Energy-dependent Efflux Mechanism Research Articles

P-Glycoprotein Activity at Diagnosis Does Not Predict Therapy Outcome and Survival in Canine B-Cell Lymphoma.

Simple SummaryClinical experience in human and canine clinics shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and eventually, most tumors become resistant to all available therapies. The most straightforward cause of therapy resistance is linked to cellular alterations that prevent drugs from acting on their target. Drug efflux mediated by the ABC transporter P-glycoprotein (P-gp) contributes to unfavorable treatment outcome in several human malignancies. Here, we characterize a large cohort of canine B-cell lymphoma patients followed for over 7 years. We show that the intrinsic P-gp activity of tumor cells characterized at the time of diagnosis is not predictive for therapy outcome. Our results highlight the complexity of clinical drug resistance mechanisms and suggests that the relevance of P-gp in acquired resistance should be further investigated by the continuous monitoring of tumor cells during treatment.Various mechanisms are known to be involved in the development of multidrug resistance during cancer treatment. P-glycoprotein (P-gp) decreases the intracellular concentrations of cytotoxic drugs by an energy-dependent efflux mechanism. The aim of this study was to investigate the predictive value of P-gp function based on the evaluation of P-gp activity in tumor cells obtained from canine B-cell lymphoma patients at diagnosis. P-gp function of 79 immunophenotyped canine lymphoma samples was determined by flow cytometry using the Calcein assay. Dogs were treated with either the CHOP or the L-CHOP protocol, a subset of relapsed patients received L-asparaginase and lomustine rescue treatments. Among the 79 dogs, the median overall survival time was 417 days, and the median relapse-free period was 301 days. 47 percent of the samples showed high P-gp activity, which was significantly higher in Stage IV cancer patients compared to Stage II + III and V. Whereas staging was associated with major differences in survival times, we found that the intrinsic P-gp activity of tumor cells measured at diagnosis is not predictive for therapy outcome. Further studies are needed to identify the intrinsic and acquired resistant mechanisms that shape therapy response and survival in B-cell canine lymphoma patients.

RLIP76: A Target for Kidney Cancer Therapy

RLIP76 is a multifunctional transporter protein that serves as an energy-dependent efflux mechanism for endogenously generated toxic metabolites as well as exogenous toxins, including chemotherapy drugs. Our recent studies in cultured cells, syngeneic animal tumor model, and in xenograft model have shown that RLIP76 serves a major cancer-specific antiapoptotic role in a wide variety of histologic types of cancer, including leukemia, melanoma, colon, lung, prostate, and ovarian cancer. Results of present studies in cell culture and xenograft model of Caki-2 cells show that RLIP76 is an important anticancer for kidney cancer because inhibition of RLIP76 function by antibody or its depletion by small interfering RNA or antisense DNA caused marked and sustained regression of established human kidney xenografts of Caki-2 cells in nude mouse.

Functional characterization of Brucella melitensis NorMI, an efflux pump belonging to the multidrug and toxic compound extrusion family.

Two putative proteins (NorMI and NorMII) similar to the multidrug efflux protein NorM of Vibrio parahaemolyticus are encoded by the Brucella melitensis 16 M genome. We show that a drug-hypersusceptible Escherichia coli strain overexpressing NorMI displays increased resistance to norfloxacin, ciprofloxacin, gentamicin, tetraphenylphosphonium ion, acriflavine, and berberine. This elevated resistance was proven to be mediated by an energy-dependent efflux mechanism. NorMI belongs to the multidrug and toxic compound extrusion family and is the first multidrug efflux protein identified in Brucella spp.

Involvement of adaptive mechanism and an energy-dependent efflux system of Pseudomonas sp. LE2 counteracting insecticide lindane

An adaptive mechanism and efflux-transport system of Pseudomonas sp. LE2 counteracting organochlorine insecticide lindane were investigated to understand the survival mechanism of this microorganism in lindane-contaminated environment. Changes in membrane fluidity and fatty acid composition of Pseudomonas sp. LE2 cells grown on glucose either in the presence or absence of lindane were determined. Considerably high ratio of total saturated to unsaturated fatty acids in cells grown on glucose in the presence of lindane was observed as compared to that in cells grown on glucose in the absence of lindane. Lindane significantly affected in vitro the membrane of Pseudomonas sp. LE2 cells by decreasing the fluorescence polarization of membrane probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) inserted into cytoplasmic membrane. Less fluidizing effect of lindane on the membranes of cells that had adapted to lindane was observed. Lindane transport was assessed by incubating cell suspensions with [ 14C]lindane either in the presence or absence of sodium azide, a membrane transport inhibitor, and by measuring the radioactivity in cell pellets. No time-dependent and steady-state increase in the radioactivity of [ 14C]lindane without sodium azide treatment was observed in cell pellets, suggesting simple diffusion mechanism for lindane accumulation in cells. Significant increase in the radioactivity was observed in cell pellets by sodium azide treatment. Sodium azide inhibited the efflux of lindane by Pseudomonas sp. LE2. These results suggest the view that Pseudomonas sp. LE2 capable of growing in the presence of lindane is able to adapt to the fluidizing action of lindane by incorporating the saturated fatty acid into membrane lipids, and eliminate lindane from cells by an energy-dependent efflux mechanism.

Characterization of the regional intestinal kinetics of drug efflux in rat and human intestine and in Caco-2 cells.

The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 microns to 1000 microns. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C4 (100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3 degrees C to 37 degrees C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Ileal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 microns. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Each of the intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Km's were in the micro-molar range, and involved an energy dependent mechanism(s).

Active efflux of bile salts by Escherichia coli.

Enteric bacteria such as Escherichia coli must tolerate high levels of bile salts, powerful detergents that disrupt biological membranes. The outer membrane barrier of gram-negative bacteria plays an important role in this resistance, but ultimately it can only retard the influx of bile salts. We therefore examined whether E. coli possessed an energy-dependent efflux mechanism for these compounds. Intact cells of E. coli K-12 appeared to pump out chenodeoxycholate, since its intracellular accumulation increased more than twofold upon deenergization of the cytoplasmic membrane by a proton conductor. Growth inhibition by bile salts and accumulation levels of chenodeoxycholate increased when mutations inactivating the acrAB and emrAB gene clusters were introduced. The AcrAB system especially appeared to play a significant role in bile acid efflux. However, another efflux system(s) also plays an important role, since the accumulation level of chenodeoxycholate increased strongly upon deenergization of acrA emrB double mutant cells. Everted membrane vesicles accumulated taurocholate in an energy-dependent manner, apparently consuming delta pH without affecting delta psi. The efflux thus appears to be catalyzed by a proton antiporter. Accumulation by the everted membrane vesicles was not decreased by mutations in acr and emrB genes and presumably reflects activity of the unknown system seen in intact cells. It followed saturation kinetics with Vmax and Km values in the neighborhood of 0.3 nmol min(-1) mg of protein(-1) and 50 microM, respectively.

The accumulation of novobiocin by Escherichia coli and Staphylococcus aureus.

The accumulation of novobiocin by Escherichia coli was shown to be a nonsaturable process. Inhibitors or uncouplers of respiration had no effect on accumulation in cells grown in Isosensitest broth, whereas, bacteria grown in nutrient broth demonstrated an energy-dependent efflux mechanism. In contrast, accumulation of novobiocin by Staphylococcus aureus was found to have a Km of 74 microM and uncouplers and inhibitors of respiration reduced the accumulation suggesting that in S. aureus novobiocin is transported actively.

Cycloheximide efflux in antibiotic-adapted cells of the fungus Mucor racemosus.

Mucor racemosus cells adapted to either cycloheximide or trichodermin were approximately 40-fold more resistant to cycloheximide than nonadapted cells. Ribosomes isolated from adapted and nonadapted cells were equally sensitive to cycloheximide in an in vitro poly(U) translation assay. There was no detectable modification of cycloheximide by adapted cells. Uptake of drug by nonadapted and adapted cells was characterized by a rapid initial accumulation during the first 2 min of incubation with [3H]cycloheximide, followed by a steady-state intracellular drug concentration well below that of the medium. The steady-state drug concentration was approximately 10-fold lower in adapted cells than in nonadapted cells. Treatment of cells with sodium azide or dinitrophenol abolished the difference between uptake of drug by nonadapted and adapted cells and resulted in intracellular drug levels equal to that of the medium. Direct efflux measurements showed that adapted cells loaded with cycloheximide were able to excrete the drug far more rapidly than nonadapted cells. These results suggest that both nonadapted and adapted cells possess an energy-dependent efflux mechanism for transporting cycloheximide and that resistance in adapted cells is due to increased efficiency of transport.

Cytofluorescence localization of adriamycin in resistant colon cancer cells.

A simple fluorescent microscopic method demonstrated that adriamycin was distributed in two cellular compartments of living rat colon cancer cells. Adriamycin accumulated slowly in cytoplasmic granules, probably lysosomes, where it persisted long after the drug was removed from the medium. On the other hand, adriamycin accumulated rapidly in the nucleus, but was rapidly cleared in adriamycin-free medium. Drug efflux from the nucleus was blocked by sodium azide in glucose-free medium or by verapamil, a calcium-blocking agent. When colon cancer cells were cultivated for 1 day or longer in adriamycin-containing medium no nuclear fluorescence was observed. However, the addition of sodium azide to glucose-free medium or verapamil restores the nuclear fluorescence. The colon cancer cells had low sensitivity to adriamycin, but the addition of verapamil strongly enhanced adriamycin toxicity. Thus adriamycin is permanently cleared from the nucleus of rat colon cancer cells through an energy-dependent efflux mechanism, which is blocked by verapamil. The efficiency of this efflux mechanism is enhanced by exposure of the cell to adriamycin. This mechanism could be involved in the resistance of colon cancer to adriamycin.

An energy-dependent efflux mechanism for fenarimol in a wild-type strain and fenarimol-resistant mutants of Aspergillus nidulans

Uptake of [ 14C]fenarimol (30 μ M) by mycelium of wild-type Aspergillus nidulans was characterized by a rapid initial accumulation during the first 10 min of incubation with the fungicide and a subsequent gradual release with time. Uptake appeared to be the result of influx and efflux. Influx of fenarimol could not be inhibited by low temperature, anaerobiosis, starvation of mycelium, or incubation with several respiratory inhibitors and is, therefore, a passive process. Under identical test conditions efflux activity was severely inhibited and should, therefore, be regarded as an energy-dependent mechanism. After prolonged incubation (90 min) an equilibrium between influx and efflux was established, resulting in an energy-dependent permeability barrier, since uptake could instantaneously be enhanced by addition of oligomycin or N,N′-dicyclohexylcarbodiimide. It also indicates that efflux activity is inducible; this hypothesis is supported by the observation that pretreatment of mycelium with unlabeled fungicide prevented subsequent uptake of [ 14C]fenarimol. Uptake by fenarimol-resistant mutants J146, M193, and R264 of A. nidulans, all possessing the imaB gene for resistance, was relatively low and almost constant in time. In this case, uptake appeared to be considerably enhanced by low temperature, anaerobiosis, starvation of mycelium, and incubation with respiratory inhibitors. Low uptake by these mutants is ascribed to a higher energy-dependent efflux activity for fenarimol compared with the wild-type strain. Upon inhibition of the barrier activity, net uptake resulted from remaining passive influx, which in that case may be as high as in the wild-type strain. The results suggest that both wild-type and fenarimol-resistant mutants possess an energy-dependent efflux mechanism with different efficiencies to excrete fenarimol and probably other chemicals to which cross-resistance or collateral sensitivity is present.