Reversal of multidrug resistance-associated protein-mediated daunorubicin resistance by camptothecin

The multidrug-resistance (MR) status of camptothecin (CPT) was investigated in colon adenocarcinoma HT29 cells, leukemia K562, and breast carcinoma MCF7 cells expressing P-glycoprotein (Pgp) and/or MR-associated protein (MRP1). The concentration that induced 50% growth inhibition (IC(50)) against CPT was 0.14 and 0.20 microM in parental K562/WT and MCF7/WT cells, respectively. The drug resistant subline KH30 and MCF7/VP cells, which both overexpress MRP1, presented IC(50) values of 0.63 and 3.10 microM, respectively. The resulting resistance indexes were 3.80 and 12.50, respectively. However, in KH300 cells, a cell line that preferentially overexpresses Pgp, the IC(50) of CPT was 0.08 microM and thus did not exhibit resistance against CPT. In MCF7/DoX cells, preferentially overexpressing Pgp, but also a significant level of MRP1, the IC(50) of CPT was 0.64 microM and thus presented a resistance index of 3.26 against CPT. The cytotoxic effect of CPT was modulated in cells expressing MRP1 (MCF7/VP, HT29 cells) by the specific MRP1 modulators, probenecid and MK571. These results led us to consider CPT as a substrate for MRP1 and a potential modulator of MRP1 activity. To test this hypothesis, we examined the ability of nontoxic concentrations of CPT to sensitize MRP1-overexpressing cells to daunorubicin (DNR). In MCF7/VP and KH30 cells, nontoxic concentrations of CPT were able to enhance cytotoxicity of DNR and its nuclear accumulation. Sequential and simultaneous associations of CPT (100 nM) and DNR provided complete reversal of resistance, thus showing a synergistic effect in KH30 cells. However, simultaneous association (with 10 or 20 nM CPT) had an additive effect in MCF7/VP. These data suggest that CPT could be proposed as a candidate for the reversal of the MRP1 phenotype at clinically achievable concentrations.     

麻黄碱逆转K562/A02细胞多药耐药性的研究

目的：观察非细胞毒性质量浓度的麻黄碱对耐药的白血病细胞株（KS62／A02）多药耐药性的逆转作用，并探讨其逆转机制。方法：用MTT法检测麻黄碱的细胞毒作用；用流式细胞仪检测非细胞毒性浓度的麻黄碱处理后K562／A02细胞膜表面糖蛋白P170表达及功能的变化。结果：麻黄碱对K562／A02有一定的细胞毒作用，其非细胞质量浓度（IC10）为75mg·L^-1，非细胞毒性质量浓度的麻黄碱对K562／A02细胞对阿霉素的耐药性有部分逆转作用（5．67倍），作用于K562／A02细胞后，细胞膜糖蛋白P170的表达从（85．3±5．5）％下调至（34．8±1．2）％，DNR外渗试验显示，细胞内化疗药物的质量浓度明显增加。结论：麻黄碱通过下调K562／A02细胞膜糖蛋白P170的表达，抑制其将化疗药物“泵”出细胞外的功能，提高化疗药物在K562／A02细胞内的有效质量浓度，能部分逆转K562／A02细胞的多药耐药性。     

洛美利嗪逆转K562/ADM细胞多药耐药性

目的研究洛美利嗪(lomerizine,Lom)逆转K562/ADM细胞多药耐药性的作用及机制。方法MTT法检测细胞毒作用,流式细胞仪研究Lom对ADM和长春新碱(vincristine,VCR)的K562/ADM细胞凋亡诱导作用的影响及对罗丹明123(rhodamine 123,Rh123)外排和P-糖蛋白(P-glycoprotein,P-gp)表达的作用。结果Lom明显提高ADM对K562/ADM多药耐药细胞的细胞毒作用及ADM和VCR的凋亡诱导作用,3,10和30 μmol·L^-1 Lom使K562/ADM对ADM的IC₅₀值由79.03 μmol·L^-1分别降至28.14,8.16和3.16 μmol·L^-1。Lom增加胞内ADM的蓄积浓度并抑制Rh123外排;但作用72 h后对K562/ADM细胞P-gp表达无影响。结论Lom通过抑制P-gp的活性逆转K562/ADM细胞的多药耐药性。     

Reversal of P-glycoprotein associated multidrug resistance by new isoprenoid derivatives

To find a drug to overcome P-glycoprotein associated multidrug resistance, we synthesized 43 new isoprenoid derivatives. Ten compounds were effective in an in vitro assay with the human MDR-type resistant carcinoma KB/VJ-300 and MRP-type KB/VP-4 cell lines. One of the most effective compounds, N-5228 [trans-N,N'-bis(3,4-dimethoxybenzyl)-N-solanesyl-1,2-diaminocyclohexane, mol. wt 1100.481, was tested in P388/VCR-bearing mice. It showed a antitumor effect on MDR-type resistant tumor cells. Moreover, N-5228 potentiated the accumulation of [3H]vincristine in drug-resistant cells and blocked [3H]azidopine photoaffinity labeling of P-glycoprotein molecules in MDR-type resistant cell membranes. We think that N-5228 is promising as a lead compound in the screening of resistance reversing drugs for multidrug resistant cancers.     

Liposome-encapsulated doxorubicin reverses drug resistance by inhibiting P-glycoprotein in human cancer cells

The most frequent drawback of doxorubicin is the onset of drug resistance, due to the active efflux through P-glycoprotein (Pgp). Recently formulations of liposome-encapsulated doxorubicin have been approved for the treatment of tumors resistant to conventional anticancer drugs, but the molecular basis of their efficacy is not known. To clarify by which mechanisms the liposome-encapsulated doxorubicin is effective in drug-resistant cancer cells, we analyzed the effects of doxorubicin and doxorubicin-containing anionic liposomal nanoparticles ("Lipodox") on the drug-sensitive human colon cancer HT29 cells and on the drug-resistant HT29-dx cells. Interestingly, we did not detect any difference in drug accumulation and toxicity between free doxorubicin and Lipodox in HT29 cells, but Lipodox was significantly more effective than doxorubicin in HT29-dx cells, which are rich in Pgp. This effect was lost in HT29-dx cells silenced for Pgp and acquired by HT29 cells overexpressing Pgp. Lipodox was less extruded by Pgp than doxorubicin and inhibited the pump activity. This inhibition was due to a double effect: the liposome shell per se altered the composition of rafts in resistant cells and decreased the lipid raft-associated amount of Pgp, and the doxorubicin-loaded liposomes directly impaired transport and ATPase activity of Pgp. The efficacy of Lipodox was not increased by verapamil and cyclosporin A and was underwent interference by colchicine. Binding assays revealed that Lipodox competed with verapamil for binding Pgp and hampered the interaction of colchicine with this transporter. Site-directed mutagenesis experiments demonstrated that glycine 185 is a critical residue for the direct inhibitory effect of Lipodox on Pgp. Our work describes novel properties of liposomal doxorubicin, investigating the molecular bases that make this formulation an inhibitor of Pgp activity and a vehicle particularly indicated against drug-resistant tumors.     

新的四氢异喹啉类化合物CPUE1逆转K562/A02细胞的多药耐药性

目的 :研究新的四氢异喹啉类化合物CPUE1逆转K5 6 2 A0 2细胞多药耐药性的作用及机制。方法 :在CPUE1的作用下 ,用MTT法检测长春新碱(vincristine ,VCR)对K5 6 2 A0 2多药耐药细胞的细胞毒作用的变化 ,使用DNA分析和Annexin Ⅴ PI双染法研究CPUE1对VCR诱导K5 6 2 A0 2细胞凋亡的影响 ,流式细胞仪检测CPUE1对P 糖蛋白 (P glycop rotein ,P gp)外排罗丹明 12 3(rhodamine12 3,Rh12 3)的作用。结果 :CPUE1能明显提高VCR对K5 6 2 A0 2多药耐药细胞的细胞毒作用以及凋亡诱导作用 ,10 μmol·L-1的CPUE1能使K5 6 2 A0 2对VCR的IC50值由 6 0 .5 4μmol·L-1降至 4 .17μmol·L-1。CPUE1还能抑制Rh12 3外排从而增加细胞内Rh12 3的蓄积浓度。结论 :CPUE1通过抑制P gp的活性逆转K5 6 2 A0 2细胞的多药耐药性。     

Reversal of acquired resistance to doxorubicin in K562 human leukemia cells by astemizole

This study demonstrates that astemizole, a non-sedating anti-histaminergic drug with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of doxorubicin in doxorubicin-resistant human leukemia cells (K562/DXR). Astemizole synergistically potentiated the cytotoxicity of doxorubicin for K562/DXR cells at a concentration of 0.1-3 microM in a dose-dependent manner, whereas they showed hardly any synthergistic effect in the parental cell line (K562) at the same concentration. Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of astemizole on P-glycoprotein activity in cytofluorographic efflux experiments with doxorubicin. Our results indicate that astemizole inhibits the P-glycoprotein pump-efflux activity in a dose-related manner. Moreover, it also inhibits the photolabeling of P-glycoprotein by [3H]azidopine in a dose-dependent manner. These findings provide a biological basis for the potential therapeutic application of astemizole as an anticancer drug either alone or in combination with doxorubicin to multidrug-resistant leukemic cells.     

Reversal of P-glycoprotein mediated multidrug resistance in K562 cell line by a novel synthetic calmodulin inhibitor, E6

The overexpression of P-glycoprotein (P-gp) is associated with multidrug resistance (MDR) of tumor cells to a number of chemotherapeutic drugs. P-gp inhibitors have been shown to effectively reverse P-gp-mediated MDR in both in vitro and in vivo. Our previous studies demonstrated that E6, a novel synthetic calmodulin inhibitor, exhibited potent inhibitory effect on P-gp in rat brain microvessel endothelial cells (RBMECs). In the present study, the effect of E6 on MDR in a K562 MDR cell line (K562/DOX) highly expressing P-gp was studied and compared with that of a conventional P-gp inhibitor, verapamil (VER). E6 at concentrations of 1, 3, 10, 30 microM reduced the IC50 value of doxorubicin in K562/DOX cells from 79.19 microM to 35.18, 21.86, 6.31 and 1.97 microM, respectively. However, the IC50 value of doxorubicin in K562 sensitive subline was not significantly changed by E6. Using a DNA content analysis and an annexin V binding assay, the effects of E6 on doxorubicin-induced apoptosis were also examined. The results indicated that E6 effectively reversed the resistance to doxorubicin-induced apoptosis in K562/DOX cells. In addition, co-treatment of E6 and doxorubicin resulted in a remarkably G2/M blocking effect in K562/DOX cells. Furthermore, the treatment of K562/DOX cells with 10 microM E6 led to increased intracellular accumulation and decreased efflux of doxorubicin. Overall, the pharmacological effects of E6 on P-gp-mediated MDR is much stronger than that of positive control drug VER. These results suggested that E6 is a novel and potent MDR reversal agent and may be a potential adjunctive agent for tumor chemotherapy.     

Impact of P-glycoprotein on intracellular drug concentration in peripheral blood mononuclear cells and K562 cells

P-glycoprotein (P-gp) expression in lymphocytes is variable and 2-fold higher in rheumatoid arthritis (RA) patients with treatment resistance than in healthy subjects. To date the information on P-gp-mediated drug interaction in lymphocyte is limited. We analyzed the importance on P-gp in lymphocytes using peripheral blood mononuclear cells (PBMCs) together with K562, K562/Adr, and K562/Vin cells, which have various P-gp levels, as cell models, and dexamethasone, nintedanib and apafant as weak to good P-gp substrates. P-gp levels in K562, K562/Adr, and K562/Vin cells were 0.3-, 20-, and 106-fold of healthy PBMCs, respectively. While cell accumulation of apafant and nintedanib decreased in all cells with increasing P-gp levels, dexamethasone accumulation in K562/Adr was comparable to that in healthy PBMCs and K562 cells. Cell accumulations of substrates in cells with low P-gp expression were not significantly changed by the P-gp inhibitors at therapeutic concentrations. However, accumulation increased to 1.4-fold at highest in K562/Adr cells with higher P-gp expression than in PBMCs of the RA patients. These results suggest P-gp controls the cellular concentration of P-gp substrates in PBMCs or K562 cells but cellular concentration of a weak P-gp substrate would not be apparently affected even in cells with a sufficient P-gp expression.     

乳腺癌耐受蛋白介导的5-氟尿嘧啶耐受及机制

目的筛选乳腺癌耐受蛋白(BCRP)介导的耐受药物,探讨BCRP介导抗肿瘤药物耐受的机制,优化以BCRP表达检测结果为评价指标,为肿瘤临床化疗方案提供有价值的资料。方法不同浓度的抗肿瘤药物分别处理BCRP呈不同程度表达的PA317/Teton/TREBCRP细胞,经MTT法筛选出BCRP介导的耐受药物。高效液相色谱仪检测耐受药物在细胞内的相对剂量。采用核DNA染料Hochest33258荧光染色技术和流式细胞术检测耐受药物诱导的细胞凋亡。结果随着细胞BCRP表达的增高,PA317/Teton/TREBCRP细胞对5氟尿嘧啶(5Fu)、甲氨蝶呤、多柔比星、吡柔比星、依托泊苷、米托蒽醌等药物的耐药指数增高(P<0.05),而对如紫杉醇、顺铂、长春碱、丝裂霉素、长春地辛等药物均敏感。细胞的BCRP表达量与胞内5Fu浓度具有负相关性(r=-0.885,P<0.05)。随着细胞BCRP表达的增高,经5Fu处理后细胞凋亡率随之降低(P<0.05),而Ko143能显著提高BCRP表达细胞的凋亡率(P<0.05)。结论BCRP能增强细胞对5Fu所致的凋亡耐受。     

4种外排泵抑制剂对鲍曼不动杆菌耐药性的抑制影响

目的 检测4种外排泵抑制剂对5种抗菌药物耐药的影响,了解主动外排泵耐药机制在鲍曼不动杆菌菌株中的作用,为外排泵抑制剂的开发和鲍曼不动杆菌感染的治疗提供实验室数据.方法 临床收集鲍曼不动杆菌116株,采用琼脂平皿二倍稀释法测定5种抗菌药物对实验菌株的最低抑菌浓度(MIC),同批测定加入外排泵抑制剂后抗菌药物的MIC值.结果 庆大霉素加羰基氰氯苯腙(cCCp)后显示出外排阳性的菌株数量最多,有87株(75％);其他显示外排阳性菌株的数量分别为:利血平+氯霉素(46.6％),奥美拉唑+氯霉素(33.6％),维拉帕米+庆大霉素(17.2％).外排泵抑制剂对头孢唑林的耐药性无影响.结论 4种所用外排泵抑制剂可不同程度逆转鲍曼不动杆菌的耐药,以CCCP筛选外排阳性菌效果最佳.当存在明显的酶水解耐药机制时,外排泵抑制剂对耐药性无影响.     

Reversal of P-glycoprotein and multidrug resistance-associated protein 1 mediated multidrug resistance in cancer cells by HZ08 Isomers, tetrataisohydroquinolin derivatives

Overexpression of P-glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1) by tumors results in multidrug resistance (MDR) to structurally unrelated anti-tumor agents. HZ08, a chiral compound, was a newly synthesized tetraisohydroquinoline derivative to reverse Pgp and MRP1 mediated MDR. In present studies, R, S-HZ08 and their racemate reversed the resistance to adriamycin and vincristine of adriamycin-selected human leukemia (K562/ADM) cells that overexpress Pgp. R, S-HZ08 and their racemate modulated adriamycin cytotoxicity when R, S-HZ08 and their racemate were removed 12 h prior to the cytotoxicity assay. In addition, R, S-HZ08 and their racemate increased intracellular accumulation of Rhodamine123 in Caco-2 cells that overexpress Pgp. Furthermore, using a DNA content analysis and an annexin V binding assay, R, S-HZ08 and their racemate effectively reversed the resistance to adriamycin-induced apoptosis in K562/ADM cells. R, S-HZ08 and their racemate also moderately reversed the resistance to adriamycin and vincristine of MCF-7/ADM cells that overexpress MRP1. However, R, S-HZ08 and their racemate hardly affected intracellular glutathione (GSH) levels and glutathione S-transferase (GST) activities in MCF-7/ADM cells. The result showed that R, S-HZ08 and their racemate possibly reverse MDR1 mediated multidrug resistance by a direct interaction with MRP1, not interaction with MRP1 via GSH. Thus, R, S-HZ08 and their racemate should be useful for treating patients with tumors that overexpress both Pgp and MRP1.     

Reversal of anticancer drug resistance by macrolide antibiotics in vitro and in vivo

1. The combined effects of the macrolide antibiotics erythromycin, josamycin, clarithromycin and YM17K (3,4'-dideoxy mycaminosyl tylonolide hydrochloride) on in vitro intracellular accumulation of vinblastine or cyclosporine (Cs)A and on the in vivo antitumour activity of vinblastine were investigated using mouse leukaemia P388 cells (P388/S) and anticancer drug-resistant (P388/ADR) cells. These effects were compared with those of a calcium antagonist (verapamil) or immunosuppressants (FK506 and CsA). 2. All tested macrolide antibiotics increased the accumulation of both vinblastine and CsA in P388/ADR cells in a dose-dependent manner, but their potency was lower than that of verapamil, CsA or FK506. 3. When vinblastine (200 microg/kg) was administered intraperitoneally with each of the macrolide antibiotics (10 or 100 mg/kg) or with verapamil (25 mg/kg) once a day for 10 days in P388/ADR-bearing mice, combined effects of vinblastine with the macrolide antibiotics (erythromycin, clarithromycin and YM17K) or verapamil were observed. 4. The present study suggests that macrolide antibiotics may overcome anticancer drug resistance by inhibiting the binding of vinblastine or CsA to P-glycoprotein in P388/ADR cells. 5. We believe that these results are encouraging for combination chemotherapy to overcome P-glycoprotein-dependent anticancer drug-resistant tumours in clinical practice.     

Reversal of multidrug resistance by methoxypolyethylene glycol-block-polycaprolactone diblock copolymers through the inhibition of P-glycoprotein function

Overexpression of P-glycoprotein (Pgp) is one of the major limitations in cancer chemotherapy. Previous work has shown that amphiphilic diblock copolymers composed of methoxypolyethylene glycol-block-polycaprolactone (MePEG-b-PCL) diblock copolymers enhanced the cellular accumulation of Pgp substrates by modulating the function of this drug efflux transporter. The objective of this work was to determine whether MePEG-b-PCL diblock copolymers modulated Pgp function in multidrug resistant (MDR) cancer cells. The diblock copolymer enhanced the accumulation of various Pgp substrates in Pgp overexpressing MDR cells but did not influence substrate accumulation in non-Pgp expressing cells. Treatment of MDR cells with the diblock copolymer enhanced paclitaxel (ptx) and doxorubicin (dox) accumulation. Following uptake, ptx was rapidly effluxed from MDR cells whereas diblock copolymer treatment retained dox inside MDR cells. Treatment of MDR cells with the diblock copolymer reversed drug resistance to dox but not ptx. However, resistance to ptx was reversed by verapamil, which indicated that a sustained inhibition of Pgp was required for ptx to induce cytotoxicity in MDR cells. Taken together, these results highlight the potential of MePEG-b-PCL diblock copolymer to reverse drug resistance in MDR cancer cells through inhibition of Pgp function, making it a promising candidate for overcoming MDR.     

Reversal of multidrug resistance in mouse lymphoma cells by phenothiazines

Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene. Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures). Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp. Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines. Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp. Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues. WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter. Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.     

补骨脂素逆转多药耐药细胞系K562/ADR耐药性研究

目的　研究补骨脂素对白血病细胞阿霉素耐药株(K5 6 2 /ADR)多药耐药 (multidrugresistance,MDR)性的逆转作用及其机制。方法　采用MTT法检测药物细胞毒性作用 ,高效液相色谱法检测细胞内阿霉素 (ADR)的浓度 ,流式细胞术测定细胞P 糖蛋白 (P gp)的表达。 结果　补骨脂素 (1～ 2 0 μmol·L-1)能不同程度地降低ADR对K5 6 2 /ADR细胞的IC50 。 2 0 μmol·L-1能显著提高ADR在K5 6 2 /ADR细胞内的浓度 ,降低K5 6 2 /ADR细胞P gp的表达。 结论　补骨脂素能逆转K5 6 2 /ADR细胞的MDR ,其机制与抑制P gp的功能及其表达 ,增加细胞内ADR的积累有关     

木香烃内酯对K562/A02细胞阿霉素耐药的逆转作用研究

目的探索木香烃内酯对白血病耐药细胞系K562/A02阿霉素耐药的逆转作用。方法将木香烃内酯与K562/A02细胞共培养72 h后,采用MTT法检测木香烃内酯对细胞生长的抑制作用。不同浓度木香烃内酯处理K562/A02细胞24 h后,采用Annexin V和PI双染法检测木香烃内酯对细胞的凋亡。不同浓度木香烃内酯与阿霉素共培养72 h后采用MTT法检测木香烃内酯对K562/A02细胞的耐药逆转情况。采用流式细胞仪检测木香烃内酯处理24 h之后K562/A02细胞阿霉素蓄积以及细胞膜表面P-糖蛋白(P-gp)的表达。结果木香烃内酯对K562/A02细胞的生长具有明显抑制作用,呈显著的剂量相关性。与对照组比较,2.5~50μmol/L木香烃内酯组K562/A02细胞存活率显著降低(P0.05、0.01、0.001)。随着木香烃内酯浓度的增加,凋亡细胞的比例明显增加。与对照组比较,不同浓度木香烃内酯组细胞凋亡比例显著升高(P0.05、0.01、0.001)。加入5μmol/L木香烃内酯后,使K562/A02细胞对阿霉素的敏感性提高12倍。木香烃内酯处理后K562/A02细胞内部阿霉素的蓄积明显增加,呈浓度相关性。与对照组比较,5、10μmol/L木香烃内酯组K562/A02细胞内部阿霉素的蓄积增加显著升高(P0.05)。细胞表面的P-gp的表达并无显著影响。结论木香烃内酯能够抑制K562/A02细胞增殖,诱导K562/A02细胞凋亡,增强阿霉素的化疗敏感性,逆转阿霉素耐药。     

Circumvention of multidrug resistance in human carcinoma KB cells by polyether antibiotics.

We examined the effect of various polyether antibiotics on colchicine resistance in multidrug-resistant KB-C4 cells which exhibit about 4,000-fold resistance to colchicine. As a result, 4 out of 14 polyether antibiotics were found to reverse colchicine resistance. Among them, laidlomycin was the most potent. It potentiated colchicine cytotoxicity on KB-C4 cells about 700-fold at 1 microgram/ml. Degree of potentiation was calculated by dividing of the IC50 value of colchicine in the absence of a polyether antibiotic by the IC50 value of colchicine in the presence of the polyether antibiotic. Monensin, dianemycin, and leuseramycin at 3 micrograms/ml also potentiated the cytotoxicity, about 100-fold. We previously reported that inostamycin is a potent chemosensitizer in KB-C4 cells. Although lysocellin has a structure very similar to that of inostamycin, it didn't reverse colchicine resistance. It slightly increased [3H]vinblastine accumulation in KB-C4 cells and weakly inhibited the [3H]vinblastine binding to KB-C4 plasma membranes.