Active Drug Efflux Research Articles

230 THE RENAL CELL CARCINOMA SIDE POPULATION: THE POTENTIAL USE OF TARGETED AGENTS AS NOVEL MDR MODULATORS TO CIRCUMVENT CHEMORESISTANCE

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011230 THE RENAL CELL CARCINOMA SIDE POPULATION: THE POTENTIAL USE OF TARGETED AGENTS AS NOVEL MDR MODULATORS TO CIRCUMVENT CHEMORESISTANCE Matthew Liew, Thomas Tawadros, Claire Hart, Michael Brown, and Noel Clarke Matthew LiewMatthew Liew Manchester, United Kingdom More articles by this author , Thomas TawadrosThomas Tawadros Manchester, United Kingdom More articles by this author , Claire HartClaire Hart Manchester, United Kingdom More articles by this author , Michael BrownMichael Brown Manchester, United Kingdom More articles by this author , and Noel ClarkeNoel Clarke Manchester, United Kingdom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.299AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Renal cell carcinoma (RCC) is the commonest malignant renal tumour accounting for 60,000 new cases and 13,000 deaths in the US per year. The only curative treatment is surgery in early stage disease, as metastatic disease is highly chemo-resistant with response rates of less than 6%. A key mechanism of chemoresistance is the ability to efflux chemotherapeutic agents via multidrug resistant pumps (MDR), where MDR expression has been correlated with a poorer prognosis. We have previously shown that RCC tumours contain a verapamil sensitive side population (SP), as defined by their ability to efflux the fluorescent dye Hoechst 33342. Moreover, recent reports have suggested that the newer ‘targeted' agents used in metastatic RCC may also target MDR. The objective of this study was to assess the ability of targeted agents to sensitise RCC to other chemotherapeutic agents. METHODS RCC SP and Non-SP populations were treated with various chemotherapeutic agents. IC50 values were determined by SRB assay and drug efflux by FACS and fluorescence microscopy. Combination studies were also conducted using verapamil, temsirolimus, sunitinib and sorafenib as potential MDR inhibitors. RESULTS Verapamil sensitive SPs were isolated from 5 out of the 8 RCC cell lines tested. The 2245R cell line had the highest SP (6.0 ± 4.6%), and was chosen for further studies. The IC50 values for the SP, compared to the non SP, was significantly higher with docetaxel, paclitaxel, adriamycin and etoposide (5.4 vs 3.3, 42 vs 13, 90 vs 48 nM, and 11.6 vs 8.8 μM respectively). There was no difference seen between sub-populations treated with cisplatin or 5-fluorouracil. Retention of adriamycin fluorescence following treatment was significantly lower in the SP using FACS and fluorescent microscopy techniques. Sunitinib, sorafenib and temsirolimus were able to block the SP at ìM concentrations with a similar efficiency to verapamil, and these agents were able to reverse the enhanced chemoresistance observed in the SP. CONCLUSIONS We have shown that the SP cells are the chemo-resistant sub-population in RCC cell lines and established that the increased resistance seen is via active drug efflux pump mechanisms. We have also demonstrated that this process can be reversed using ‘targeted' agents as novel MDR modulators. This data indicate that the use of conventional chemotherapy should be considered in future RCC trials using ‘targeted' agents. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e94 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew Liew Manchester, United Kingdom More articles by this author Thomas Tawadros Manchester, United Kingdom More articles by this author Claire Hart Manchester, United Kingdom More articles by this author Michael Brown Manchester, United Kingdom More articles by this author Noel Clarke Manchester, United Kingdom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells

Human hepatic cancer cells, HepG2, formed spheroids on a poly-L-glutamic acid-coated dish. Doxorubicin (DOX) efflux activity of the cells in spheroid culture was higher than that in monolayer culture due to the higher expression of MDR1 protein of the cells in spheroids compared with those in monolayer. The amount of MDR1 per cell in spheroids was similar to that of hepatic tumor tissue in vivo. Consequently, it was suggested that the drug efflux activity of cells in spheroid culture reflected the activity of hepatic cancer cells. Furthermore, the IC(50) of DOX and epirubicin (EPI) in HepG2 cells, both of which are known to be exported by MDR1, were higher in spheroid compared with monolayer cells, while IC(50) of 5-fluorouracil (5-FU), which is not exported by MDR1 protein, was almost the same in both types of culture. The higher IC(50) of DOX and EPI in HepG2 cells in spheroid culture was associated with a higher efflux activity of the drugs in the spheroid-cultured cells, which appeared to reflect the IC(50) of DOX and EPI in cancer cells in vivo. Therefore, a spheroid culture of hepatic cancer cells seems to provide a promising cell-based in vitro assay system for examining the proper IC(50) values of anticancer agents that would reflect the drug resistance of cancer cells in vivo. In addition, the system would be useful in screening for inhibitors of MDR1 activity, which will help to overcome the multidrug resistance of cancer cells.

Different Transport Activity of Human Triallelic MDR1893Ala/Ser/Thr Variant and its Association with Herb Extracts

Individual pharmacokinetic differences for herb-drug interaction have been associated with genetic variations of the multidrug resistance (MDR) gene. A high level expression of MDR protein increases cellular efflux and might decrease drug sensitivity. This study investigated the drug efflux activity difference of human MDR1 triallelic variant 2677G/T/A (rs2032582), as a nonsynonymous 893Ala/Ser/Thr, using Xenopus laevis oocytes and MDR1 overexpressing LLC-PK1 cells. Two MDR1 variants (2667T/893Ser and 2667A/893Thr) were generated using human MDR1 cDNA (2677G/893Ala). No significant difference in the expression of MDR1 893Ala/Ser/Thr was found in X. laevis oocytes. However, the MDR1 2667A/893Thr variant interestingly showed a significant decrease of efflux activity for both digoxin and daunorubicin compared with those of 893Ala and 893Ser variants. In further investigation assessing the inhibitory effects of three herbal extracts on MDR1, 893Ala and 893Ser showed significant decreases of efflux activities in treatments with P. cocos (p = 0.005 for 893Ser) and D. dasycarpus (p = 0.0009 for 893Ala; p = 0.002 for 893Ser) in X. laevis oocytes. The results in this study suggest that herbal medicines could interact with other drugs and change the therapeutic effects depending on the genetic polymorphisms of individuals.

Molecular mechanisms of antibiotic resistance

Over the past decade, resistance to antibiotics has emerged as a crisis of global proportion. Microbes resistant to many and even all clinically approved antibiotics are increasingly common and easily spread across continents. At the same time there are fewer new antibiotic drugs coming to market. We are reaching a point where we are no longer able to confidently treat a growing number of bacterial infections. The molecular mechanisms of drug resistance provide the essential knowledge on new drug development and clinical use. These mechanisms include enzyme catalyzed antibiotic modifications, bypass of antibiotic targets and active efflux of drugs from the cell. Understanding the chemical rationale and underpinnings of resistance is an essential component of our response to this clinical challenge.

Modulatory effects of curcumin on multi-drug resistance-associated protein 5 in pancreatic cancer cells

Chemotherapy of pancreatic cancer often fails due to the development of intrinsic and acquired resistance during drug treatment. Recent studies have suggested that MRP5 conferred resistance to first-line drugs 5-fluorouracil and gemcitabine by active efflux of drugs from the cell. Our aim was to evaluate whether curcumin could reverse this multi-drug resistance by inhibition of MRP5-mediated efflux. MRP5 protein was detected and localized by immunocytochemistry using a monoclonal antibody in MRP5 over-expressing HEK293 (HEK293/MRP5) cells and two pancreatic cancer cell lines PANC-1 and MiaPaCa-2. The cellular accumulation of a specific MRP5 fluorescent substrate 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) into these cells was measured by flow cytometry and the cell proliferation determined by a 72-h CyQuant assay. The cellular accumulation of BCECF in HEK293/MRP5 cells and in PANC-1 and MiaPaCa-2 cells was significantly increased by curcumin in a concentration-dependent manner. Curcumin and a MRP5 inhibitor MK571 had no apparent effects on cellular accumulation of BCECF in parental HEK293 cells. In the proliferation assays, curcumin caused a concentration-dependant increase in the sensitivity to the cytotoxic drug 5-fluorouracil in HEK293/MRP5 cells, PANC-1 and MiaPaCa-2 pancreatic cancer cells, but not in parental HEK293 cells. Our results suggest that curcumin is an inhibitor of MRP5 and may be useful in the reversal of multi-drug resistance in pancreatic cancer chemotherapy.

Image-Based Chemical Screening Identifies Drug Efflux Inhibitors in Lung Cancer Cells

Cancer cells with active drug efflux capability are multidrug resistant and pose a significant obstacle for the efficacy of chemotherapy. Moreover, recent evidence suggests that high drug efflux cancer cells (HDECC) may be selectively enriched with stem-like cancer cells, which are believed to be the cause for tumor initiation and recurrence. There is a great need for therapeutic reagents that are capable of eliminating HDECCs. We developed an image-based high-content screening (HCS) system to specifically identify and analyze the HDECC population in lung cancer cells. Using the system, we screened 1,280 pharmacologically active compounds that identified 12 potent HDECC inhibitors. It is shown that these inhibitors are able to overcome multidrug resistance (MDR) and sensitize HDECCs to chemotherapeutic drugs, or directly reduce the tumorigenicity of lung cancer cells possibly by affecting stem-like cancer cells. The HCS system we established provides a new approach for identifying therapeutic reagents overcoming MDR. The compounds identified by the screening may potentially be used as potential adjuvant to improve the efficacy of chemotherapeutic drugs.

ChemInform Abstract: Efflux Pump Inhibitors: A Strategy to Combat P‐Glycoprotein and the NorA Multidrug Resistance Pump

AbstractReview: 84 refs.

Efflux Pump Inhibitors: A Strategy to Combat P‐Glycoprotein and the NorA Multidrug Resistance Pump

Multidrug resistance (MDR) is the cause of an ever-increasing number of problems in the treatment of cancers and bacterial infections. The active efflux of drugs contributes significantly to this phenomenon. This minireview summarizes recent advances in combating MDR, with particular emphasis on natural and synthetic efflux pump inhibitors of P-glycoprotein in resistant tumor cells and of the NorA MDR pump in Staphylococcus aureus.

Abstract 1534: Bioassay-guided identification of novel P-glycoprotein inhibitors from traditional Chinese medicines

Abstract Multi-drug resistance (MDR) is an obstacle for therapeutic success of anticancer agents. Overexpression of P-glycoprotein (P-gp), encoded by ABCB1 gene, plays a key role in conferring MDR via active efflux of anticancer drugs from cells. Previous efforts with several generations of P-gp inhibitors had not been successful in modulating clinical MDR. Recently, an increasing number of cancer patients are using complementary and alternative medicines, in particular, traditional Chinese medicines (TCMs), in combination with conventional anticancer agents. The purpose of this study is to examine the effects of TCM extracts on modulating P-gp function. We initiated our study from four single herbal products (Glycyrrhiza uralensis, Hibiscus syriacus, Tripterygium wilfordii, and Cephalotaxus sinensis) and one formula of herbal mixture (Si-Wu-Tang), using a bio-assay guided fractionation method. Crude extracts were firstly prepared with ethanol. The most active extracts identified were further extracted with hexane, chloroform, acetone and ethanol sequentially to obtain fractions of increasing polarity. The MDR modulating effects of the crude extracts and individual fractions were evaluated based on their capability to enhance daunorubicin (DNR) accumulation in P-gp over-expressing human leukemia cell line K562/DOX. The intracellular fluorescence intensity in the presence or absence of test drugs or known P-gp inhibitor cyclosporine A (10 µM) was measured by flow cytometer. The crude extracts were also tested for cytotoxicity against K562/DOX cells by the MTS cell proliferation assay. This study revealed that among the five TCMs, the crude extracts of Tripterygium wilfordii demonstrated a dose-dependent effect on increasing the DNR accumulation, indicating the presence of novel P-gp inhibitor(s). Upon further separation, the chloroform fractions of this herb showed the highest P-gp inhibitory activity. However, known compounds present in this herb, celastrol, triptolide and myristic acid did not show effects on DNR accumulation. Most of the plant extracts tested were able to inhibit the growth of K562/DOX cells with Cephalotaxus sinensis being the most effective, consistent with literature reports that this herb contains highly cytotoxic compound homoharringtonine and congeners. The potency of P-gp inhibition for the five TCMs was not correlated with their cytotoxicity. In conclusion, our results demonstrated that the chloroform fractions of Tripterygium wilfordii contain unknown components for P-gp inhibition. Our current efforts are focused on isolation and identification of active compounds from it. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1534.

Microarray Analysis of Efflux Pump Genes in Multidrug-ResistantMycobacterium tuberculosisDuring Stress Induced by Common Anti-Tuberculous Drugs

Treatment of multidrug-resistant tuberculosis has become one of the major problems in public health. Understanding the molecular mechanisms of drug resistance has been central to tuberculosis research in recent times. DNA microarray technology provides the platform to study the genomic variations related to these mechanisms on a comprehensive level. To investigate the role of efflux pumps in drug resistance, we have constructed a custom DNA microarray containing 25 drug efflux pump genes of Mycobacterium tuberculosis (Indian Patent file no. 2071/DEL/2007) and monitored changes in the expression of these genes on exposure of common anti-tuberculous drugs. Expression profiling of efflux pump genes in multidrug-resistant M. tuberculosis isolates showed overexpression of 10 genes following exposure to various anti-tuberculous drugs. Although two of these genes (Rv3065 and Rv2938) have already been reported to be active drug efflux pumps in M. tuberculosis in earlier studies, the increased activities of other eight efflux pump genes (Rv1819, Rv2209, Rv2459, Rv2477c, Rv2688, Rv2846, Rv2994, and Rv3728) have been demonstrated in multidrug-resistant isolates by us for the first time. After confirmation of differential expressions of these genes by real-time reverse transcription polymerase chain reaction, it was observed that a simultaneous overexpression of efflux pump genes Rv2459, Rv3728, and Rv3065 was associated with resistance to the combination of isoniazid and ethambutol, and these drugs, along with streptomycin, were identified to group together, where efflux-mediated drug resistance appears to be important in M. tuberculosis and follows a constant pattern of induction in multidrug-resistant isolates. Isoniazid and ethambutol combination was also found to be affected in 10% (6/60) of the clinical isolates in the presence of carbonyl cyanide m-chloro phenylhydrazone in resazurin microtitre plate assay, supporting the role of efflux pumps in the resistance to these drugs. Overexpression of two of the genes (Rv2477 and Rv2209) has also been observed with ofloxacin stress in M. tuberculosis.

Human Skeletal Muscle Drug Transporters Determine Local Exposure and Toxicity of Statins

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance-associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

P-glycoprotein (ABCB1) modulates collateral sensitivity of a multidrug resistant cell line to verapamil

P-glycoprotein (or P-gp1, ABCB1) expression in tumor cells is causative of multidrug resistance through the active efflux of drugs across the cell membrane. However, the over-expression of P-glycoprotein in some tumor cells has been associated with increased sensitivity, or “collateral sensitivity”, of multidrug resistant cells to specific drugs, including the calcium channel blocker verapamil. We previously demonstrated that collateral sensitivity to verapamil correlates with the effect of this drug on P-gp1 ATPase, and is reversed by inhibitors of P-gp1 ATPase (e.g., PSC 833 and Ivermectin). In this report, we expand on our earlier study and demonstrate that P-gp1 expression in drug-resistant cells modulates collateral sensitivity. Using P-gp1-specific siRNA, P-gp1 expression in the multidrug resistant CH RC5 cells was significantly down-regulated beginning on day 2 post-transfection of siRNA. Furthermore, down-regulation of P-gp1 led to increased sensitivity of CH RC5 cells to paclitaxel and doxorubicin, but not to cis-platinum, due to inhibition of P-gp1 drug efflux pump. Down-regulation of P-gp1 expression completely reversed collateral sensitivity to verapamil. Moreover, known inhibitors of ETC, rotenone and antimycin A which cause an increase in reactive oxygen species, synergized with verapamil-induced collateral sensitivity leading to increased cell death as determined by MTT cell survival assay. Similarly, the addition of hydrogen peroxide also synergized with verapamil. Taken together, the results of this study demonstrate a direct link between P-gp1 expression and collateral sensitivity of drug-resistant cells, possibly due to an increase in reactive oxygen species.

Influence of P-glycoprotein (P-gp) induction on the pharmacokinetics of risperidone and paliperidone in mouse brain

P-glycoprotein (P-gp) is responsible for active efflux of drugs at the blood-brain barrier (BBB). With this study we wanted to investigate the consequences of P-gp induction on the pharmacokinetics of the known P-gp substrates risperidone and its active metabolite 9-OH-risperidone (paliperidone) in P-gp wildtype (WT) mice (FVB/N) and double-knockout (KO) mice (mdr1a/1b -/-/-/-). We used the known P-gp inducing substances dexamethasone (50mg/kg/d) and 5-pregnene-3beta-ol-20-one-16alpha-carbonitrile (PCN) (25mg/kg/d) for our study and injected them i.p. for 4 days. As controls solvent only was injected. On day 5 risperidone (3mg/kg) was i.p. injected. 1, 3 and 6h after the risperidone injection brain levels of risperidone and paliperidone were analysed using a reversed phase high performance liquid chromatography (RP-HPLC) method. The active moiety, sum of risperidone and paliperidone, were calculated due to their similar receptor profile and pharmacological effect with respect to therapeutic drug monitoring (TDM) standards. The results demonstrate the outstanding influence of transporter proteins in the disposition of risperidone and paliperidone. Brain levels of the active moiety were significantly changed in both induction groups. Both inducers showed distinct decreases in the area-under-the-data (AUD) and the brain versus plasma (B/P) ratios. But the results of the KO mice and the risperidone vs. 9-OH-risperidone (RIS:9-OH-RIS) ratios suggest that other mechanisms must be involved.

Effect of sinomenine on the in vitro intestinal epithelial transport of selected compounds

Herbal products can interfere with allopathic medicinal treatment through pharmacokinetic and pharmacodynamic interactions. Although pharmacokinetic interactions that alter drug absorption may cause variable and unsatisfactory drug bioavailability, a drug absorption enhancement effect of a herb may be used to ensure sufficient absorption of poorly absorbable drugs. The effect of the hydrochloride salt of sinomenine, an alkaloid obtained from the plant Sinomenium acutum, on the transepithelial electrical resistance and transport of different compounds (including cimetidine, vitamin C, rutin, luteolin and insulin) across Caco-2 epithelial cell monolayers was investigated in this study. Sinomenine HCl induced a concentration dependent lowering effect on the transepithelial electrical resistance of Caco-2 cell monolayers, which was completely reversible. Sinomenine HCl significantly increased the transport of all the test compounds in the apical-to-basolateral direction compared with the control group and decreased the transport of cimetidine, a P-glycoprotein substrate, in the basolateral-to-apical direction. From these results it can be concluded that sinomenine HCl increases drug absorption across the intestinal epithelium by means of one or more mechanisms including a transient opening of the tight junctions (as indicated by a reduction in transepithelial electrical resistance) to allow for paracellular transport and/or inhibition of active drug efflux transport (as indicated by inhibition of basolateral-to-apical transport of cimetidine).

Trinity revealed: Stoichiometric complex assembly of a bacterial multidrug efflux pump

The capacity of bacteria to expel from themselves a broad diversity of compounds confers upon them resistance to drugs and other bacteriocidal agents (1). In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa multidrug efflux is achieved by energy-powered macromolecular machinery that extrudes cytotoxic substances like multiple anti biotics, dyes, bile salts, and detergents from the inner membrane of the cell directly into the medium, bypassing the periplasm and the outer membrane. Known complexes of such multidrug efflux pumps are composed for three types of components: an outer membrane channel, a periplasmic bridge, and an inner membrane energy-transducer. There are a variety of possible inner membrane components, which can include members of the ATP binding cassette transporter superfamily, the major facilitator superfamily, or the resistance nodulation cell division (RND) superfamily. These inner membrane-located units are the major sites for substrate recognition and energy transduction of the entire tripartite system and function in conjunction with the 2 other pump components. The periplasmic bridge component often goes by the name of “membrane fusion protein” (MFP) for historical reasons and is represented by the AcrA protein. Well-characterized representatives of the outer membrane factor (OMF) are channel-like proteins such as TolC. Both the outer membrane and tripartite efflux pumps are considered to be the main barrier for drugs on their way to the inside of the bacterial cell (2). Importantly, only assembly of all 3 components into a trinity results in an active and effective drug efflux phenotype. In this issue of PNAS, Symmons et al. (3) complete the picture of an E. coli 3-component multidrug efflux system with a well-tested and insightful model.

Role of the AraC–XylS family regulator YdeO in multi-drug resistance of Escherichia coli

Multi-drug efflux pumps contribute to the resistance of Escherichia coli to many antibiotics and biocides. In this study, we report that the AraC-XylS family regulator YdeO increases the multi-drug resistance of E. coli through activation of the MdtEF efflux pump. Screening of random fragments of genomic DNA for their ability to increase beta-lactam resistance led to the isolation of a plasmid containing ydeO, which codes for the regulator of acid resistance. When overexpressed, ydeO significantly increased the resistance of the E. coli strain to oxacillin, cloxacillin, nafcillin, erythromycin, rhodamine 6G and sodium dodecyl sulfate. The increase in drug resistance caused by ydeO overexpression was completely suppressed by deleting the multifunctional outer membrane channel gene tolC. TolC interacts with different drug efflux pumps. Quantitative real-time PCR showed that YdeO activated only mdtEF expression and none of the other drug efflux pumps in E. coli. Deletion of mdtEF completely suppressed the YdeO-mediated multi-drug resistance. YdeO enhances the MdtEF-dependent drug efflux activity in E. coli. Our results indicate that the YdeO regulator, in addition to its role in acid resistance, increases the multi-drug resistance of E. coli by activating the MdtEF multi-drug efflux pump.

Multidrug resistant P-glycoprotein positive L1210/VCR cells are also cross-resistant to cisplatin via a mechanism distinct from P-glycoprotein-mediated drug efflux activity

P-glycoprotein (P-gp, a drug transporter found in the plasma membrane)-mediated multidrug resistance of leukemia cells represents a real obstacle in the effective chemotherapeutic treatment of leukemia. While cisplatin (CisPt) is known to be a substance that is untransportable by P-gp, P-gp positive cells were often found to be resistant to CisPt. The aim of the current paper is to study this phenomenon using P-gp positive mouse leukemia cells L1210/VCR in which the overexpression of P-gp was induced by its ability to adapt to growth on vincristine (VCR). L1210/VCR cells are also resistant to CisPt. However, resistance to this substance could not be reversed by addition of the known P-gp inhibitor verapamil. CisPt induced more pronounced entry into apoptosis, as measured using the annexin V/propidium iodide kit, in sensitive L1210 cells than in resistant L1210/VCR cells. In addition, CisPt induced an increase in the proportion of L1210 cells that were in the g2 phase of the cell cycle when compared to L1210/VCR cells, as measured by staining with propidium iodide. Similarly, a higher release of cytochrome c from the mitochondria to the cytosol was induced by CisPt treatment in L1210 than in L1210/VCR cells. While similar levels of Bax and Bcl-2 proteins were observed in sensitive and resistant cells, CisPt induced a more pronounced decrease of the Bcl-2 levels in L1210 cells than in L1210/VCR cells. Consistent with this observation, CisPt induced a larger decrease of the Bcl-2 content in the Bcl-2:Bax heterooligomer in L1210 cells than in L1210/VCR cells. Moreover, CisPt induced a similar apoptotic DNA fragmentation pattern in both resistant and sensitive cells. All of the above observations indicated that L1210/VCR cells are also resistant to CisPt and that this resistance is related to the differences in the regulatory mechanisms responsible for CisPt-induced apoptosis in L1210/VCR cells without any contribution from the drug efflux activity of P-gp.

Effects of the High-Affinity Peptide Reversin 121 on Multidrug Resistance Proteins in Experimental Pancreatic Cancer

Objectives: Multidrug resistance (MDR) is the major obstacle in the treatment of pancreatic carcinoma. Recent studies indicate resistance-modulating effects of high-affinity peptides. Material and Methods: Pancreatic cancer was induced in an ultrasound-guided orthotopic mouse model. Expression and function of MDR proteins (MRPs) P-glycoprotein MRP1 and MRP3 were analyzed in vitroandin vivo by multicolor flow-cytometric analysis. Proliferation of tumor cells was evaluated with a nonradioactive proliferation assay. Animals and cells received standard chemotherapy or chemotherapy plus the high-affinity peptide reversin 121 (R121). Results: The proportions of P-glycoprotein-, MRP1- and MRP3-positive tumor cells increased significantly after chemotherapy in vitro and in vivo. Drug efflux activity increased after chemotherapy. Addition of R121 to chemotherapeutic regimens significantly reduced the proportions of tumor cells positive for MRPs in vitroandin vivo. Tumor size and prevalence of metastases decreased significantly compared to untreated controls. Conclusion: Beneficial effects of high-affinity peptide R121 on reversing chemotherapy-induced MDR were demonstrated in pancreatic cancer.

Analysis of ABCG2 expression and side population identifies intrinsic drug efflux in the HCC cell line MHCC-97L and its modulation by Akt signaling

Active drug efflux by the adenosine triphosphate-binding cassette (ABC) transporter ABCG2 is one of the common mechanisms causing multiple drug resistance in various human cancers. In the intrinsic drug resistance of hepatocellular carcinoma (HCC), the role of ABCG2 is closely associated with 'side population (SP)', a minor subset of cancer stem-like cells with unique capacity to extrude lipophilic dye Hoechst 33342 and many chemotherapeutic agents. In this study, we showed that ABCG2 was intrinsically expressed in a subgroup of HCC tissues and its expression pattern significantly influenced the levels of drug efflux from HCC cell lines. In MHCC-97L HCC cell line with intrinsic ABCG2 expression, we confirmed the importance of SP cells to the drug efflux-related chemotherapy resistance and found that the SP analysis provided an efficient method to evaluate the functional activity of ABCG2 transporter. In this cell line, we discovered that the SP proportion was modulated by the treatments of Akt signaling inhibitors and serum supplement, which led to the finding that Akt signaling was able to regulate the SP cells' efflux activity via altering the subcellular localization of ABCG2 transporter. We further demonstrated that the Akt signaling inhibition attenuated the doxorubicin efflux from MHCC-97L cells and increased the drug efficacy. Our results indicate the protective role of intrinsic ABCG2 expression in HCC cells and suggest that suppressing Akt signaling could help overcome the drug efflux by ABCG2 transporter.

Inducing remission in drug resistant acute myeloid leukaemia with cyclosporin A

Recent research suggests that over expression of P-glycoprotein is involved in the resistance of some malignancies to structurally unrelated cytotoxic agents (Pastan & Gottesman, 1987; Kaye & Kerr 1991). It is postulated that P-glycoprotein decreases intracellular concentrations of cytotoxic agents by promoting their active efflux across the cell membrane (Musto et al. 1991). Modulating agents such as cyclosporin A and its analogues, (Sonneveld & Nooter 1990), verapamil, quinidine, the newer cephalosporines and torenifene have been put forward to inhibit resistance, by inhibiting active drug efflux. We report a case of acute myeloid leukaemia which was resistant to standard induction therapy. In vitro tests showed cyclosporin A to increase the sensitivity of the patient's myeloblasts to daunorubicin by threefold. Remission was successfully induced when cyclosporin A was combined with daunorubicin, cytosine, arabinoside and etoposide.