eradication of drug-resistant cancer cells expressing P-glycoprotein through ADCC reaction.     

白血病细胞耐药基因mdr1和多药耐药相关蛋白的表达水平的研究

用ＲＴ－ＰＣＲ方法对５４例不同时期不同类型的白血病患者进行ｍｄｒ１及多药耐药相关蛋白（ＭＲＰ）的检测及１２例Ｐ－ｇｐ免疫组化检测，结果表明：在初发组三者的阳性表达分别为３３．３％、３３．３％和２５％。复发组三者的阳性表达分别为７９．１％、５４．２％和６２．５％。提示复发患者的耐药现象是与ｍｄｒ１、ＭＰＲ和Ｐ－ｇｐ有密切相关的，同时也说明人体内ｍｄｒ１及ＭＲＰ存在一定的内在表达性     

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     

MDR1基因下调逆转人白血病阿霉素耐药细胞株K562/ADM的耐药性

目的：探讨RNA干扰（RNAi）人MDR1基因对人白血病阿霉素耐药细胞株K562/ADM耐药性的影响。方法：应用针对人MDR1基因的RNAi质粒pENTRTM/U6-MDR1转染人白血病阿霉素耐药细胞株K562/ADM和亲本细胞株K562,48 h后实时荧光定量PCR检测MDR1 mRNA表达,流式细胞术检测P-gp蛋白表达和P-gp功能,MTT法检测细胞对ADM的耐药性。结果：与未转染细胞相比,K562/ADM耐药细胞pENTRTM/U6-MDR1组的MDR1 mRNA和P-gp蛋白表达和功能均显著下降（ P <0.05）,对阿霉素的耐药性显著降低（ P <0.05）。结论：MDR1基因下调可逆转人白血病阿霉素耐药细胞株对阿霉素的耐药性。     

Resistance to Lipophilic Cationic Compounds in Multidrug Resistant Leukemia Cells

Previously we have shown that multidrug resistant cells are cross-resistant to certain permanently charged cationic lipophilic compounds. In the present study we extend these observations to additional cationic lipophilic compounds with unrelated chemical structures. Study of the growth inhibitory activity of series of triphenylalkyl-phosphonium and alkyl-ammonium compounds revealed that cross-resistance to these compounds in multidrug resistant P-388 murine leukemia cells, was related to the presence of cationic charge but not to the molecular size/degree of lipophilicity.