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.     

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.     

Cepharanthine potently enhances the sensitivity of anticancer agents in K562 cells

A major impediment to cancer treatment is the development of resistance by the tumor. P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) are involved in multidrug resistance. In addition to the extrusion of chemotherapeutic agents through these transporters, it has been reported that there are differences in the intracellular distribution of chemotherapeutic agents between drug resistant cells and sensitive cells. Cepharanthine is a plant alkaloid that effectively reverses resistance to anticancer agents. It has been previously shown that cepharanthine is an effective agent for the reversal of resistance in P-gp-overexpressing cells. Cepharanthine has also been reported to have numerous pharmacological effects besides the inhibition of P-gp. It has also been found that cepharanthine enhanced sensitivity to doxorubicin (ADM) and vincristine (VCR), and enhanced apoptosis induced by ADM and VCR of P-gp negative K562 cells. Cepharanthine changed the distribution of ADM from cytoplasmic vesicles to nucleoplasm in K562 cells by inhibiting the acidification of cytoplasmic organelles. Cepharanthine in combination with ADM should be useful for treating patients with tumors.     

K562多药耐药细胞系中肿瘤干细胞样细胞对伊马替尼耐药机制的初步研究

Objective： To characterize a novel chronic myeloid leukemia （CML） cell line and to further elucidate the mechanisms of resistance to STI571. Methods： A novel K562 cell line （K562NP16） was achieved after exposure of the K562 cells to VP16. A small subpopulation （K562NP16 SP） that was capable of excluding Hoechst 33342 in the K562NP16 cell line was isolated by fiow cytometry sorting. The rest of the K562NP16 cells were classified as non-SP K562NP16. The mechanisms involved in K562NP16 SP cells which became resistant to STI571 were studied. Results： The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562NP16 and K562NP16 SP cells. The multidrug-resistant gene 1 （MDR1） expression of the 170 kDa P-glycoprotein （P-gp） was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562NP16 cell lines were similar. Compared with non-SP K562/ VP16, the K562NP16 SP cells were more resistant to STI571. This resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced tumorigenesis in mice, while the K562NP16 non-SP cells failed to do so. Conclusion： A novel K562 cell line, K562NP16, was generated. A small side population K562NP16 SP cells, had high resistance to STI571 treatment and more tumorigenic than the K562 cells. It may represent the cancer stem cells of the K562NP16 cell line.     

Reversal of multidrug resistance by novel nitrophenyl pyrones, SNF4435C and D.

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis, were examined for their reversing effects in vitro on various multidrug-resistant (MDR) tumor cells overexpressing P-glycoprotein. These two compounds in the range of 3-10 microM completely reversed the resistance of MDR variant cells, mouse leukemia P388 cells [vincristine (VCR)-resistant P388/VCR and adriamycin (ADM)-resistant P388/ADM], human myelogenous leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) and human ovarian cancer A2780 cells (ADM-resistant AD(10)), against VCR. Both compounds moderately potentiated the sensitivity of the MDR cells to ADM but the reversal was not complete. SNF4435C and D significantly increased the intracellular accumulation of VCR in AD(10) cells as potently as verapamil, cyclosporin A (CysA) and FK506, whereas the compounds exerted no effect on the accumulation of VCR in the drug-sensitive parent cells. Moreover, SNF4435C improved the chemotherapeutic efficacy of VCR in the treatment of P388/VCR-bearing mice. When 10 mg/kg SNF4435C was administered intraperitoneally to the mice concurrently with 0.2 mg/kg VCR for every 5 days, a treated/control (T/C) value of 143% was obtained. These results suggest that the compounds are useful candidates or tools for MDR modification in cancer chemotherapy.     

Reversal of Multidrug Resistance by Novel Nitrophenyl Pyrones, SNF4435C and D

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis , were examined for their reversing effects in vitro on various multidrug-resistant (MDR) tumor cells overexpressing P-glycoprotein. These two compounds in the range of 3–10 [jM completely reversed the resistance of MDR variant cells, mouse leukemia P388 cells [vincristine (VCR)-resistant P388/ VCR and adriamycin (ADM)-resistant P388/ADM], human myelogenous leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) and human ovarian cancer A2780 cells (ADM-resistant AD10), against VCR. Both compounds moderately potentiated the sensitivity of the MDR cells to ADM but the reversal was not complete. SNF4435C and D significantly increased the intracellular accumulation of VCR in AD10 cells as potently as verapamil, cyclosporin A (CysA) and FK506, whereas the compounds exerted no effect on the accumulation of VCR in the drug-sensitive parent cells. Moreover, SNF4435C unproved the chemotherapeutic efficacy of VCR in the treatment of P388/VCR-bearing mice. When 10 mg/kg SNF4435C was administered intra-peritoneally to the mice concurrently with 0.2 mg/kg VCR for every 5 days, a treated/control (T/C) value of 143% was obtained. These results suggest that the compounds are useful candidates or tools for MDR modification in cancer chemotherapy.     

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。     

Synergistic effect of cyclosporin A and verapamil in overcoming vincristine resistance of multidrug-resistant cultured human leukemia cells

Reversal of vincristine (VCR) resistance by cyclosporin A (CyA) or the combination of CyA and verapamil (VER) was investigated by using four P-glycoprotein (P-gp)-associated human multidrug-resistant (MDR) cell lines (K562/ADM, KYO-1, HEL and CMK). Drug sensitivity was expressed as 50% inhibitory concentration (IC50). The degree of reversal of resistance was expressed as x-fold decrease by dividing the IC50 value without modifier(s) by that with modifier(s). CyA overcame P-gp-associated MDR significantly in all four MDR cell lines. Reversal of VCR resistance by CyA appeared to be dose-dependent. In the case of low-grade MDR cell lines (KYO-1, HEL and CMK), CyA at the low concentration of 0.5 microgram/ml was still effective. The degree of reversal of VCR resistance in this condition was greater (6.3- to 16-fold decrease) in the low-grade MDR cell lines than in a high-grade MDR cell line (K562/ADM) (2.9-fold decrease). At a high concentration (5 micrograms/ml) of CyA, however, it was greater (240-fold decrease) in the high-grade MDR cell lines than in the low-grade MDR cell line (20- to 100-fold decrease). This indicates that concentration of CyA required for overcoming drug resistance in MDR cells was dependent on the degree of drug resistance. CyA overcame VCR resistance more efficiently than VER. The combination of CyA and VER enhanced reversal of VCR resistance in a supra-additive or at least an additive manner and overcame VCR resistance at low concentrations of both modifiers that are clinically achievable with safety.     

Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways

A tyrosine kinase inhibitor, STI571, has been demonstrated to be effective for the treatment of chronic myelogenous leukemia (CML). STI571 inhibits tyrosine kinase activity of ABL and induces apoptosis of CML cells. However, drug resistance develops commonly in patients with blast phase CML, and has become a significant therapeutic problem. We examined the effects of aminopeptidase inhibitors on CML cell line (K562) and a STI571-resistant subline of K562. Ubenimex and the more potent aminopeptidase inhibitor, actinonin, inhibited proliferation of both K562 cells and STI571-resistant K562 cells and also induced their apoptosis in dose- and time-dependent manners. Ubenimex and actinonin induced the activation of caspase-3, and the induction of apoptosis was inhibited by pan-caspase inhibitor, indicating this apoptosis is caspase-dependent. We found that serine phosphorylation of both MAPK and glycogen synthase kinase-3β were suppressed by aminopeptidase inhibitors in parent K562 and STI571-resistant K562 cells. The expression level of cyclin D1 protein was also reduced by ubenimex and actinonin in both cell lines. These results indicated STI571-resistance does not confer the cross-resistance to aminopeptidase inhibitors in K562 cells and revealed the new findings of aminopeptidase inhibitor-induced intracellular signaling pathways.     

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.     

Synergistic Effect of Cyclosporin A and Verapamil in Overcoming Vincristine Resistance of Multidrug-resistant Cultured Human Leukemia Cells

Reversal of vincristine (VCR) resistance by cyclosporin A (CyA) or the combination of CyA and verapamil (VER) was investigated by using four P-glycoprotein (P-gp)-associated human multidrug-resistant (MDR) cell lines (K562/ADM, KYO-1, HEL and CMK). Drug sensitivity was expressed as 50% inhibitory concentration (IC₅₀). The degree of reversal of resistance was expressed as x -fold decrease by dividing the IC₅₀ value without modifier(s) by that with modifier(s). CyA overcame P-gp-associated MDR significantly in all four MDR cell lines. Reversal of VCR resistance by CyA appeared to be dose-dependent. In the case of low-grade MDR cell lines (KYO-1, HEL and CMK), CyA at the low concentration of 0.5 μg/ml was still effective. The degree of reversal of VCR resistance in this condition was greater (6.3- to 16-fold decrease) in the low-grade MDR cell lines than in a high-grade MDR cell line (K562/ADM) (2.9-fold decrease). At a high concentration (5 μg/ml) of CyA, however, it was greater (240-fold decrease) in the high-grade MDR cell lines than in the low-grade MDR cell line (20- to 100-fold decrease). This indicates that concentration of CyA required for overcoming drug resistance in MDR cells was dependent on the degree of drug resistance. CyA overcame VCR resistance more efficiently than VER. The combination of CyA and VER enhanced reversal of VCR resistance in a supra-additive or at least an additive manner and overcame VCR resistance at low concentrations of both modifiers that are clinically achievable with safety.     

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.     

A new quinoline derivative MS-209 reverses multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells.

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.     

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。     

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。     

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。     

The Synergistic Inhibitory Effect of STI571 in Combination with Arsenic Trioxide (As2O3) on Multidrug-Resistant Leukemic Cells

OBJECTIVE To study the synergistic effect of STI571, an inhibitor of tyrosine kinase, in combination with arsenic trioxide As2O3 on a multidrug-resistant leukemia cell line expressing bcr-abl.METHODS The cytotoxic effect of STI571 alone or in combination with different concentrations of As2O3 on the bcr-abl and mdr1 -positive leukemia cell line, K562-n/VCR, was examined by the MTT method.RESULTS One μmol/L of STI571 alone had no significant cytotoxic effect on K562-n/VCR cells. However the cytotoxic effect increased markedly when combined with As2O3 at concentrations of 10-5, 10-6, 10-7 and 10-8 mol/L. The IC50 of K562-n/VCR cells in As2O3 group was 1.879 μmol/L, with. Upon addition of STI571, the IC50 decreased to 0.155 μmol/L resulting in a synergistic cytotoxic effect on K562-n/VCR ceils that was increased 12.1 times.CONCLUSION A combination of STI571 with As2O3 has a more powerful inhibitory effect on leukemia cells expressing positive bcr-abl and positive mdrl compared to the effect with As2O3 alone.     

Decitabine (5-Aza-2'-deoxycytidine) decreased DNA methylation and expression of MDR-1 gene in K562/ADM cells.

Multidrug resistance (MDR) is a major problem in patients with hematological malignancies. Although drug-resistance is known to be induced by the expression of P-glycoprotein (P-gp) encoded by the MDR-1 gene, little is known about the mechanisms regulating this gene. Herein, we studied the DNA methylation patterns at the enhancer and repressor binding sites of the MDR-1 gene using the human erythroleukemia cell line K562 and its multidrug resistant derivative K562/ADM (adriamycin). Direct DNA sequence analysis demonstrated methylation to be present at the repressor site (minus 110 GC-box) of the MDR-1 gene in K562/ADM cells, but not in parental K562 cells. Methylation-specific PCR (MSP) analysis yielded similar results. Treatment of K562/ADM cells with 5-Aza-2'-deoxycytidine (decitabine; DAC), an inhibitor of DNA methyltransferase, caused demethylation of the repressor binding site of MDR-1 gene, as assessed by MSP, and also decreased P-gp expression, as assessed by flow cytometric and Northern blot analysis. Although it is generally accepted that DAC upregulates gene expression by demethylating the activator binding sites, our present results suggest that DAC induces down-regulation of P-gp expression as a result of demethylation at the repressor binding site in K562/ADM cells. In this regard, methylation-dependent regulation of the MDR-1 gene in K562/ADM cells is unique.     

Bromocriptine reverses P-glycoprotein-mediated multidrug resistance in tumor cells.

One of the most important causes of anticancer treatment failure is the development of multidrug resistance (MDR). The main characteristics of tumor cells displaying the MDR phenomena are cross-resistance to structurally unrelated cytotoxic drugs having different mechanisms of action and the overexpression of the MDR1 gene, which encodes a transmembrane glycoprotein named P-glycoprotein (P-gp). This study evaluated whether bromocriptine, a D2 dopaminergic receptor agonist, influenced anticancer drug cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. The K(i) values for P-gp of cyclosporine and verapamil were 1.09 and 540 microM, respectively, and that of bromocriptine was 6.52 microM in a calcein-AM efflux assay using porcine kidney epithelial LLC-PK1 and L-MDR1 cells, overexpressing human P-gp. Bromocriptine at 10 microM reduced the IC50 of doxorubicin (DXR) in K562-DXR from 9000 to 270 ng/ml and that of vincristine (VCR) in K562-VCR from 700 to 0.30 ng/ml, whereas the IC50 values of DXR and VCR in the K562 subline were only marginally affected by these drugs. Bromocriptine restored the anticancer effect of DXR, VCR, vinblastine, vinorelbine and etoposide on MDR-tumor cells overexpressing P-gp. These observations suggest that bromocriptine has the potential to reverse tumor MDR involving the efflux protein P-gp in the clinical situation.     

Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells

Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP). The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB). Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16. The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine. ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB. When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased. Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells. Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced. PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei. These findings suggest that at least in part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei. PAK-104P may be useful for reversing MDR in tumors that overexpress LRP.