Multidrug-resistant Human Cells Research Articles

Increased expression of the pathological O-glycosylated form of oncofetal fibronectin in the multidrug resistance phenotype of cancer cells

Changes in protein glycosylation are a hallmark of transformed cells and modulate numerous phenomena associated with cancer progression, such as the acquisition of multidrug resistance (MDR) phenotype. Different families of glycosyltransferases and their products have already been described as possible modulators of the MDR phenotype. Among the glycosyltransferases intensively studied in cancer research, UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (pp-GalNAc-T6), which is widely expressed in many organs and tissues, stands out. Its influence in several events associated with kidney, oral, pancreatic, renal, lung, gastric and breast cancer progression has already been described. However, its participation in the MDR phenotype has never been studied. Here, we demonstrate that human breast adenocarcinoma MCF-7 MDR cell lines, generated by chronic exposure to doxorubicin, in addition to exhibiting increased expression of proteins belonging to the ABC superfamily (ABCC1 and ABCG2), and anti-apoptotic proteins (Blcl-2 and Bcl-xL), also present high expression of pp-GalNAc-T6, the enzyme currently proposed as the main responsible for the biosynthesis of oncofetal fibronectin (onf-FN), a major extracellular matrix component expressed by cancer cells and embryonic tissues, but absent in healthy cells. Our results show that onf-FN, which is generated by the addition of a GalNAc unit at a specific threonine residue inside the type III homology connective segment (IIICS) domain of FN, is strongly upregulated during the acquisition of the MDR phenotype. Also, the silencing of pp-GalNAc-T6, not only compromises the expression of the oncofetal glycoprotein, but also made the MDR cells more sensitive to all anticancer drugs tested, partially reversing the MDR phenotype. Taken together, our results demonstrate for the first time the upregulation of the O-glycosylated oncofetal fibronectin, as well as the direct participation of pp-GalNAc-T6 during the acquisition of a MDR phenotype in a breast cancer model, giving credence to the hypothesis that in transformed cells, glycosyltransferases and/or their products, such as unusual extracellular matrix glycoproteins can be used as potential therapeutic targets for the treatment of cancer.

Reversal of drug resistance by JS-K and nitric oxide in ABCB1- and ABCG2-expressing multi-drug resistant human tumor cells

Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of ABC transporter proteins, including P-170 glycoprotein (P-gp), and breast cancer resistance protein (BCRP, ABCG2) have been implicated in this multi-drug resistance (MDR). These ABC transporters are ATP-dependent efflux proteins. We have recently shown that nitric oxide (NO) inhibits the ATPase activities of P-gp, resulting in a significant enhancement of drug accumulation and the reversal of multi-drug resistance in NCI/ADR-RES cells, a P-gp-overexpressing human MDR cell line. In this study, we used [O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1 yl]-diazene-1-ium-1-2-diolate] (JS-K), a tumor-specific NO-donor to study the reversal of drug resistance in both P-gp- and BCRP-overexpressing human tumor cells. We report here that while JS-K was extremely effective in reversing adriamycin resistance in the P-gp-overexpressing tumor cells (NCI/ADR-RES); it was significantly resistant to BCRP-overexpressing (MCF-7/MX) tumor cells, suggesting that JS-K may be a substrate for BCRP. Using another NO-donor (DETNO), we show that NO directly inhibits the ATP activities of BCRP, inducing significant increases in the accumulations of both Hoechst 33342 dye and topotecan, substrates for BCRP. Furthermore, NO treatment significantly reversed topotecan and mitoxantrone resistance to MCF-7/MX tumor cells. Molecular docking studies indicated that while DETNO and JS-K bind to ATP binding site in both ABC proteins, binding score was significantly reduced, compared to the ATP binding. Our results indicate that appropriately designed NO donors may find success in reversing multidrug resistance in the clinic.

The reversal effect of abnormal savda munziq total flavonoids on leukemia cell lines

Objective To investigate the reversal effect of abnormal savda munziq total flavonoids on leukemic cell lines. Methods CCK-8 method was used to detect the cytotoxicity of abnormal savda munziq total flavonoids on leukemia cells, by multivariate analysis of variance in the chemotherapy drug intervention, further calculated the reversal fold. Results CCK-8 results showed that with the increase of concentration of ASMq800(μg/mL) compared with 200(μg/mL) and 400(μg/mL), the A value of K562/ADR cells gradually decreased, the growth curve suggested that with the increase of ASMq concentration in K562/ADR cell, survival time was shortened, the inhibition of the cells was significantly increased.Different concentrations of ASMq on the adriamycin toxicity with increase of concentration of adriamycin, the inhibition of ASMq on multidrug resistance cell line was significantly higher.The reversal multiples calculated that with the increase of ASMq concentration 600 μg/mL and 900 μg/mL, the reversal index increased significantly 2.485 times and 3.651 times. Conclusion ASMq on human leukemia multidrug resistance cell line K562/ADR could reverse the drug resistance, the reversal mechanism needs further study. Key words: Abnormal savda munziq total flavonoids; Leukemia; Multidrug resistance cell; Reversal

NVP-TAE684 reverses multidrug resistance (MDR) in human osteosarcoma by inhibiting P-glycoprotein (PGP1) function.

Increased expression of P-glycoprotein (PGP1) is one of the major causes of multidrug resistance (MDR) in cancer, including in osteosarcoma, which eventually leads to the failure of cancer chemotherapy. Thus, there is an urgent need to develop effective therapeutic strategies to override the expression and function of PGP1 to counter MDR in cancer patients. In an effort to search for new chemical entities targeting PGP1-associated MDR in osteosarcoma, we screened a 500+ compound library of known kinase inhibitors with established kinase selectivity profiles. We aimed to discover potential drug synergistic effects among kinase inhibitors and general chemotherapeutics by combining inhibitors with chemotherapy drugs such as doxorubicin and paclitaxel. The human osteosarcoma MDR cell lines U2OSR2 and KHOSR2 were used for the initial screen and secondary mechanistic studies. After screening 500+ kinase inhibitors, we identified NVP-TAE684 as the most effective MDR reversing agent. NVP-TAE684 significantly reversed chemoresistance when used in combination with doxorubicin, paclitaxel, docetaxel, vincristine, ET-743 or mitoxantrone. NVP-TAE684 itself is not a PGP1 substrate competitive inhibitor, but it can increase the intracellular accumulation of PGP1 substrates in PGP1-overexpressing cell lines. NVP-TAE684 was found to inhibit the function of PGP1 by stimulating PGP1 ATPase activity, a phenomenon reported for other PGP1 inhibitors. The application of NVP-TAE684 to restore sensitivity of osteosarcoma MDR cells to the cytotoxic effects of chemotherapeutics will be useful for further study of PGP1-mediated MDR in human cancer and may ultimately benefit cancer patients.

Two New Diketopiperazines from ARUM Palaestinum

The phytochemical investigation of the water extract of Arum palaestinum led to the isolation of three compounds identified as isoorientin (luteolin 6-C-β-D-glucopyranoside), vitexin (apigenin 8-C-β-Dglucopyranoside), and vicenin II (apigenin 6,8-di-C-β-D-glucopyranoside) as well as two new diketopiperazines (DKP), namely 3-hydroxypiperazine-2,5-dione (1) and piperazine-2,5-dione (2). The chemical structures of the isolated compounds were established by spectroscopic methods including IR, MS, and NMR. The new diketopiperazine (1) exhibited mild cytotoxic activity against human multidrug-resistant cell line MES-SA/DX5 with ED50 3.44 μM.

A-770041 reverses paclitaxel and doxorubicin resistance in osteosarcoma cells.

BackgroundReversing multidrug resistance (MDR) has been an important goal for clinical and investigational oncologists. In the last few decades, significant effort has been made to search for inhibitors to reverse MDR by targeting ATP-binding cassette (ABC) transporters (Pgp, MRP) directly, but these efforts have achieved little clinical success. Protein kinases play important roles in many aspects of tumor cell growth and survival. Combinations of kinase inhibitors and chemotherapeutics have been observed to overcome cancer drug resistance in certain circumstances.MethodsWe screened a kinase specific inhibitor compound library in human osteosarcoma MDR cell lines to identify inhibitors that were capable of reversing chemoresistance to doxorubicin and paclitaxel.ResultsWe identified 18 small molecules that significantly increase chemotherapy drug-induced cell death in human osteosarcoma MDR cell lines U-2OSMR and KHOSR2. We identified A-770041 as one of the most effective MDR reversing agents when combined with doxorubicin or paclitaxel. A-770041 is a potent Src family kinase (Lck and Src) inhibitor. Western blot analysis revealed A-770041 inhibits both Src and Lck activation and expression. Inhibition of Src expression in U-2OSMR and KHOSR2 cell lines using lentiviral shRNA also resulted in increased doxorubicin and paclitaxel drug sensitivity. A-770041 increases the intracellular drug accumulation as demonstrated by calcein AM assay.ConclusionsThese results indicate that small molecule inhibitor A-770041 may function to reverse ABCB1/Pgp-mediated chemotherapy drug resistance. Combination of Src family kinase inhibitor with regular chemotherapy drug could be clinically effective in MDR osteosarcoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-681) contains supplementary material, which is available to authorized users.

Vitamin E reverses multidrug resistance in vitro and in vivo

Multidrug resistance (MDR) is a major obstacle to successful and effective chemotherapeutic treatments of cancers. This study explored the reversal effects of vitamin E on MDR tumor cells in vitro and in vivo, elucidating the potential mechanism of this reversal. VE at a concentration of 50μM exhibited a significant reversal of the MDR effect (compared to only PTX in DMSO, p<0.05) in two human MDR cell lines (H460/taxR and KB-8-5). The MDR cell xenograft model was established to investigate the effect of VE on reversing MDR in vivo. Mice intravenously injected with Taxol (10mg/kg) with VE (500mg/kg, IP) showed an ability to overcome the MDR. VE and its derivatives can significantly increase intracellular accumulation of rhodamine 123 and doxorubicin (P-gp substrate), but not alter the levels of P-gp expression. These treatments also did not decrease the levels of intracellular ATP, but were still able to inhibit the verapamil-induced ATPase activity of P-gp. The new application of VE as an MDR sensitizer will be attractive due to the safety of this treatment.

Evaluation of Sanazole Cytotoxicity in Human Drug-Sensitive and MDR Uterine Sarcoma Cells

Chemotherapy is one of the principle modes of cancer treatment. However, cells adapt to repeated chemotherapeutic exposures in several ways resulting in multidrug resistance (MDR). Therefore, it is mandatory to search for effective alternatives that are refractory to resistant mechanisms. In this study, we investigated the cytotoxicity of sanazole, a nitrotriazole derivative known for its hypoxic radio-sensitization, against a human MDR cell line highly expressing P-glycoprotein that functions as an efflux pump to lipophilic drugs. The results showed that MDR cells exhibited initial sensitivity to the drug compared to the parent sensitive cells. Sanazole action was independent of the degree of resistance and P-glycoprotein expression. Prolonged exposures (48 h) to the drug affected both cell phenotypes viability to a similar extent. However, cell cycle analysis revealed that the underlying pathways differed between cells with respect to their p53 status. For instance, cell cycle was halted in G1- and S-phase in parent and MDR cells, respectively. The (apoptotic) DNA fragmentation in MDR cells was dose-dependently higher compared to the parent cells. Sanazole treatment decreased polyploidy in a dose-dependent manner in MDR cells. The present study provides evidence on the possible potential of sanazole multidrug resistant cancer cells.

Synthesis and MDR inhibitory activity evaluation of derivatives of schizandrin A

Eighteen schizandrin A derivatives, possessing an acyl group at 7-OH and/or halogen(s) at C-4 and C-11, were designed and synthesized for evaluation of their in vitro ability to inhibit multidrug resistance (MDR). They exhibit weak ability to restore the intracellular Rhodamine 123 in human hepatocarcinoma MDR cell lines Bel7402 and HCT8 relative to the reference drug verapamil.

Establishment of human multidrug resistant hepatocarcinoma cell line transfected human multidrug resistance-associated protein gene and its biological features

Objective Treatment of hepatocellular carcinoma(HCC)is hampered by resistance to chemotherapy,which might be mediated by muhidrug resistance-associated proteins(MRP).The recombinant mammalian expression vector(pCI-neo)carrying human full lengh MRP cDNA was construtted and transfected into human hepatocellular carcinoma cell line HeDG2.The transfergene multidrug resistant cell Iine(HepG2/mrp1)was established.Methods The human full-length MRP cDNA was obtained from cloning vector pGEM-mrp1,which was cloned into pCI-neo multiple cloning site and the recombinant expression vector(pCI-mrp1)was constructed,which transfected into HepG2 and established the multidrug resistant cell line HepG2/mrp1.The chemosensitivity of HepG2/mrp1 was determined bv MTT assay.The intracellular concentration of DNR and MRP was determined by flow cytometry.The MRP mRNA was assessed with reverse transcriptase polymerase chain reaction(RTPCR).Results The resistance index of ADM and DNR was 11.4 and 8 times in HepG2/mrpl cell line,higher than that of parent cell line HeDG2.The MRP and MRP mRNA positive expression increased and the introcellular concentration of DNR decreased compared with parent cell line HepG2 respectively.Conclusion Hepatocarcinoma cell line HepG2/mrpl transfected MRP cDNA with stably over-expressed MRP presents typical MDR phenotype.It provides a good model for the study of MRP mediated MDR. Key words: Carcinoma,hepatocellular; Multidrug resistance; Multidrug resistance-associated proteins; Plasmid; Transfer-gene

Antineoplastic activity of 2‐methoxyestradiol in human pancreatic and gastric cancer cells with different multidrug‐resistant phenotypes

Chemoresistance often leads to loss of the last treatment option for cancer. 2-Methoxyestradiol (2-ME2) has been shown to inhibit tumor growth. The aim was to examine the efficacy of 2-ME2 on multidrug-resistant human cells from pancreatic and gastric cancer. We investigated the impact of 2-ME2 on multidrug-resistant cells derived from human pancreatic and gastric cancer cells that were positive or negative for the MDR1-gene. In pancreatic cancer cells, growth inhibition was 57% in parental, 72% in MDR1-negative and 87% in MDR1-positive cells after 1 micromol/L 2-ME2. In gastric cancer cells we found a growth inhibition of 75% in parental, 82% in MDR1-positive and 95% in MDR1-negative cells. Strong induction of apoptosis was induced after a low dose of 2-ME2. No significant difference in the amount of apoptotic cells was observed between parental and multidrug-resistant cells of both tumor types. The number of apoptotic cells after 2-ME2 ranged from 7.5% in parental gastric cancer cells to 20.1% in MDR1-negative gastric cancer cells. 2-ME2 may therefore have clinical application for chemoresistant cancer.

Anti-Cripto Mab inhibit tumour growth and overcome MDR in a human leukaemia MDR cell line by inhibition of Akt and activation of JNK/SAPK and bad death pathways

Doxorubicin (DOX) selection of CCRF-CEM leukaemia cell line resulted in multidrug resistance (MDR) CEM/A7R cell line, which overexpresses MDR, 1 coded P-glycoprotein (Pgp). Here, we report for the first time that oncoprotein Cripto, a founding member of epidermal growth factor-Cripto-FRL, 1-Criptic family is overexpressed in the CEM/A7R cells, and anti-Cripto monoclonal antibodies (Mab) inhibited CEM/A7R cell growth both in vitro and in an established xenograft tumour in severe combined immunodeficiency mice. Cripto Mab synergistically enhanced sensitivity of the MDR cells to Pgp substrates epirubicin (EPI), daunorubicin (DAU) and non-Pgp substrates nucleoside analogue cytosine arabinoside (AraC). In particular, the combination of anti-Cripto Mab at less than 50% of inhibition concentrations with noncytotoxic concentrations of EPI or DAU inhibited more than 90% of CEM/A7R cell growth. Cripto Mab slightly inhibited Pgp expression, and had little effect on Pgp function, indicating that a mechanism independent of Pgp was involved in overcoming MDR. We demonstrated that anti-Cripto Mab-induced CEM/A7R cell apoptosis, which was associated with an enhanced activity of the c-Jun N-terminal kinase/stress-activated protein kinase and inhibition of Akt phosphorylation, resulting in an activation of mitochondrial apoptosis pathway as evidenced by dephosphorylation of Bad at Ser136, Bcl-2 at Ser70 and a cleaved caspase-9.

LY294002, an inhibitor of phosphatidylinositol-3-kinase, causes preferential induction of apoptosis in human multidrug resistant cells

Protein kinase B (PKB), a kinase downstream of phosphatidylinositol 3-kinase (PI3-kinase) provides anti-apoptotic and survival signals via phosphorylation of various targets. Inhibiting PI3-kinase with a 12 h exposure to 10 μM LY294002 induces levels of apoptosis of 30.39±1.53% in the KB-V1 multidrug resistant (MDR) cell line compared to 4.54±1.00% in drug sensitive KB-3-1 cells (P<0.001). This occurred in conjunction with a preferential reduction in activated PKB in MDR cells. These results suggest the PI3-kinase/PKB signalling pathway is important for the survival of MDR cells and inhibition of this pathway results in the selective induction of apoptosis in MDR cells.

Alu-associated interstitial deletions and chromosomal re-arrangement in 2 human multidrug-resistant cell lines.

Previous studies have shown that gene re-arrangements play a significant role in tumorigenesis. Gene re-arrangements involving the human multidrug resistance-1 (MDR1) gene have been identified as a mechanism for MDR1 over-expression in human malignant cells. In 2 multidrug-resistant human cancer sublines with high levels of MDR1 and P-glycoprotein (MCF7/TX400 and S48-3s/Adr10), hybrid mRNAs containing sequences from MDR1 and an unrelated gene have previously been identified. To characterize and determine the site of the re-arrangements resulting in generation of hybrid mRNAs, we first constructed a lambda phage library extending over a contiguous genomic region of 100 kb and containing the region upstream of MDR1. In MCF7/TX400 cells, homologous recombination was observed involving an Alu repeat 80 kb upstream of the MDR1 gene, with a 79 bp intra-Alu deletion flanked by chi-like sequences at the re-arrangement junction. By contrast, non-homologous recombination was observed in S48-3s/Adr10 cells with Alu repeats near the junction sequence. While the specific features of the breakpoints appear to be different, Alu repeats might be involved in both gene re-arrangements. The gene re-arrangements at or near the Alu sequence should be regarded as potentially involved in the transcriptional activation of human MDR1.

Mechanism of action of the dual topoisomerase-I and -II inhibitor TAS-103 and activity against (multi)drug resistant cells

TAS-103 is a recently developed dual inhibitor of topoisomerase-I (topo-I) and topoisomerase-II (topoII). TAS-103 has documented cytotoxicity in vitro and antitumor activity against a variety of mouse, rat, and human xenografts in vivo. To determine TAS-103 activity against (multi)drug resistant cells in vitro and to delineate its mechanism of action. TAS-103 was evaluated for activity against three human multidrug-resistant cell lines representing resistance mediated by P-glycoprotein (Pgp)-, multidrug resistance protein (MRP), and lung resistance protein (LRP) as well as one camptothecin-resistant cell line associated with a mutated topo-I enzyme. Drug sensitivity following short (2 h), intermediate (6-8 h) and long term (24 h) exposures were compared. The mechanism of action was studied by evaluating inhibition of topoisomerase-I and -II specific DNA relaxation assays, drug-induced DNA/protein cross-link formation, and competitive DNA intercalation with ethidium bromide. Increasing the exposure time only modestly potentiated TAS-103 cytotoxicity (3-5 fold) demonstrating a lack of strong exposure duration dependency. TAS-103 cytotoxicity was not affected by the presence of any of the drug resistance mechanisms studied. TAS-103 inhibits topo-I and -II activity in DNA relaxation assays, but in our assay system TAS-103 was found to have only a weak ability to induce DNA-protein crosslinks. DNA migration patterns in agarose gel electrophoresis indicate that TAS-103 can interact directly with DNA. Also its ability to displace ethidium bromide which has intercalated into the DNA provides an indication on the nature of drug-DNA interaction. TAS-103 cytotoxicity is not affected by the presence of Pgp, MRP, LRP or mutations in the CAM binding region of the topo-I enzyme and its growth-inhibitory effect appears to be weakly dependent on exposure duration. The presented evidence suggest that the inhibitory effects of TAS-103 on topo-I and -II may in part be related to its DNA binding rather than primarily through stabilization of topo-I or -II intermediates with DNA through specific binding to the enzymes.

In vivo efficacy of XR9051, a potent modulator of P-glycoprotein mediated multidrug resistance.

Overexpression of P-glycoprotein (P-gp) is a potential cause of multidrug resistance (MDR) in tumours. We have previously reported that XR9051 (N-(4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phenyl)-3-((3Z,6Z)-6-benzylidene-1-methyl-2,5-dioxo-3-piperazinylidene)methylbenzamide) is a potent and specific inhibitor of P-gp, which reverses drug resistance in several murine and human MDR cell lines. In this study we have evaluated the in vivo efficacy of this novel modulator in a panel of murine and human tumour models and examined its pharmacokinetic profile. Efficacy studies in mice bearing MDR syngeneic tumours (P388/DX Johnson, MC26) or human tumour xenografts (A2780AD, CH1/DOXr, H69/LX) demonstrated that co-administration of XR9051 significantly potentiated the anti-tumour activity of a range of cytotoxic drugs. This modulatory activity was observed following parenteral and oral co-administration of XR9051. In addition, the combination schedules were well-tolerated. Following intravenous administration in mice, XR9051 is rapidly distributed and accumulates in tumours and other tissues. In addition, the compound is well-absorbed after oral administration. These data suggest that XR9051 has the potential for reversing clinical MDR mediated by P-glycoprotien. © 1999 Cancer Research Campaign

Experimentally induced changes in the endocytic traffic of P-glycoprotein alter drug resistance of cancer cells.

The MDR-1 gene product, plasma membrane glycoprotein or P-glycoprotein (PGP), has been shown to confer drug resistance to cancer cells by acting as an energy-dependent drug-efflux pump. We have examined the endocytic traffic of PGP in human multidrug-resistant cells and tested whether the traffic and the steady-state intracellular localization of PGP can be experimentally modulated. Here we show that 1) under steady state approximately 70% of cellular PGP is on the surface whereas approximately 30% is intracellular, 2) surface PGP undergoes constitutive endocytosis and recycling, 3) endocytosis of PGP involves clathrin and adaptin complex 2-dependent mechanism, and 4) PGP cycles through a Rab5-responsive endosomal compartment. Biochemical (such as antibody crosslinking of PGP or treatment of cells with chloroquine) and molecular (such as overexpression of Rab5) treatments were used to modulate the endocytic/ recycling traffic of PGP. Such treatments resulted in the redistribution of PGP from the cell surface to intracellular compartments. Cells with such "mislocalized" PGP showed a decrease in multidrug resistance, suggesting that clinically relevant strategies can be attempted by modulating PGP's temporal and spatial distribution within cancer cells.

Clinical Significance of Multi-Drug Resistance Associated Protein and P-Glycoprotein in Patients with Bladder Cancer

The clinical significance of multi-drug resistant proteins, such as multi-drug resistance associated protein and P-glycoprotein, in terms of prognostic value was determined in patients with bladder cancer. The expression of multi-drug resistance associated protein and P-glycoprotein was investigated immunohistochemically before and after chemotherapy. The relationship between expression of these multi-drug resistant proteins and clinical outcome assessed by tumor recurrence rate, cystectomy rate and 5-year survival rate was also investigated in 33 patients with bladder cancer. Before chemotherapy multi-drug resistance associated protein expression was observed in 1 of 28 patients (4%) while P-glycoprotein expression was observed in 22 of 33 (67%). Multi-drug resistance associated protein induction by chemotherapy was observed in 6 of 28 patients (21%), whereas P-glycoprotein induction was noted in 4 (14%). Multi-drug resistance associated protein in this disease is induced more frequently by high dose (more than 300 mg.) than low dose (less than 300 mg.) anthracyclines (p < 0.01). Immunohistochemical analysis also revealed co-expression of multi-drug resistance associated protein and P-glycoprotein in 5 of 28 patients (18%) after chemotherapy. However, there was no significant correlation between positive P-glycoprotein expression before chemotherapy and clinical outcome. Multi-drug resistance associated protein as well as P-glycoprotein mediated multi-drug resistance may be induced after chemotherapy for bladder tumors. However, the presence of P-glycoprotein before chemotherapy does not predict clinical outcome in patients with bladder cancer.

Indomethacin-mediated reversal of multidrug resistance and drug efflux in human and murine cell lines overexpressing MRP, but not P-glycoprotein.

Decreased accumulation of the fluorescent dye BCECF [2', 7'-bis-(2-carboxyethyl)-5-(6)- carboxyfluorescein] characterized murine and human multidrug-resistant cell lines overexpressing the multidrug resistance protein (MRP). Indomethacin (10 microM), a known cyclo-oxygenase and glutathione-S-transferase inhibitor as well as a modulator of anion transport, increased accumulation and blocked efflux of BCECF in MRP-expressing murine and human cells. The drug did not affect P-glycoprotein (P-gp)-mediated export of rhodamine 123. The indomethacin effect on BCECF efflux was not reversed by the addition of exogenous prostaglandins, suggesting that the drug acts by a mechanism other than decreasing prostaglandin synthesis. Indomethacin also increased multidrug susceptibility of both murine and human cell lines overexpressing MRP, but not those displaying P-gp-associated resistance. In addition, indomethacin modulated the decreased vincristine accumulation in cells expressing MRP, but not in those expressing P-gp. These data suggest that indomethacin is a specific inhibitor of MRP, possibly functioning by inhibition of glutathione-S-transferase or, alternatively, by direct competition with the drug at the transport site.

Aureobasidin A, an antifungal cyclic depsipeptide antibiotic, is a substrate for both human MDR1 and MDR2/P-glycoproteins

The human MDR1 gene encodes the multidrug transporter P-glycoprotein (Pgp). Although the MDR2/Pgp shares about 80% identity at the amino acid level with the MDRI/Pgp, the MDR2/Pgp cannot act as a multidrug transporter. We examined the drug sensitivity of Saccharomyces cerevisiae expressing either the human MDR1/Pgp or MDR2/Pgp. The human MDR1/Pgp conferred about 4-fold resistance to aureobasidin A, a cyclic depsipeptide antifungal antibiotic, on the drug-sensitive yeast strains. Interestingly the human MDR2/Pgp also conferred about 2.5-fold resistance to aureobasidin A. The resistance to aureobasidin A conferred by the MDR2/Pgp as well as by the MDR1/Pgp was overcome by vinblastine, verapamil, and cyclosporin A, depending on their concentrations, but not by colchicine. Aureobasidin A probably interacts directly with Pgps, because it overcame multidrug resistance of human cells and inhibited azidopine photoaffinity labeling of MDRI/Pgp in human cell membranes. These results suggest the possibility that the human MDR1 and MDR2/Pgps have conserved domain(s) for drug recognition.