Spatiotemporal expression of the putative MdtABC efflux pump of Phtotorhabdus luminescens occurs in a protease-dependent manner during insect infection.

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.

Increase of zinc resistance in German human derived livestock-associated MRSA between 2000 and 2014.

PROBLEM ADDRESSED: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), particularly of the clonal complex (CC) 398, emerged as zoonotic pathogens predominantly among humans with direct or indirect livestock contact, but also in healthcare settings. The factors contributing to the success of LA-MRSA are only poorly understood. OBJECTIVE: During the past years, the use of heavy metal compounds as feed-supplements was found to influence the co-selection of LA-MRSA in pig herds. This study aimed to determine the prevalence of zinc resistance among MRSA CC398 isolated from patients of a German university hospital located in a pig farming-dense area. METHODS AND APPROACH: In comparison to concurrent healthcare-associated MRSA (HA-MRSA), LA-MRSA CC398 comprising isolates from their first appearance in 2000 to recent isolates from 2014 were included. RESULTS: Among MRSA CC398, the overall resistance rate towards zinc chloride was 57% compared to only 3% among concurrently isolated HA-MRSA. Zinc resistance correlated with the presence of the czrC gene in 100% of the MRSA CC398 and in 67% of the HA-MRSA. CONCLUSIONS: The zinc resistance rate in MRSA CC398 significantly increased from 2009 to 2014 with a maximum in 2014. Alarmingly, zinc resistance has become a frequent phenotype of human LA-MRSA in Germany potentially facilitating co-selection of antibiotic resistance genes.

Inhibition of the multidrug efflux pump LmrS from Staphylococcus aureus by cumin spice Cuminum cyminum.

Staphylococcus aureus is a serious causative agent of infectious disease. Multidrug-resistant strains like methicillin-resistant S. aureus compromise treatment efficacy, causing significant morbidity and mortality. Active efflux represents a major antimicrobial resistance mechanism. The proton-driven multidrug efflux pump, LmrS, actively exports structurally distinct antimicrobials. To circumvent resistance and restore clinical efficacy of antibiotics, efflux pump inhibitors are necessary, and natural edible spices like cumin are potential candidates. The mode of cumin antibacterial action and underlying mechanisms behind drug resistance inhibition, however, are unclear. We tested the hypothesis that cumin inhibits LmrS drug transport. We found that cumin inhibited bacterial growth and LmrS ethidium transport in a dosage-dependent manner. We demonstrate that cumin is antibacterial toward a multidrug-resistant host and that resistance modulation involves multidrug efflux inhibition.

Linezolid-resistant Staphylococcus aureus strain 1128105, the first known clinical isolate possessing the cfr multidrug resistance gene.

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZD(r) clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination.

Effect of therapeutic moderate hypothermia on multi-drug resistance protein 1-mediated transepithelial transport of drugs.

To clarify the effect of therapeutic moderate hypothermia on drug distribution, transepithelial transport via multi-drug resistance protein 1 (MDR1) (also called P-glycoprotein or ABCB1) was evaluated at various temperatures in vitro using LLC-GA5-COL150 cells, which were established by transfecting human MDR1 complementary deoxyribonucleic acid into kidney epithelial LLC-PK(1) cells and express MDR1 on the apical membrane. MDR1 is expressed in the blood-brain barrier to limit drug distribution to the brain by exporting exogenous substances including calcium blockers and antiarrhythmic drugs. Digoxin was used as a typical substrate, as well as the non-substrate tetracycline and paracellular marker inulin. MDR1-mediated transport of digoxin decreased at lower temperatures. Transport of tetracycline also decreased at lower temperatures, probably due to changes in membrane fluidity. However, no change was found at over 32 degrees C, suggesting that passive diffusion does not change during moderate hypothermia. The distribution of MDR1 substrates should be considered during hypothermic conditions, as the clinical outcome could be affected.

Effect of milk thistle (Silybum marianum) and black cohosh (Cimicifuga racemosa) supplementation on digoxin pharmacokinetics in humans.

Phytochemical-mediated modulation of P-glycoprotein (P-gp) and other drug transporters may underlie many herb-drug interactions. Serial serum concentration-time profiles of the P-gp substrate, digoxin, were used to determine whether supplementation with milk thistle or black cohosh modified P-gp activity in vivo. Sixteen healthy volunteers were randomly assigned to receive a standardized milk thistle (900 mg daily) or black cohosh (40 mg daily) supplement for 14 days, followed by a 30-day washout period. Subjects were also randomized to receive rifampin (600 mg daily, 7 days) and clarithromycin (1000 mg daily, 7 days) as positive controls for P-gp induction and inhibition, respectively. Digoxin (Lanoxicaps, 0.4 mg) was administered orally before and at the end of each supplementation and control period. Serial digoxin serum concentrations were obtained over 24 h and analyzed by chemiluminescent immunoassay. Comparisons of area under the serum concentration time curves from 0 to 3 h (AUC(0-3)), AUC(0-24), Cmax, apparent oral clearance of digoxin (CL/F), and elimination half-life were used to assess the effects of milk thistle, black cohosh, rifampin, and clarithromycin on digoxin pharmacokinetics. Rifampin produced significant reductions (p < 0.01) in AUC(0-3), AUC(0-24), and Cmax, whereas clarithromycin increased these parameters significantly (p < 0.01). Significant changes in digoxin half-life and CL/F were also observed with clarithromycin. No statistically significant effects on digoxin pharmacokinetics were observed following supplementation with either milk thistle or black cohosh, although digoxin AUC(0-3) and AUC(0-24) approached significance (p = 0.06) following milk thistle administration. When compared with rifampin and clarithromycin, supplementation with these specific formulations of milk thistle or black cohosh did not appear to affect digoxin pharmacokinetics, suggesting that these supplements are not potent modulators of P-gp in vivo.

Reversal of cancer multidrug resistance by green tea polyphenols.

The aim of this study was to examine the effect and mechanism of green tea polyphenols (TP) on reversal of multidrug resistance (MDR) in a carcinoma cell line. Using the MTT assay, TP was examined for its modulating effects on the drug-resistant KB-A-1 cells and drug-sensitive KB-3-1 cells. When 10 microg mL(-1) (-)-epigallocatechin gallate (EGCG) or 40 microg mL(-1) TP were present simultaneously with doxorubicin (DOX), the IC50 of DOX on KB-A-1 cells decreased from 10.3 +/- 0.9 microg mL(-1) to 4.2 +/- 0.2 and 2.0 +/- 0.1 microg mL(-1), respectively. TP and EGCG enhanced the DOX cytotoxicity on KB-A-1 cells by 5.2- and 2.5-times, respectively, but did not show a modulating effect on KB-3-1 cells. This indicated that both TP and EGCG had reversal effects on the MDR phenotype in-vitro. A KB-A-1 cell xenograft model was established, and the effect of TP on reversing MDR in-vivo was determined. Mechanistic experiments were conducted to examine the uptake, efflux and accumulation of DOX. Cloning and expression of the nucleotide binding domain of the human MDR1 gene in Escherichia coli was established, and by using colorimetry to examine the activity of ATPase to hydrolyse ATP, the ATPase activity of target nucleotide binding domain protein was determined. TP exerted its reversal effects through the inhibition of ATPase activity, influencing the function of P-glycoprotein, and causing a decreased extrusion of anticancer drug and an increased accumulation of anticancer drug in drug resistant cells. Using reverse transcription-polymerase chain reaction, the inhibitory effect of TP on MDR1 gene expression was investigated. Down-regulation of MDR1 gene expression was the main effect, which resulted in the reversal effect of TP on the MDR phenotype. TP is a potent MDR modulator with potential in the treatment of P-glycoprotein mediated MDR cancers.

[Reversal effect of hyperthemia on multidrug resistant phenomena].

OBJECTIVE: The purpose of this study was investigate the effect of hyperthemia on multidrug resistance in K562/ADM cell. METHODS: The MDR1 (mulitdrug resistance gene) and MRP (multidrug resistant associated gene) gene expressions in Tca8113 and K562/ADM cell lines were analyzed by RT-PCR after treated with different cytotoxic drugs and different temperature (37 degrees C and 41 degrees C). The function and expression of Pgp and MRP were detected by fluorescence photometeric assays. RESULTS: Inhibition rate of both cells was significantly enhanced by exposure to chemotherapeutic drugs and 41 degrees C temperature; Exposing to 41 degrees C hyperthemia reduced MDR1 and MRP expression and enhanced intracellular drug concentration as well in K562/ADM. CONCLUSION: 41 degrees C hyperthemia could effectively enhance the inhibition rate of chemotherapeutic drugs and partially reverse the multidrug resistance. It is suggested that hyperthemia could be used as a method to overcome multidrug resistance.

Expression of multidrug resistance genes MVP, MDR1, and MRP1 determined sequentially before, during, and after hyperthermic isolated limb perfusion of soft tissue sarcoma and melanoma patients.

PURPOSE: Isolated, hyperthermic limb perfusion (ILP) with recombinant human tumor necrosis factor alpha and melphalan is a highly effective treatment for advanced soft tissue sarcoma (STS) and locoregional metastatic malignant melanoma. Multidrug resistance (MDR)-associated genes are known to be inducible by heat and drugs; expression levels of the major vault protein (MVP), MDR1, and MDR-associated protein 1 (MRP1) were determined sequentially before, during, and after ILP of patients. PATIENTS AND METHODS: Twenty-one STS or malignant melanoma patients were treated by ILP. Tumor tissue temperatures were recorded continuously and ranged from 33.4 degrees C initially to peak values of 40.4 degrees C during ILP. Serial true-cut biopsy specimens from tumor tissues were routinely microdissected. Expression analyses for MDR genes were performed by real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. RESULTS: In 83% of the patients, MVP expression was induced during hyperthermic ILP. MVP-mRNA inductions often paralleled the increase in temperature during ILP. Increased MVP protein expressions either were observed simultaneously with the MVP-mRNA induction or were delayed until after the induction at the transcriptional level. Inductions of MDR1 and MRP1 were observed in only 13% and 27% of the specimens analyzed. Temperatures and drugs applied preferentially led to an induction of MVP and were not sufficient to induce MDR1 and MRP1 in the majority of tumors. CONCLUSION: This study is the first to analyze the expression of MDR-associated genes sequentially during ILP of patients and demonstrates that treatment might lead to increased levels of MVP, whereas enhanced levels of MDR1 and MRP1 remain rare events.

Are MDCK cells transfected with the human MDR1 gene a good model of the human intestinal mucosa?

PURPOSE: To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. METHODS: P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDRI cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K(M),Vmax) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K(I)) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. RESULTS: MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [3H]-digoxin than did Caco-2 and MDCK-WT cells. K(M) values of vinblastine in Caco-2. MDCK-WT, and MDCK-MDR1 cells were 89.2+/-26.1, 24.5+/-1.1, and 252.8+/-134.7 microM, respectively, whereas Vmax values were 1.77+/-0.22, 0.42+/-0.01, and 2.43+/-0.86 pmolcm(-2)s(-1), respectively. Known P-gp substrates/inhibitors showed, in general, lower K(I) values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. CONCLUSIONS: These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.

Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for antigene radiotherapy.

PURPOSE: Antigene radiotherapy (AR) is based on targeting localized radiodamage to specific sites in the genome by using sequence-specific triplex-forming oligonucleotides (TFO) to carry Auger-electron-emitters (A-Ettr) such as Iodine-125 (125I) to the target gene sequence. The radiodecay of an A-Ettr produces a cascade of low-energy electrons and creates a highly positively-charged daughter atom; delivered by a TFO, it should produce double-strand breaks (dsb) localized to the specific DNA target sequence. The result should be a "knock-out" of the targeted gene. METHODS AND MATERIALS: As a model, we used the MDR1 gene amplified nearly 100 times in the human KB-V1 carcinoma cell line. Chemically modified TFO complementary to the polypurine/polypyrimidine region of the MDR1 gene were synthesized and radiolabeled with 125I-dCTP by the primer extension method. Purified plasmid and genomic DNA and extracted nuclei were treated with 125I-TFO and analyzed for sequence-specific cleavage by electrophoresis in agarose gel and Southern hybridization. RESULTS: We created 125I-TFO that could effectively recognize, bind, and cleave the target sequence in plasmid and genomic DNA. We showed that these 125I-TFO in nanomolar concentrations were able to cleave the target MDR1 gene sequence in a natural environment, i.e., within the eucaryotic nucleus. CONCLUSION: 125I-TFO can effectively introduce sequence-specific dsb to a target within the MDR1 gene, both in purified DNA and inside intact nuclei. Chemically modified TFO conjugated with nuclear localization signal appear to be a promising delivery vehicle for future in vivo trials of AR.

In vitro evaluation of newly developed chalcone analogues in human cancer cells.

PURPOSE: Among flavonoids, chalcones have been identified as interesting compounds having chemopreventive and antitumor properties. We studied a panel of newly developed chalcone analogues (S1-S10) using MDA-MB 231 and MCF-7 ADRr breast cancer cells and the T-leukemic Jurkat cell line. Quercetin was used as the reference compound. METHODS: Antiproliferative activity was evaluated by cell counts performed after 72 h of exposure to the drugs. DNA analysis and redox activity were evaluated using flow cytometry. Apoptosis was assessed by morphological analysis, using YOYO-1 as DNA dye; p-glycoprotein function was ascertained by quantitating the efflux of rhodamine 123. RESULTS: All cells were sensitive to chalcone analogues yielding IC50 in micromolar concentrations with the following order regardless of the multidrug resistance (MDR) status: S1 > S2 > quercetin. S1 and S2, the most active compounds, were selected to evaluate their effect on the cell cycle, apoptosis, redox activity, and modulation of the p-glycoprotein function. No significant perturbation in cell cycle was seen with concentration up to 1 microM after 24 h. After 72 h a slight increase in G2/M block and DNA fragmentation occurred at 10 microM. Morphological analysis of apoptosis showed that chalcone analogues induced apoptosis to a higher extent than quercetin. Redox analysis demonstrated that all substances were able to increase intracellular thiol levels, which returned to baseline value after 24 h for all drugs except quercetin. Production of reactive oxygen species was essentially unaffected by all compounds. Finally, in MDR-positive MCF-7 ADRr cells chalcone analogues were unable to modulate p-glycoprotein function while quercetin was able to. CONCLUSIONS: Newly developed S1 and S2 chalcones have a different but higher antitumor activity than quercetin and could be considered as potential new anticancer drugs.

Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein.

P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the K(d) (412 nM) of the substrate analogue [(125)I]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5'-adenylylimididiphosphate and vanadate (K(d) = 403 nM). Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [(125)I]iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp x ADP x vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis. Additionally, there is an inverse correlation (R(2) = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.

Establishment of a myeloid cell line, YM711, characterized by retinoid resistance.

A myeloid cell line (YM711) was established by transfecting exogenous PML/RARalpha cDNA into peripheral blood stem cells. The cells were positive for CD33, CD34, CD38, CD13, CD14, and CD11b. Cytochemical examination revealed YM711 cells to be positive for peroxidase, alpha-naphtyl butyrate esterase, and acid phosphatase as well. Karyotypic analysis showed them to be nearly tetraploid (92 XXYY). Reverse-transcription polymerase chain reaction showed that, although PML/RARalpha mRNA was detected in YM711, these cells could not be differentiated by all-trans retinoic acid (ATRA). We therefore designated the YM711 cell line as being ATRA resistant. Because YM711 expressed multi drug resistance 1 (MDR-1) mRNA and p-glycoprotein cell surface protein, we assessed whether verapamil and ATRA would induce the differentiation of YM711 cells; they did not. An increased expression of cellular retinoic acid-binding protein (CRABP)-II was also detected on YM711 cells compared with that of HL-60. These results suggest that high level of expression of CRABP-II may contribute to be the mechanism of ATRA resistance. This cell line may be useful in evaluating the mechanism of resistance to retinoid.