Reversal of P-glycoprotein and multidrug-resistance protein-mediated drug resistance in KB cells by 5-O-benzoylated taxinine K

A newly synthesized taxoid originally from the Japanese yew Taxus cuspidata, 5-O-benzoylated taxinine K (BTK) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein (MRP)-mediated multidrug resistance. BTK reversed the resistance to paclitaxel, doxorubicin (ADM), and vincristine (VCR) of KB-8-5 and KB-C2 cells that overexpress P-gp by directly interacting with P-gp. BTK also moderately reversed the resistance to ADM of KB/MRP cells that overexpress MRP. However, BTK neither inhibited the transporting activity of MRP nor reduced intracellular glutathione levels in KB/MRP cells. BTK shifted the distribution of ADM in KB/MRP cells from punctate cytoplasmic compartments to the nucleoplasm and cytoplasm by inhibiting acidification of cytoplasmic organelles. These two functions of BTK make it able to reverse both P-gp- and MRP-mediated MDR. BTK in combination with ADM should be useful for treating patients with tumors that overexpress both P-gp and MRP.     

Effects of the multidrug resistance modulator HZ08 on the apoptosis pathway in human chronic leukaemia cell line K562/A02

ōancer falls to respond to chemotherapy by acquiring multidrug resistance in over 90% of patients. A previous study revealed that multidrug resistance modulator HZ08 had great multidrug resistance reversal effect in vitro and in vivo. It could enhance adriamycin (doxorubicin) induced intrinsic apoptosis pathway and rectify cell cycle and some apoptosis related proteins in human breast resistant cancer MCF-7/ADM cells. This study detected Rh123 accumulation to assess the effect of HZ08 on P-glycoprotein function in human chronic leukaemia cell line K562/A02. Moreover, mitochondria membrane potential, cytochrome c release and caspase-3 activity were analyzed for HZ08 treatment with or without vincristine. Since pretreatment with HZ08 could also reverse the multidrug resistance to vincristine in K562/A02 cells, the individual influence of HZ08 was further detected on apoptotic regulator like Bcl-2, Bax, p53, cell cycle checkpoints and proliferation regulatory factors like survivin, hTERT, c-Myc, c-Fos, c-Jun. Finally, it revealed that HZ08 increased vincristine induced activation in intrinsic apoptosis pathway by inhibition of P-gp mediated efflux. In addition, the outstanding reversal effect of HZ08 should also attribute to its individual effect on apoptosis and proliferation related regulatory factors. It renders HZ08 possibility of application in pretreatment to reverse multidrug resistance while avoiding unexpected drug interactions and accumulative toxicity.     

The multidrug resistant modulator HZ08 reverses multidrug resistance via P-glycoprotein inhibition and apoptosis sensitization in human epidermoid carcinoma cell line KBV200

Previous studies have demonstrated that the multidrug resistance modulator HZ08 has a strong multidrug resistance reversal effect in vitro and in vivo by inhibiting P-glycoprotein and multidrug resistance-associated protein 1 in K562/A02 and MCF-7/ADM cells, respectively. However, there are many other mechanisms responsible for resistance. In this study, MTT assay was used to examine the cytotoxicity and multidrug resistance reversal of HZ08 in KBV200 cells. It was also used to detect Rh123 and adriamycin accumulation in the presence of HZ08 to assess the effect on P-glycoprotein. Caspase-3 activity was analyzed under the incubation of HZ08 per se and in combination with vincristine. Results showed that HZ08 could increase the activity of caspase-3 with P-glycoprotein inhibition. Further studies revealed that HZ08 increased vincristine-induced apoptosis, characterized as an intrinsic apoptosis pathway with enhanced G2/M phase arrest, since HZ08 had an effect on the intrinsic apoptotic regulator Bcl-2 and Bax. Therefore, the outstanding reversal effect of HZ08 occurs not only through suppressing the P-glycoprotein function but also through activating the intrinsic apoptosis pathway.     

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。     

黄芩苷逆转人肝癌细胞耐药株Bel-7402/ADM多药耐药性的相关机制研究

目的 考察黄芩苷对人肝癌耐药细胞Bel-7402/ADM 多药耐药性的逆转机制.方法 MTT法考察Baicalin对人肝癌多药耐药细胞的逆转作用.结果 Baicalin逆转Bel-7402/ADM多药耐药性的机制可能与抑制MDR1部分基因产物P-gp、MRP、GSH/GST的表达和诱导细胞凋亡有关.结论 Baicalin可能通过抑制MDR1部分基因产物P-gp、MRP、GSH/GST的表达和诱导细胞凋亡逆转Bel-7402/ADM的多药耐药性.     

抗多药耐药基因肽核酸与反义寡脱氧核苷酸增强K562／ADM细胞的敏感性和促进凋亡

AIM: To investigate the reversal effect and apoptosis enhancement of peptide nucleic acid (PNA) and antisenseoligodeoxyribonucleotide (ASODN) targeted to multidrug resistance gene (mdrl) on human multidrug resistantleukemia K562/ADM cells. METHODS: A 15-mer PNA and the same sequence of ASODN, complementary to the5' end of the AUG initiator codon-containing region of mdrl messenger RNA (MDR1-PNA, MDR1-ASODN), weredesigned and synthesized. Proliferation and sensitivity to adriamycin of K562/ADM cells treated with MDRI-PNAand MDR1-ASODN were analyzed with a MTT colorimetric assay. Apoptotic morphologies, P-glycoprotein (P-gp)expression, intracellular adriamycin accumulation, and cell cycle were measured. RESULTS: MDRI-PNA 1 to 10μmol/L and MDR1-ASODN 2 to 20 μmol/L alone had no inhibitory effects on the proliferation of K562/ADM cells,but significantly inhibited the growth of K562/ADM cells cultured in adriamycin-containing medium. After treatment with MDRI-PNA and MDRI-ASODN, intracellular adriamycin accumulation in K562/ADM cells increasedgreatly and P-gp synthesis was strikingly reduced. The resistance to adriamycin of the drug-resistant cells waspartly reversed and the cells were induced to apoptosis by adriamycin. The reversal efficacy of MDR1-PNA was3.1-fold higher than that of the same sequence of MDR-ASODN, but neither MDRI-PNA nor MDRI-ASODNcould completely block the mdrllP-gp expression. CONCLUSION: Sequence-special PNA targeted to mdr1 genemore effectively than the same sequence of MDR1-ASODN inhibited the expression of P-glycoprotein to overcomethe drug-resistance.     

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1

The present study was performed to investigate the ability of the multidrug resistance protein (MRPI) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4',4'-azo-in-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations > or = 10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 microM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1.     

The effect of survivin on multidrug resistance mediated by P-glycoprotein in MCF-7 and its adriamycin resistant cells

Although anticancer chemotherapeutic drugs have been designed to inhibit the growth of tumor cells, chemotherapy frequently fails due to the development of multidrug resistance (MDR). In this paper, the effect of survivin on multidrug resistance mediated by P-glycoprotein (Pgp) was investigated in breast cancer cells. Overexpression of survivin in MCF-7 cells transfected with survivin expression vector pEGFP/survivin results in decreasing sensitivity to anticancer drugs and activation of Pgp to export drug out of cells. Down regulation of survivin in MCF-7/adriamycin (ADR) transfected with RNAi directed against survivin vector psh1/survivin could increase the drug accumulation in cells by inhibiting Pgp. Downregulation of the expression of the Pgp with the specific inhibitor verapamil could markedly suppress the survivin mRNA expression, whereas the reverse impact was not observed. Survivin might modulate the turnover of Pgp or transport by Pgp in cells, which result in anti-apoptosis and drug resistance. Our results suggest that survivin might play a key role in MDR in the presence of Pgp, and this might represent a novel strategy for modulating MDR in cancer cells.     

Diverse effects of P-glycoprotein inhibitory agents on human leukemia cells expressing the multidrug resistance protein (MRP)

Multidrug resistance (MDR) to cancer chemotherapy is frequently associated with decreased drug accumulation in cancer cells due to drug expulsion by multidrug transporters such as P-glycoprotein (Pgp) and multidrug resistance protein (MRP). The novel resistance modifying agents PSC 833, 280-446, and LY 335979 are primarily targeted at inhibition of Pgp, and their MRP inhibitory potential is largely unknown. OBJECTIVE: In the present study we addressed the effect of these agents on MRP-derived drug resistance. MATERIALS: Drug-resistant human leukemia cells with Pgp+/MRP- (KG1a/200, K562/150) and Pgp-/MRP+ (HL60/130) phenotypes were maintained in suspension cultures for experimental studies of drug accumulation and drug sensitization by Pgp inhibitors. METHODS: Intracellular accumulation of the fluorescent anthracycline daunorubicin was measured by flow cytometry and fluorescence detection. Daunorubicin dose-response curves were generated by non-linear regression of electronically measured cell counts of 72- - 96-h cultures. The half-maximal growth inhibitory dose (GI50) was used as measure of growth inhibition. RESULTS: All MDR phenotypes studied exercised significant resistance to daunorubicin. PSC 833, 280-446 and LY335979 were equal in sensitizing Pgp+/MRP- cells to daunorubicin-induced growth inhibition (p < 0.0001). The Pgp-/MRP+ cells responded to PSC 833 and 280-446 by increased accumulation of daunorubicin (p = 0.0022 and p = 0.0005, respectively) and sensitization to the drug (p = 0.0009 and p = 0.0007, respectively). Conversely, LY335979 did not affect accumulation of daunorubicin in Pgp-/MRP+ cells nor sensitize these cells to daunorubicin. CONCLUSION: Pgp inhibitory agents have differential effects on MRP-derived drug resistance which could be exploited in treatment of multidrug resistance in cancer patients.     

Reversal of P-glycoprotein-mediated multidrug resistance by protopanaxatriol ginsenosides from Korean red ginseng

The overexpression of P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP) confers multidrug resistance (MDR) to cancer cells. MDR cells can be sensitized to anticancer drugs when treated concomitantly with an MDR modulator. In this study, we investigated whether or not ginseng saponins could reverse MDR mediated by Pgp or MRP. The chemosensitization and drug accumulation effects of ginseng saponins such as the total saponin, protopanaxadiol ginsenosides (PDG), protopanaxatriol ginsenosides (PTG), ginsenosides-Rb 1, -Rb 2, -Rc, -Rg 1 and -Re were tested on the daunorubicin- and doxorubicin-resistant acute myelogenous leukemia sublines (AML-2/D100 and AML-2/DX100), which overexpress Pgp and MRP, respectively. PTG showed cytotoxicity in both sublines and was able to reverse resistance in the AML-2/D100 subline in a concentration-dependent manner. Conversely, other ginseng saponins at concentrations less than 300 microg/mL showed neither cytotoxicity nor chemosensitizing activity in both resistant sublines. Flow cytometry analysis showed that the effect of PTG (100 microg/mL) on drug accumulation of daunorubicin in the AML-2/D100 subline was 2-fold higher than that observed in the presence of verapamil (5 microg/mL) and 1.5 times less than cyclosporin A (3 microg/mL). The maximum non-cytotoxic concentrations of PTG did not appear to increase the Pgp levels, which is in contrast to verapamil and cyclosporin A. PTG at 200 microg/mL or more completely inhibited the azidopine photolabeling of Pgp. The results suggest that PTG has a chemosensitizing effect on Pgp-mediated MDR cells by increasing the intracellular accumulation of drugs through direct interaction with Pgp at the azidopine site. In addition, PTG may have a beneficial effect on cancer chemotherapy with respect to the possibility of long-term use without the concern of Pgp activation.     

异喹啉类的新化合物CPUC1对K562/A02细胞多药耐药的逆转作用

目的　研究新化合物CPUC1对K5 6 2 /A0 2细胞多药耐药的逆转作用。方法　MTT法检测长春新碱细胞毒作用。流式细胞仪测定细胞内罗丹明 12 3的累积。DNA含量分析和AnnexinV/PI双染测定长春新碱诱导的细胞凋亡作用。结果　CPUC1可以明显逆转K5 6 2 /A0 2细胞对长春新碱的耐药性。CPUC1可以浓度依赖性增加K5 6 2 /A0 2细胞内罗丹明12 3的积累。CPUC1可明显增强长春新碱诱导的K5 6 2 /A0 2细胞凋亡。结论　CPUC1通过抑制P 糖蛋白 (P glycoprotein ,P gp)功能逆转了P gp介导的K5 6 2 /A0 2细胞的多药耐药性     

自噬水平与白血病K562/ADM细胞耐药的关系研究

目的采用阿霉素(adriamycin,ADM)“逐步提高药物浓度+间歇性诱导”的方式体外诱导建立稳定耐受15μmol·L^-1 ADM的白血病K562/ADM细胞,观察该细胞对其它化疗药物的敏感性以及细胞自噬水平与耐药的关系。方法MTT法检测细胞对几种化疗药物的敏感性;用透射电镜、荧光显微镜观察细胞自噬形态学改变;Annexin-V/PI双染流式细胞仪检测细胞凋亡;Western blot检测自噬和耐药相关蛋白的表达水平。结果K562/ADM细胞除了对ADM产生明显耐药外,还对多种化疗药物如:吡柔比星、柔红霉素、5-FU和长春新碱等有交叉耐药,但对三氧化二砷较敏感。K562/ADM细胞内自噬体数量、MDC荧光强度以及LC3Ⅰ/Ⅱ、Beclin-1蛋白表达水平均高于亲本细胞。用3-MA抑制自噬可明显增加K562/ADM细胞对ADM的敏感性,同时也能有效抑制K562/ADM细胞内耐药相关蛋白的表达。结论K562/ADM细胞出现多药耐药现象,且耐药性与细胞自噬水平有密切关系。     

Modulation of P-glycoprotein-mediated multidrug resistance by flavonoid derivatives and analogues

Flavonoid derivatives were synthesized and tested for their ability to modulate P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) in vitro. These compounds belong to various flavonoid subclasses, namely: chromones, azaisoflavones, and aurones. Among the investigated compounds, three showed potent reversing activity. 2-(4-methylpiperazin-1-ylcarbonyl)-5-hydroxychromone (4a), 5,7-dimethoxy-3-phenyl-4-quinolone (5), and 4,6-dimethoxyaurone (6) potentiated daunorubicin cytotoxicity on resistant K562 cells. They were also able to increase the intracellular accumulation of rhodamine-123, a fluorescent molecule which acts as a probe of P-glycoprotein-mediated MDR. This suggests that these compounds act, at least in part, by inhibiting P-glycoprotein activity. The most active compound, 5-hydroxy-2-(4-methylpiperazin-1-ylcarbonyl)chromone (4a) was found to be a powerful reversal agent, more potent than cyclosporin A, used as the reference molecule. No effect was observed on MRP transport nor on cell proliferation. Little apoptosis was induced on K562S cells with 4a compared to K562R, probably due to the extrusion of the compound by Pgp.     

Substituted tetrahydroisoquinoline compound B3 inhibited P-glycoprotein-mediated multidrug resistance in-vitro and in-vivo

P-glycoprotein (P-gp) mediated multidrug resistance (MDR) is one of the main obstacles in tumour chemotherapy. A promising approach to reverse MDR is the combined use of nontoxic and potent P-gp inhibitor with conventional anticancer drugs. We have examined the potential of a newly synthesized tetrahydroisoquinoline derivative B3 as a MDR-reversing agent. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to examine the effect of B3 on the cytotoxicity in K562/A02 and MCF-7/ADM cells caused by doxorubicin (adriamycin). Accumulation and efflux of P-gp substrate rhodamine123 in K562/A02 and primary cultured rat brain microvessel endothelial cells (RBMECs) were measured to evaluate the inhibitory effect of B3 on P-gp. The K562/A02 xenograft model in nude mice was established to examine MDR-reversing efficacy of B3 in-vivo. The results indicated that co-administration of B3 resulted in an increase on chemosensitivity of K562/A02 and MCF-7/ADM cells to doxorubicin in a dose-dependent manner. Rhodamine123 accumulation in K562/A02 cells and RBMECs were significantly enhanced after the incubation with various concentrations of B3. Furthermore, B3 inhibited the efflux of rhodamine123 from RBMECs. Co-administration of B3 with doxorubicin significantly decreased weight and volume of tumour in nude mice. In conclusion, B3 is a novel and potent MDR reversal agent with the potential to be an adjunctive agent for tumour chemotherapy.     

A review of selected anti-tumour therapeutic agents and reasons for multidrug resistance occurrence

It is assumed that proteins from the ABC family (i.e., glycoprotein P (Pgp)) and a multidrug resistance associated protein (MRP) play a main role in the occurrence of multidrug resistance (MDR) in tumour cells. Other factors that influence the rise of MDR are mechanisms connected with change in the effectiveness of the glutathione cycle and with decrease in expression of topoisomerases I and II. The aim of this review is to characterize drugs applied in anti-tumour therapy and to describe the present state of knowledge concerning the mechanisms of MDR occurrence, as well as the pharmacological agents applied in reducing this phenomenon.     

苦瓜蛋白逆转K562／AO2细胞多药耐药性的研究

目的：研究非细胞毒性质量浓度苦瓜蛋白对耐阿霉素的人红白血病细胞株K562／AO2多药耐药性的逆转作用和促凋亡作用。方法：采用CCK-8法测定苦瓜蛋白的细胞毒性及其对K562／AO2细胞敏感性的影响,用流式细胞仪检测K562／AO2细胞经不同药物处理后细胞的凋亡情况。结果：苦瓜蛋白对K562／AO2细胞有一定的细胞毒作用,其非细胞毒性质量浓度为5μg／mL,非细胞毒性质量浓度苦瓜蛋白对K562／AO2细胞对阿霉素、长春新碱（VCR）和柔红霉素的耐药性都有部分逆转作用（分别为5．4、6．5和4．0倍）;5μg／mL苦瓜蛋白联合VCR诱导K562／AO2细胞凋亡,凋亡率为（19．38±1．06）％,而对照组为（1．64±0．27）％,单一苦瓜蛋白组为（3．79±0．82）％,单一VCR组为（9．83±0．98）％。结论：苦瓜蛋白能部分逆转人红白血病K562／AO2细胞对阿霉素、VCR和柔红霉素的耐药,一定剂量的苦瓜蛋白与VCR联合应用可增加肿瘤细胞凋亡率。     

Acquired multidrug resistance in human K562/ADM cells is associated with enhanced autophagy

Abstract

Autophagy, as a necessary process for survival in mammalian cells deprived of nutrients or growth factors, will be activated in many tumor cells while treated with chemotherapeutic drugs, but the role of autophagy in acquired multidrug resistance of human acute myelogenous leukemia to adriamycin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental acute myelogenous leukemia cell line K562 and resistant sub cell line, K562/ADM, which were obtained by treating adriamycin with increasing concentrations. K562/ADM and K562 cells were exposed to PBS culture medium for 3 hours, then the stress-induced autophagy was measured. Real-time quantitative RT-PCR revealed the expression of LC3 mRNA was higher in K562/ADM than in K562 cells. LC3-II, as an autophagosomal marker, was more abundant in K562/ADM than in K562 cells measured by Western blotting. To determine the effect of 3-MA, a known specific inhibitor of autophagy, on overcoming acquired multidrug resistance induced by adriamycin, the MTT assay and flow cytometry were performed. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of adriamycin in acquired resistant cells (K562/ADM). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in multidrug resistance of K562/ADM cells induced by adriamycin.     

Synthesis and evaluation of dihydropyrroloquinolines that selectively antagonize P-glycoprotein

In a search for improved multiple drug resistance (MDR) modulators, we identified a novel series of substituted pyrroloquinolines that selectively inhibits the function of P-glycoprotein (Pgp) without modulating multidrug resistance-related protein 1 (MRP1). These compounds were evaluated for their toxicity toward drug-sensitive tumor cells (i.e. MCF-7, T24) and for their ability to antagonize Pgp-mediated drug-resistant cells (i.e. NCI/ADR) and MRP1-mediated resistant cells (i.e. MCF-7/VP). Cytotoxicity and drug accumulation assays demonstrated that the dihydropyrroloquinolines inhibit Pgp to varying degrees, without any significant inhibition of MRP1. The compound termed PGP-4008 was the most effective at inhibiting Pgp in vitro and was further evaluated in vivo. PGP-4008 inhibited tumor growth in a murine syngeneic Pgp-mediated MDR solid tumor model when given in combination with doxorubicin. PGP-4008 was rapidly absorbed after intraperitoneal administration, with its plasma concentrations exceeding the in vitro effective dose for more than 2 h. PGP-4008 did not alter the plasma distribution of concomitantly administered anticancer drugs and did not cause systemic toxicity as was observed for cyclosporin A. Because of their enhanced selectivity toward Pgp, these substituted dihydropyrroloquinolines may be effective MDR modulators in a clinical setting.     

华蟾素对耐阿霉素人乳腺癌细胞内阿霉素蓄积的影响

目的：测定华蟾素（cinobufacine,Cino）对耐阿霉素人乳腺癌细胞MCF-7/ADM内阿霉素（ADM）积累的影响,以探索Cino逆转MCF-7/ADM多药耐药（MDR）的可能机制。方法：MTT法检测CinoMDR的逆转作用,HPLC法检测Cino作用MCF-7/ADM后细胞内ADM的浓度的变化。结果：Cino15mg/L能增加MCF-7/ADM细胞对ADM的敏感性,使ADM的半数抑制浓度（IC50）由38.14mg/L降至12.93mg/L,显著增加MCF-7/ADM株细胞内ADM的浓度。结论：Cino能明显增加MCF-7/ADM株细胞内ADM的含量,部分逆转MCF-7/ADM的MDR。