Up-regulation of P-glycoprotein expression by catalase via JNK activation in HepG2 cells.

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Li, Lin; Xu, Jianfeng; Min, Taishan; Huang, Weida

2006-01-01

Overexpression of the MDR1 gene is one of the reasons for multidrug resistance (MDR). Some studies suggested that antioxidants could down-regulate MDR1 expression as a possible cancer treatment. In this report, we try to determine the effects of antioxidants (catalase or N-acetylcysteine [NAC]) on the regulation of intrinsic MDR1 overexpression in HepG2 cells. Adding catalase or N-acetylcysteine to the HepG2 culture led to a significant increase of MDR1 mRNA and P-glycoprotein drug transporter activity. After catalase or NAC treatment, a reduced intracellular reactive oxygen species (ROS) was observed. The JNK inhibitor SP600125 abolished the positive effects of catalase on drug transporter activity in a dose-dependent manner. Furthermore, the up-regulation of P-glycoprotein functions by catalase was only observed in HepG2 cells but not in other cell lines tested (MCF-7, A549, A431). These data suggested that catalase can up-regulate P-glycoprotein expression in HepG2 cells via reducing intracellular ROS, and JNK may mediate this process.

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

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Bebawy, M; Combes, V; Lee, E; Jaiswal, R; Gong, J; Bonhoure, A; Grau, G E R

2009-09-01

Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation

International Nuclear Information System (INIS)

Nielsen, Dorte; Maare, Christian; Eriksen, Jens; Litman, Thomas; Skovsgaard, Torben

2001-01-01

Purpose: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. Methods and Materials: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3 H-vincristine (VCR), and 3 H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method. Results: Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr. In EHR2/irr, the steady-state accumulation of 3 H-VCR and 3 H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPase activity was neither stimulated by vinblastine nor VER. Conclusion: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was

Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation

DEFF Research Database (Denmark)

Nielsen, D; Maare, C; Eriksen, J

2001-01-01

PURPOSE: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. METHODS...... AND MATERIALS: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT......-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity...

Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

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Mahsa Mohseni

2016-03-01

Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1 to (15±2.6 nM and for vinblastine from (30±5.9 to (5±5.6 nM (P≤0.05.               P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001. Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05. Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents.  Conclusion:Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression.

Expression of P-glycoprotein, multidrug resistance-associated protein, glutathione-S-transferase pi and p53 in canine transmissible venereal tumor

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Daniel G. Gerardi

2014-01-01

Full Text Available The overexpression of proteins P-glycoprotein (P-gp, multidrug resistance-associated protein (MRP1, mutant p53, and the enzyme glutathione-S-transferase (GSTpi are related to resistance to chemotherapy in neoplasms. This study evaluated the expression of these markers by immunohistochemistry in two groups of canine TVT, without history of prior chemotherapy (TVT1, n=9 and in TVTs presented unsatisfactory clinical response to vincristine sulfate (TVT2, n=5. The percentage of specimens positively stained for P-gp, MRP1, GSTpi and p53 were, respectively 88.8%, 0%, 44.5% and 22.2% in TVT1 and 80%, 0%, 80% and 0% in TVT2. In TVT1, one specimen presented positive expression for three markers and four specimens for two markers. In TVT2, three specimens expressed P-gp and GSTpi. In conclusion, the canine TVTs studied expressed the four markers evaluated, but just P-gp and GSTpi were significantly expressed, mainly at cytoplasm and cytoplasm and nuclei, respectively, either before chemotherapy as after vincristine sulfate exposure. Future studies are needed to demonstrate the function of these two markers in conferring multidrug resistance (MDR or predict the response to chemotherapy in canine TVT.

In vivo imaging and specific targeting of P-glycoprotein expression in multidrug resistant nude mice xenografts with [125I]MRK-16 monoclonal antibody

International Nuclear Information System (INIS)

Scott, Andrew M.; Rosa, Eddie; Mehta, Bippin M.; Divgi, Chaitanya R.; Finn, Ronald D.; Biedler, June L.; Tsuruo, Takashi; Kalaigian, Hovannes; Larson, Steven M.

1995-01-01

Multidrug resistance (MDR) in tumors is associated with P-glycoprotein (Pgp) expression. In vivo quantitation of Pgp may allow MDR to be evaluated noninvasively prior to treatment planning. The purpose of this study was to radiolabel MRK-16, a monoclonal antibody that targets an external epitope of P-glycoprotein, and perform in vivo quantitation of P-glycoprotein in a MDR xenograft nude mouse model. MRK-16 was labeled with 125 I by the iodogen method, with subsequent purification by size exclusion chromatography. Groups of 10 Balb/c mice were each xenografted with colchicine-resistant or -sensitive neuroblastoma cell lines, respectively. Whole body clearance and tumor uptake over time was quantitated by gamma camera imaging, and biodistribution studies were performed with [ 125 ]MRK-16 and an isotype matched control antibody, A33. Quantitative autoradiography and immunohistochemistry analysis of tumors was also evaluated to confirm specific targeting of [ 125 I]MRK-16. Peak tumor uptake was at 2-3 days post-injection, and was significantly greater in resistance compared to sensitive tumors (mean % injected dose/g ± SD) (18.76 ± 2.94 vs 10.93 ± 0.96; p 125 I]MRK-16 was confirmed by comparison to [ 131 I]A33 in biodistribution studies, and localized to cellular components of tissue stroma by comparison of histologic and autoradiographic sections of sensitive and resistant tumors. Immunoblot analysis demonstrated a 4.5-fold difference in P-glycoprotein expression between sensitive and resistant cell lines without colchicine selective pressure. We conclude that in vivo quantitation of P-glycoprotein in MDR tumors can be performed with [ 125 I]MRK-16. These findings suggest a potential clinical application for radiolabeled MRK-16 in the in vivo evaluation of multidrug resistance in tumors

Multiple Drug Transport Pathways through Human P-Glycoprotein.

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McCormick, James W; Vogel, Pia D; Wise, John G

2015-07-21

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

Expression and localization of p-glycoprotein, multidrug resistance protein 4, and breast cancer resistance protein in the female lower genital tract of human and pigtailed macaque.

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Zhou, Tian; Hu, Minlu; Pearlman, Andrew; Patton, Dorothy; Rohan, Lisa

2014-11-01

Antiretroviral drug absorption and disposition in cervicovaginal tissue is important for the effectiveness of vaginally or orally administered drug products in preexposure prophylaxis (PrEP) of HIV-1 sexual transmission to women. Therefore, it is imperative to understand critical determinants of cervicovaginal tissue pharmacokinetics. This study aimed to examine the mRNA expression and protein localization of three efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated protein 4 (MRP4), and breast cancer resistance protein (BCRP), in the lower genital tract of premenopausal women and pigtailed macaques. Along the human lower genital tract, the three transporters were moderately to highly expressed compared to colorectal tissue and liver, as revealed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). In a given genital tract segment, the transporter with the highest expression level was either BCRP or P-gp, while MRP4 was always expressed at the lowest level among the three transporters tested. The immunohistochemical staining showed that P-gp and MRP4 were localized in multiple cell types including epithelial cells and vascular endothelial cells. BCRP was predominantly localized in the vascular endothelial cells. Differences in transporter mRNA level and localization were observed among endocervix, ectocervix, and vagina. Compared to human tissues, the macaque cervicovaginal tissues displayed comparable expression and localization patterns of the three transporters, although subtle differences were observed between the two species. The role of these cervicovaginal transporters in drug absorption and disposition warrants further studies. The resemblance between human and pigtailed macaque in transporter expression and localization suggests the utility of the macaque model in the studies of human cervicovaginal transporters.

iP-gp , a novel cell line with tight barrier function and expression of human P-glycoprotein (ABCB1) for drug screening

DEFF Research Database (Denmark)

Brodin, Birger; Ozgür, Burak; Saaby, Lasse

that new API's are evaluated with respect to P-gp interactions.  Aim : The aim of the present work was to validate the suitability of the newly developed iP-gp cell line for investigating P-gp interactions with human P-gp. Methods: IPEC-J2 MDR1 (iP-gp) cells were cultured on permeable supports for 17......Background : The efflux transporter P-glycoprotein (P-gp, product of the MDR1/ABCB1 gene) hinders uptake of drug compounds to the brain, limits intestinal uptake, is a cause of resistance to chemoterapeutics and a potential "site" for drug-drug interaction. Regulatory agencies therefore recommend.......04 +/- 0.01 µM in transport experiments including digoxin and rhodamine 123, respectively. Summary/Conclusion : The iP-gp cell line may become a useful screening tool for interactions between drug compounds and human P-gp....

CORRELATION BETWEEN CHEMOTHERAPY RESPONSE AND EXPRESSION PROFILES OF TRANSMEMBRANE PROTEINS: P-GLYCOPROTEIN (ABCB1, MRP2 (ABCC2, BCRP (ABCG2 IN PATIENTS WITH INVASIVE BREAST CANCER

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К. Yu. Khristenko

2016-01-01

Full Text Available Overexpression of ABC drug transporters can cause multidrug resistance (MDR in cancer cells, which is a major obstacle in the success of cancer chemotherapy. Our study revealed a correlation between the expression of invasive breast cancer resistance-associated proteins, such as P-glycoprotein (ABCB1, MRP2 (ABCC2, BCRP (ABCG2 in tumor cells and pathologic response to neoadjuvant chemotherapy. The response to neoadjuvant chemotherapy was shown to be associated with a lack of BCRP expression in tumor cells. The pathologic tumor response was correlated with the presence of positive MRP2 expression and the expression level of P-glycoprotein in cells of invasive breast cancer. 

Multiple resistance to carcinogens and xenobiotics: P-glycoproteins as universal detoxifiers.

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Efferth, Thomas; Volm, Manfred

2017-07-01

The detoxification of toxic substances is of general relevance in all biological systems. The plethora of exogenous xenobiotic compounds and endogenous toxic metabolic products explains the evolutionary pressure of all organisms to develop molecular mechanisms to detoxify and excrete harmful substances from the body. P-glycoprotein and other members of the ATP-binding cassette (ABC) transporter family extrude innumerous chemical compounds out of cells. Their specific expression in diverse biological contexts cause different phenotypes: (1) multidrug resistance (MDR) and thus failure of cancer chemotherapy, (2) avoidance of accumulation of carcinogens and prevention of carcinogenesis in healthy tissues, (3) absorption, distribution, metabolization and excretion (ADME) of pharmacological drugs in human patients, (4) protection from environmental toxins in aquatic organisms (multi-xenobiotic resistance, MXR). Hence ABC-transporters may have opposing effects for organismic health reaching from harmful in MDR of tumors to beneficial for maintenance of health in MXR. While their inhibition by specific inhibitors may improve treatment success in oncology and avoid carcinogenesis, blocking of ABC-transporter-driven efflux by environmental pollutants leads to ecotoxicological consequences in marine biotopes. Poisoned seafood may enter the food-chain and cause intoxications in human beings. As exemplified with ABC-transporters, joining forces in interdisciplinary research may, therefore, be a wise strategy to fight problems in human medicine and environmental sciences.

eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

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Xing Hua Tang

Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

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Wang, W.; Takezawa, D.; Poovaiah, B. W.

1996-01-01

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

HIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-glycoprotein.

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Jianfang Chen

Full Text Available Multidrug resistance (MDR is one of the major reasons chemotherapy-based treatments fail. Hypoxia is generally associated with tumor chemoresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1 and the multidrug resistance (MDR1 gene/transporter P-glycoprotein (P-gp remains unclear. This study aims to explore the molecular mechanisms of reversing colon cancer MDR by focusing on the target gene HIF-1α.A chemotherapeutic sensitivity assay was used to observe the efficiency of MDR reversal in LoVo multicellular spheroids (MCS. The apoptotic level induced by different drugs was examined by flow cytometry (FCM. Binding of HIF-1α to the MDR1 gene promoter was evaluated by Chromatin immunoprecipitation (ChIP. The relationship between HIF-1α/P-gp expression and sensitivity to chemotherapy was analyzed.The sensitivity of LoVo MCS to all four chemotherapy drugs was decreased to varying degrees under hypoxic conditions. After silencing the HIF-1α gene, the sensitivities of LoVo MCS to all four chemotherapy drugs were restored. The apoptotic levels that all the drugs induced were all decreased to various extents in the hypoxic group. After silencing HIF-1α, the apoptosis level induced by all four chemotherapy drugs increased. The expression of HIF-1α and P-gp was significantly enhanced in LoVo MCS after treatment with hypoxia. Inhibiting HIF-1α significantly decreased the expression of MDR1/P-gp mRNA or protein in both the LoVo monolayers and LoVo MCS. The ChIP assay showed that HIF-1α was bound to the MDR1 gene promoter. Advanced colon carcinoma patients with expression of both HIF-1α and P-gp were more resistant to chemotherapy than that with non expression.HIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-gp. The expression of HIF-1α and MDR1/P-gp can be used as a predictive marker for chemotherapy resistance in colon cancer.

Efficacy of Intravenous Cyclophosphamide Pulse Therapy for P-Glycoprotein-expressing B Cell-associated Active True Renal Lupus Vasculitis in Lupus Nephritis

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Kawabe, Akio; Tsujimura, Shizuyo; Saito, Kazuyoshi; Tanaka, Yoshiya

2017-01-01

True renal lupus vasculitis (TRLV), a vascular lesion usually associated with proliferative lupus nephritis (LN), is resistant to conventional treatments. The expression of P-glycoprotein (P-gp) on activated lymphocytes causes drug resistance. We herein report a patient with TRLV, minimal change LN, overexpression of P-gp on peripheral B cells, and accumulation of P-gp+ B cells at the site of TRLV. High-dose corticosteroids combined with intravenous cyclophosphamide pulse therapy resulted in clinical remission and the long-term normal renal function. PMID:28626187

Functional analysis of P-glycoprotein and multidrug resistance associated protein related multidrug resistance in AML-blasts.

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Brügger, D; Herbart, H; Gekeler, V; Seitz, G; Liu, C; Klingebiel, T; Orlikowsky, T; Einsele, H; Denzlinger, C; Bader, P; Niethammer, D; Beck, J F

1999-05-01

Despite the high effectiveness of various P-glycoprotein (P-gp) modulating substances in vitro their clinical value e.g. for combination treatment of acute myelogenous leukemias (AML) remains still unclear. This might be explainable by recent findings that other factors than P-gp (e.g. the multidrug resistance associated protein (MRP)) may also be involved in clinical occurring drug resistance. To study P-gp and MRP mediated MDR in AML blasts from patients with relapses at the functional level we measured rhodamine 123 (RHO) efflux in combination with a P-gp specific (SDZ PSC 833) or a MRP specific (MK571) modulator, respectively. Furthermore, direct antineoplastic drug action was monitored by determination of damaged cell fraction of a blast population using flow cytometry. We generally found strongly modulated RHO efflux by SDZ PSC 833 but slight RHO-efflux modulation by MK571 in blasts from relapsed states of AML expressing MDR1 or MRP mRNA at various levels. We could not demonstrate, though, significant PSC 833 or MK571 mediated modulation of the cytotoxic effects of etoposide. The results point to the possibility that combination of etoposide and a modulator might not improve responses to chemotherapy by targeting P-gp or MRP exclusively.

Multiple Drug Transport Pathways through human P-Glycoprotein(†)

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McCormick, James W.; Vogel, Pia D.; Wise, John G.

2015-01-01

P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

Circumvention of multi-drug resistance of cancer cells by Chinese herbal medicines.

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Chai, Stella; To, Kenneth Kw; Lin, Ge

2010-07-25

Multi-drug resistance (MDR) of cancer cells severely limits therapeutic outcomes. A proposed mechanism for MDR involves the efflux of anti-cancer drugs from cancer cells, primarily mediated by ATP-binding cassette (ABC) membrane transporters including P-glycoprotein. This article reviews the recent progress of using active ingredients, extracts and formulae from Chinese medicine (CM) in circumventing ABC transporters-mediated MDR. Among the ABC transporters, Pgp is the most extensively studied for its role in MDR reversal effects. While other MDR reversal mechanisms remain unclear, Pgp inhibition is a criterion for further mechanistic study. More mechanistic studies are needed to fully establish the pharmacological effects of potential MDR reversing agents.

P-glycoprotein activity and biological response

International Nuclear Information System (INIS)

Vaalburg, W.; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

2005-01-01

P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators

Expression of multidrug resistance proteins in retinoblastoma

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Swati Shukla

2017-11-01

Full Text Available AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1 expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

Expression of multidrug resistance proteins in retinoblastoma.

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Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

2017-01-01

To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

Expression of multidrug resistance proteins in retinoblastoma

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Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

2017-01-01

AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

Mechanisms of drug resistance in cancer cells

International Nuclear Information System (INIS)

Iqbal, M.P.

2003-01-01

Development of drug resist chemotherapy. For the past several years, investigators have been striving hard to unravel mechanisms of drug resistance in cancer cells. Using different experimental models of cancer, some of the major mechanisms of drug resistance identified in mammalian cells include: (a) Altered transport of the drug (decreased influx of the drug; increased efflux of the drug (role of P-glycoprotein; role of polyglutamation; role of multiple drug resistance associated protein)), (b) Increase in total amount of target enzyme/protein (gene amplification), (c) alteration in the target enzyme/protein (low affinity enzyme), (d) Elevation of cellular glutathione, (e) Inhibition of drug-induced apoptosis (mutation in p53 tumor suppressor gene; increased expression of bcl-xl gene). (author)

Circumvention of multi-drug resistance of cancer cells by Chinese herbal medicines

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Lin Ge

2010-07-01

Full Text Available Abstract Multi-drug resistance (MDR of cancer cells severely limits therapeutic outcomes. A proposed mechanism for MDR involves the efflux of anti-cancer drugs from cancer cells, primarily mediated by ATP-binding cassette (ABC membrane transporters including P-glycoprotein. This article reviews the recent progress of using active ingredients, extracts and formulae from Chinese medicine (CM in circumventing ABC transporters-mediated MDR. Among the ABC transporters, Pgp is the most extensively studied for its role in MDR reversal effects. While other MDR reversal mechanisms remain unclear, Pgp inhibition is a criterion for further mechanistic study. More mechanistic studies are needed to fully establish the pharmacological effects of potential MDR reversing agents.

Curcumin as a Modulator of P-Glycoprotein in Cancer: Challenges and Perspectives

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Vanessa Lopes-Rodrigues

2016-11-01

Full Text Available Multidrug resistance (MDR presents a serious challenge to the efficiency of cancer treatment, and may be associated with the overexpression of drug efflux pumps. P-glycoprotein (P-gp is a drug efflux pump often found overexpressed in cases of acquired MDR. Nevertheless, there are no P-gp inhibitors being used in the current clinical practice, due to toxicity problems, drug interactions, or pharmacokinetic issues. Therefore, it is important to identify novel inhibitors of P-gp activity or expression. Curcumin is a secondary metabolite isolated from the turmeric of Curcuma longa L. which has been associated with several biological activities, particularly P-gp modulatory activity (by inhibiting both P-gp function and expression. However, curcumin shows extensive metabolism and instability, which has justified the recent and intensive search for analogs of curcumin that maintain the P-gp modulatory activity but have enhanced stability. This review summarizes and compares the effects of curcumin and several curcumin analogs on P-glycoprotein function and expression, emphasizing the potential of these molecules for the possible development of safe and effective inhibitors of P-gp to overcome MDR in human cancer.

P-glycoprotein is expressed and causes resistance to chemotherapy in EBV-positive T-cell lymphoproliferative diseases

International Nuclear Information System (INIS)

Yoshimori, Mayumi; Takada, Honami; Imadome, Ken-Ichi; Kurata, Morito; Yamamoto, Kouhei; Koyama, Takatoshi; Shimizu, Norio; Fujiwara, Shigeyoshi; Miura, Osamu; Arai, Ayako

2015-01-01

Epstein–Barr virus-positive T-cell lymphoproliferative diseases (EBV-T-LPDs) are rare lymphomas with poor prognosis. Although chemotherapeutic strategies such as CHOP have been often selected, they have exhibited only limited efficacy. To clarify the mechanism of chemoresistance, we examined P-glycoprotein (P-gp) expression. P-gp acts as an energy-dependent efflux pump that excretes drugs from the cytoplasm, resulting in low-intracellular drug concentrations and poor sensitivity to chemotherapy. We examined P-gp expression in EBV-positive cells by immunohistochemistry staining in three patients of EBV-T-LPDs and the expression was detected in all patients. We also examined mdr1 mRNA expression by reverse-transcriptase polymerase-chain reaction (RT-PCR) in EBV-positive tumor cells from these patients and additional three patients. The expression was detected in all examined patients. In five EBV-T-LPDs patients, P-gp function was detected by Rhodamine-123 efflux assay in these cells. The efflux was inhibited by treatment with a P-gp inhibitor, cyclosporine A (CsA). We also examined and detected P-gp expression in EBV-positive T-cell lines SNT8 and SNT16 established from EBV-T-LPDs patients, by RT-PCR and western blotting. The function was also detected by Rhodamine-123 efflux in these cell lines. Inhibition and knock down of P-gp by CsA and siRNA, respectively, enhanced etoposide- and doxorubicin-induced cell death in the EBV-positive T-cell lines. Finally, we infected the T-cell line MOLT4 with EBV, and found that mdr1 mRNA expression and Rhodamine 123 efflux were upregulated after infection. These results indicated that enhanced P-gp expression contributed to the chemoresistance of EBV-T-LPDs

Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition.

Science.gov (United States)

Morales, Eva; Cots, Francesc; Sala, Maria; Comas, Mercè; Belvis, Francesc; Riu, Marta; Salvadó, Margarita; Grau, Santiago; Horcajada, Juan P; Montero, Maria Milagro; Castells, Xavier

2012-05-23

We aimed to assess the hospital economic costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. A retrospective study of all hospital admissions between January 1, 2005, and December 31, 2006 was carried out in a 420-bed, urban, tertiary-care teaching hospital in Barcelona (Spain). All patients with a first positive clinical culture for P. aeruginosa more than 48 h after admission were included. Patient and hospitalization characteristics were collected from hospital and microbiology laboratory computerized records. According to antibiotic susceptibility, isolates were classified as non-resistant, resistant and multi-drug resistant. Cost estimation was based on a full-costing cost accounting system and on the criteria of clinical Activity-Based Costing methods. Multivariate analyses were performed using generalized linear models of log-transformed costs. Cost estimations were available for 402 nosocomial incident P. aeruginosa positive cultures. Their distribution by antibiotic susceptibility pattern was 37.1% non-resistant, 29.6% resistant and 33.3% multi-drug resistant. The total mean economic cost per admission of patients with multi-drug resistant P. aeruginosa strains was higher than that for non-resistant strains (15,265 vs. 4,933 Euros). In multivariate analysis, resistant and multi-drug resistant strains were independently predictive of an increased hospital total cost in compared with non-resistant strains (the incremental increase in total hospital cost was more than 1.37-fold and 1.77-fold that for non-resistant strains, respectively). P. aeruginosa multi-drug resistance independently predicted higher hospital costs with a more than 70% increase per admission compared with non-resistant strains. Prevention of the nosocomial emergence and spread of antimicrobial resistant microorganisms is essential to limit the strong economic impact.

Overcoming STC2 mediated drug resistance through drug and gene co-delivery by PHB-PDMAEMA cationic polyester in liver cancer cells.

Science.gov (United States)

Cheng, Hongwei; Wu, Zhixian; Wu, Caisheng; Wang, Xiaoyuan; Liow, Sing Shy; Li, Zibiao; Wu, Yun-Long

2018-02-01

Stanniocalcin 2 (STC2) overexpression in hepatocellular carcinoma (HCC) could lead to poor prognosis, which might be due to its induced P-glycoprotein and Bcl-2 protein expression level increase. P-glycoprotein or membrane pump induced drug efflux and altered prosurvival Bcl-2 expression are key mechanisms for drug resistance leading to failure of chemotherapy in HCC. However, current strategy to overcome both P-glycoprotein and Bcl-2 protein induced drug resistance was rarely reported. In this work, we utilized an amphiphilic poly[(R)-3-hydroxybutyrate] (PHB)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) cationic polyester to encapsulate chemotherapeutic paclitaxel (PTX) in hydrophobic PHB domain and Bcl-2 convertor Nur77/ΔDBD gene (Nur77 without DNA binding domain for mitochondria localization) by formation of polyplex due to cationic PDMAEMA segment, to effectively inhibit the drug resistant HepG2/STC2 and SMCC7721/STC2 liver cancer cell growth. Thanks to the cationic nanoparticle complex formation ability and high transfection efficiency to express Bcl-2 conversion proteins, PHB-PDMAEMA/PTX@polyplex could partially impair P-glycoprotein induced PTX efflux and activate the apoptotic function of previous prosurvival Bcl-2 protein. This is the pioneer report of cationic amphiphilic polyester PHB-PDMAEMA to codeliver anticancer drug and therapeutic plasmid to overcome both pump and non-pump mediated chemotherapeutic resistance in liver cancer cells, which might be inspiring for the application of polyester in personalized cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

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A. V. Shulkin

2015-09-01

Full Text Available On the basis of the analysis of published data the role of P-glycoprotein, carrier protein, in rational pharmacotherapy in cardiology was shown on the example of its substrates – digoxin, antiplatelet agents and anticoagulants. Determination of C3435T polymorphism of multidrug resistance gene (MDR1, encoding P-glycoprotein, in pharmacotherapy with digoxin, antiplatelet drugs (clopidogrel tikagrelol, prasugrel and anticoagulants (dabigatran etexilate, rivaroxaban, edoxaban is not feasible in routine practice. Drug in- teractions have clinical implications for the efficacy and safety of pharmacotherapy in coadministration of these drugs with P-glycoprotein substrates, inducers and inhibitors.

Detection of expression and modulation of multidrug-resistance (MDR) and establishment of a new bioassay

International Nuclear Information System (INIS)

Berger, W.

1993-08-01

The present thesis deals with the resistance of human malignant cells against cellular toxicity of anticancer drugs, a phenomenon representing one of the major obstacles to successful chemotherapy. One mechanism underlying a cross-resistance to different drugs called multidrug resistance (MDR) is characterized by the expression of an active transport protein (P-glycoprotein), causing decreased intracellular drug retention and cytotoxicity. The main subjects of the present work were to establish different detection methods for MDR and its modulation (by substances blocking activity of P-glycoprotein) including immunological methods (immunocytochemistry, radioimmunoassay), molecular biology (slot-blot analysis, in-situ hybridization) and functional assays (drug-accumulation analysis, drug-cytotoxicity analysis). The methods were evaluated and compared using human and mouse MDR control cell lines and human tumor cell lines established in our laboratory. In cell lines derived from human melanoma - a malignancy insensitive to chemotherapy - expression of P-glycoprotein of relatively low transporting activity was detected by different methods in 8 of 33 cases. Furthermore a new sensitive in vitro assay for the functional detection of MDR was established using the biological features of cytochalasins, a microfilament disrupting substance group. These compounds were shown to be substrates for the P-glycoprotein efflux pump and their effects on cell division (blockade of cytokinesis resulting in multinucleate cells) correlated with MDR-activity of the tested cells. With this new assay P-glycoprotein activity can be demonstrated and analysed over a wide range of resistance against different cytotoxic drugs. Therefore it may by a suitable tool for research and diagnosis in the field of drug resistance

Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition

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Morales Eva

2012-05-01

Full Text Available Abstract Background We aimed to assess the hospital economic costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. Methods A retrospective study of all hospital admissions between January 1, 2005, and December 31, 2006 was carried out in a 420-bed, urban, tertiary-care teaching hospital in Barcelona (Spain. All patients with a first positive clinical culture for P. aeruginosa more than 48 h after admission were included. Patient and hospitalization characteristics were collected from hospital and microbiology laboratory computerized records. According to antibiotic susceptibility, isolates were classified as non-resistant, resistant and multi-drug resistant. Cost estimation was based on a full-costing cost accounting system and on the criteria of clinical Activity-Based Costing methods. Multivariate analyses were performed using generalized linear models of log-transformed costs. Results Cost estimations were available for 402 nosocomial incident P. aeruginosa positive cultures. Their distribution by antibiotic susceptibility pattern was 37.1% non-resistant, 29.6% resistant and 33.3% multi-drug resistant. The total mean economic cost per admission of patients with multi-drug resistant P. aeruginosa strains was higher than that for non-resistant strains (15,265 vs. 4,933 Euros. In multivariate analysis, resistant and multi-drug resistant strains were independently predictive of an increased hospital total cost in compared with non-resistant strains (the incremental increase in total hospital cost was more than 1.37-fold and 1.77-fold that for non-resistant strains, respectively. Conclusions P. aeruginosa multi-drug resistance independently predicted higher hospital costs with a more than 70% increase per admission compared with non-resistant strains. Prevention of the nosocomial emergence and spread of antimicrobial resistant microorganisms is essential to limit the strong economic impact.

Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

Science.gov (United States)

Seebacher, Nicole A; Lane, Darius J R; Jansson, Patric J; Richardson, Des R

2016-02-19

Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

NARCIS (Netherlands)

Schinkel, A. H.; Mayer, U.; Wagenaar, E.; Mol, C. A.; van Deemter, L.; Smit, J. J.; van der Valk, M. A.; Voordouw, A. C.; Spits, H.; van Tellingen, O.; Zijlmans, J. M.; Fibbe, W. E.; Borst, P.

1997-01-01

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (-/-) mice] and in both the mdr1a and mdr1b genes

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

Science.gov (United States)

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential

Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells.

Science.gov (United States)

Abdelfatah, Sara A A; Efferth, Thomas

2015-02-15

The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to explain its activity toward drug-resistant tumor cells. Sensitive and drug-resistant tumor cell lines overexpressing P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53-knockout cells as well as the NCI panel of cell lines from different tumor origin were analyzed. Reserpine's cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions. P-glycoprotein- or BCRP overexpressing tumor cells did not reveal cross-resistance to reserpine. EGFR-overexpressing cells were collateral sensitive and p53- Knockout cells cross-resistant to this drug compared to their wild-type parental cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-overexpressing cells, indicating that reserpine inhibited the efflux function of P-glycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). COMPARE and cluster analyses of microarray data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling. The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild

Esters of the Marine-Derived Triterpene Sipholenol A Reverse P-GP-Mediated Drug Resistance

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Yongchao Zhang

2015-04-01

Full Text Available Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1. Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers.

Impact of BCRP/MXR, MRP1 and MDR1/P-Glycoprotein on thermoresistant variants of atypical and classical multidrug resistant cancer cells

DEFF Research Database (Denmark)

Stein, Ulrike; Lage, Hermann; Jordan, Andreas

2002-01-01

The impact of the ABC transporters breast cancer resistance protein/mitoxantrone resistance associated transporter (BCRP/MXR), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance gene-1/P-glycoprotein (MDR1/PGP) on the multidrug resistance (MDR) phenotype in chemoresistance...... expression of BCRP/MXR and of MRP1 were clearly enhanced (vs. parental and classical MDR lines). MDR1/PGP expression was distinctly elevated in the classical MDR subline EPG85-257RDB (vs. parental and atypical MDR sublines). In all thermoresistant counterparts basal expression of BCRP/MXR, MRP1 and MDR1/PGP...... was increased relative to thermosensitive sublines. Although it could be shown that the overexpressed ABC transporters were functionally active, however, no decreased drug accumulations of doxorubicin, mitoxantrone and rhodamine 123 were observed. Thus, expression of BCRP/MXR, MRP1 and MDR1/PGP was found...

Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

Science.gov (United States)

Yuan, Wei-Qi; Zhang, Rong-Rong; Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

2016-05-24

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

Higher Desolvation Energy Reduces Molecular Recognition in Multi-Drug Resistant HIV-1 Protease

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Ladislau C. Kovari

2012-05-01

Full Text Available Designing HIV-1 protease inhibitors that overcome drug-resistance is still a challenging task. In this study, four clinical isolates of multi-drug resistant HIV-1 proteases that exhibit resistance to all the US FDA-approved HIV-1 protease inhibitors and also reduce the substrate recognition ability were examined. A multi-drug resistant HIV-1 protease isolate, MDR 769, was co-crystallized with the p2/NC substrate and the mutated CA/p2 substrate, CA/p2 P1’F. Both substrates display different levels of molecular recognition by the wild-type and multi-drug resistant HIV-1 protease. From the crystal structures, only limited differences can be identified between the wild-type and multi-drug resistant protease. Therefore, a wild-type HIV-1 protease and four multi-drug resistant HIV-1 proteases in complex with the two peptides were modeled based on the crystal structures and examined during a 10 ns-molecular dynamics simulation. The simulation results reveal that the multi-drug resistant HIV-1 proteases require higher desolvation energy to form complexes with the peptides. This result suggests that the desolvation of the HIV-1 protease active site is an important step of protease-ligand complex formation as well as drug resistance. Therefore, desolvation energy could be considered as a parameter in the evaluation of future HIV-1 protease inhibitor candidates.

Development of PET and SPECT radiopharmaceuticals to study multi-drug resistance (MDR)

International Nuclear Information System (INIS)

Katsififs, A.; Dikic, B.; Greguric, I.; Knott, R.; Mattner, F.

2002-01-01

Full text: Cellular resistance or Multidrug Resistance (MDR) to cytotoxic agents is the major cause of treatment failure in many human cancers. P-glycoprotein (Pgp), a Mr 17,0000 transmembrane protein and Multi Resistance Protein (MRP) are two proteins that are over expressed and confer resistance to a large number of chemotherapeutic agents by enhancing their extracellular transport. P-glycoprotein is expressed at a relative high level in treated and untreated human malignant tumours, including renal, colonic, adrenal, hepatocellular carcinoma and a considerable percentage of breast carcinomas. 99m Tc-Sestamibi, a lipophilic cationic complex is a transport substrate for Pgp. In clinical studies of human neoplasms it was found that tumour uptake and clearance of this tracer correlate with Pgp expression and may be used for the phenotypic assessment of MDR. However, new tracers with better substrate specificity for Pgp and other drug transporters would greatly assist in optimising chemotherapeutic treatment and improving patient management by predicting tumour response to therapy and to assist in the development of antagonists, which may reverse or halt MDR. The aim of this project is therefore to develop PET and SPECT radiopharmaceuticals with improved affinity and selectivity for Pgp and MRP for the clinical evaluation of MDR in cancer patients. To optimise cellular transport characteristics, a number of chemical families that have been found to be substrates of Pgp and other drug efflux pumps, will be investigated. In the first instance, a series of drugs based on the flavonol natural product, Quercetin will be developed, screened for MDR and radiolabelled with PET and SPECT isotopes. Quercetin and related flavonol derivatives have been selected for this project because of their moderate to good affinity for Pgp. With the assistance of molecular modeling and in vitro studies, structural modification will be undertaken to improve the specificity and affinity for

Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression

International Nuclear Information System (INIS)

Tian, Kegui; Wang, Yuezeng; Huang, Yu; Sun, Boqiao; Li, Yuxin; Xu, Haopeng

2008-01-01

Previous results showed that over-expression of the WTH3 gene in MDR cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the WTH3 gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. WTH3 was also found to be directly targeted and up regulated by the p53 gene. Furthermore, over expression of the WTH3 gene promoted the apoptotic phenotype in various host cells. To further confirm WTH3's drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the WTH3 promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the p53 transcription factor relative to WTH3 expression. To do so, the in vitro methylation method was utilized to examine the p53 transgene's influence on either the methylated or non-methylated WTH3 promoter. The results generated from the gene knockdown strategy showed that reduction of WTH3 expression increased MDR1 expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of p53 on WTH3 promoter activity. Taken together, our studies provided further evidence that WTH3 played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized p53's positive impact on WTH3 expression

Treatment strategy based on targeting P-glycoprotein on peripheral lymphocytes in patients with systemic autoimmune disease.

Science.gov (United States)

Tsujimura, Shizuyo; Tanaka, Yoshiya

2012-02-01

Although corticosteroids, immunosuppressants and disease-modifying antirheumatic drugs (DMARDs) are widely used in the treatment of various systemic autoimmune diseases such as systemic lupus erythematosus (SLE), we often experience patients with systemic autoimmune diseases who are resistant to these treatments. P-glycoprotein (P-gp) of membrane transporters, a product of the multiple drug resistance (MDR)-1 gene, is known to play a pivotal role in the acquisition of drug resistance to chemotherapy in malignancy. However, the relevance of MDR-1 and P-gp to resting and activated lymphocytes, which are the major target in the treatment of systemic autoimmune diseases, remains unclear. Studies from our laboratories found surface expression of P-gp on peripheral lymphocytes in patients with SLE and a significant correlation between the expression level and disease activity. Such expression is induced not only by genotoxic stresses but also by various stimuli including cytokines, resulting in active efflux of drugs from the cytoplasm of lymphocytes, resulting in drug-resistance and high disease activity. However, the use of both P-gp antagonists (e.g., cyclosporine) and inhibition of P-gp synthesis with intensive immunosuppressive therapy successfully reduces the efflux of corticosteroids from lymphocytes in vitro, suggesting that P-gp antagonists and P-gp synthesis inhibitors could be used to overcome drug-resistance in vivo and improve outcome. In conclusion, lymphocytes activated by various stimuli in patients with highly active disease apparently acquire MDR-1-mediated multidrug resistance against corticosteroids and probably some DMARDs, which are substrates of P-gp. Inhibition/reduction of P-gp could overcome such drug resistance. The expression of P-gp on lymphocytes is a promising marker of drug resistance and a suitable target to combat drug resistance in patients with active systemic autoimmune diseases.

Involvement of CD147 on multidrug resistance through the regulation of P-glycoprotein expression in K562/ADR leukemic cell line

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Aoranit Somno

2016-01-01

Full Text Available The relationship between P-gp and CD147 in the regulation of MDR in leukemic cells has not been reported. This study aimed to investigate the correlation between CD147 and P-gp in the regulation of drug resistance in the K562/ADR leukemic cell line. The results showed that drug-resistant K562/ADR cells expressed significantly higher P-gp and CD147 levels than drug-free K562/ADR cells. To determine the regulatory effect of CD147 on P-gp expression, anti-CD147 antibody MEM-M6/6 significantly decreased P-gp and CD147 mRNA and protein levels. This is the first report to show that CD147 mediates MDR in leukemia through the regulation of P-gp expression.

P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with an ALK rearrangement

International Nuclear Information System (INIS)

Kim, Hee Joung; Lee, Kye Young; Kim, Young Whan; Choi, Yun Jung; Lee, Jung-Eun; Choi, Chang Min; Baek, In-Jeoung; Rho, Jin Kyung; Lee, Jae Cheol

2015-01-01

Because anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement. Resistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123. The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp. P-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement. The online

Modulation of P-glycoprotein-mediated multidrug resistance in K562 leukemic cells by indole-3-carbinol

International Nuclear Information System (INIS)

Arora, Annu; Seth, Kavita; Kalra, Neetu; Shukla, Yogeshwer

2005-01-01

Resistance to chemotherapeutic drugs is one of the major problems in the treatment of cancer. P-glycoprotein (P-gp) encoded by the mdr gene is a highly conserved protein, acts as a multidrug transporter, and has a major role in multiple drug resistance (MDR). Targeting of P-gp by naturally occurring compounds is an effective strategy to overcome MDR. Indole-3-carbinol (I3C), a glucosinolates present in cruciferous vegetables, is a promising chemopreventive agent as it is reported to possess antimutagenic, antitumorigenic, and antiestrogenic properties in experimental studies. In the present investigation, the potential of I3C to modulate P-gp expression was evaluated in vinblastine (VBL)-resistant K562 human leukemic cells. The resistant K562 cells (K562/R10) were found to be cross-resistant to vincristine (VCR), doxorubicin (DXR), and other antineoplastic agents. I3C at a nontoxic dose (10 x 10 -3 M) enhanced the cytotoxic effects of VBL time dependently in VBL-resistant human leukemia (K562/R10) cells but had no effect on parent-sensitive cells (K562/S). The Western blot analysis of K 562/R 10 cells showed that I3C downregulates the induced levels of P-gp in resistant cells near to normal levels. The quantitation of immunocytochemically stained K562/R10 cells showed 24%, 48%, and 80% decrease in the levels of P-gp by I3C for 24, 48, and 72 h of incubation. The above features thus indicate that I3C could be used as a novel modulator of P-gp-mediated multidrug resistance in vitro and may be effective as a dietary adjuvant in the treatment of MDR cancers

P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments

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Ejsing, Thomas B.; Pedersen, Anne D.; Linnet, Kristian

2005-01-01

P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice......P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice...

Increased p38-MAPK is responsible for chemotherapy resistance in human gastric cancer cells

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Guo, Xianling; Zhang, Baihe; Wu, Mengchao; Wei, Lixin; Ma, Nannan; Wang, Jin; Song, Jianrui; Bu, Xinxin; Cheng, Yue; Sun, Kai; Xiong, Haiyan; Jiang, Guocheng

2008-01-01

Chemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with Multidrug resistance (MDR). Many different mechanisms have been suggested to explain the development of an MDR phenotype in cancer cells. One of the most studied mechanisms is the overexpression of P-glycoprotein (P-gp), which is a product of the MDR1 gene. Tumor cells often acquire the drug-resistance phenotype due to upregulation of the MDR1 gene. Overexpression of MDR1 gene has often been reported in primary gastric adenocarcinoma. This study investigated the role of p38-MAPK signal pathway in vincristine-resistant SGC7901/VCR cells. P-gp and MDR1 RNA were detected by Western blot analysis and RT-PCR amplification. Mitgen-activated protein kinases and function of P-gp were demonstrated by Western blot and FACS Aria cytometer analysis. Ap-1 activity and cell apoptosis were detected by Dual-Luciferase Reporter Assay and annexin V-PI dual staining. The vincristine-resistant SGC7901/VCR cells with increased expression of the multidrug-resistance 1 (MDR1) gene were resistant to P-gp-related drug and P-gp-unrelated drugs. Constitutive increases of phosphorylated p38-MAPK and AP-1 activities were also found in the drug-resistant cells. Inhibition of p38-MAPK by SB202190 reduced activator protein-1 (AP-1) activity and MDR1 expression levels and increased the sensitivity of SGC7901/VCR cells to chemotherapy. Activation of the p38-MAPK pathway might be responsible for the modulation of P-glycoprotein-mediated and P-glycoprotein-unmediated multidrug resistance in the SGC7901/VCR cell line

A novel PET imaging protocol identifies seizure-induced regional overactivity of P-glycoprotein at the blood-brain barrier

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Bankstahl, Jens P.; Bankstahl, Marion; Kuntner, Claudia; Stanek, Johann; Wanek, Thomas; Meier, Martin; Ding, Xiao-Qi; Müller, Markus; Langer, Oliver; Löscher, Wolfgang

2013-01-01

About one third of epilepsy patients are pharmacoresistant. Overexpression of P-glycoprotein and other multidrug transporters at the blood-brain barrier is thought to play an important role in drug-refractory epilepsy. Thus, quantification of regionally different P-glycoprotein activity in the brain in vivo is essential to identify P-glycoprotein overactivity as the relevant mechanism for drug-resistance in an individual patient. Using the radiolabeled P-glycoprotein substrate (R)-[11C]verapamil and different doses of co-administered tariquidar, which is an inhibitor of P-glycoprotein, we evaluated whether small-animal positron emission tomography (PET) can quantify regional changes in transporter function in the rat brain at baseline and 48 h after a pilocarpine-induced status epilepticus. P-glycoprotein expression was additionally quantified by immunohistochemistry. To reveal putative seizure-induced changes in blood-brain barrier integrity, we performed gadolinium-enhanced magnetic resonance scans on a 7.0 Tesla small-animal scanner. Before P-glycoprotein modulation, brain uptake of (R)-[11C]verapamil was low in all regions investigated in control and post-status epilepticus rats. After administration of 3 mg/kg tariquidar, which inhibits P-glycoprotein only partially, we observed increased regional differentiation in brain activity uptake in post-status epilepticus versus control rats, which diminished after maximal P-glycoprotein inhibition. Regional increases in the efflux rate constant k2, but not in distribution volume VT or influx rate constant K1, correlated significantly with increases in P-glycoprotein expression measured by immunohistochemistry. This imaging protocol proves to be suitable to detect seizure-induced regional changes in P-glycoprotein activity and is readily applicable to humans, with the aim to detect relevant mechanisms of pharmacoresistance in epilepsy in vivo. PMID:21677164

P-glycoprotein in autoimmune rheumatic diseases.

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García-Carrasco, M; Mendoza-Pinto, C; Macias Díaz, S; Vera-Recabarren, M; Vázquez de Lara, L; Méndez Martínez, S; Soto-Santillán, P; González-Ramírez, R; Ruiz-Arguelles, A

2015-07-01

P-glycoprotein (Pgp) is a transmembrane protein of 170 kD encoded by the multidrug resistance 1 (MDR-1) gene, localized on chromosome 7. More than 50 polymorphisms of the MDR-1 gene have been described; a subset of these has been shown to play a pathophysiological role in the development of inflammatory bowel disease, femoral head osteonecrosis induced by steroids, lung cancer and renal epithelial tumors. Polymorphisms that have a protective effect on the development of conditions such as Parkinson disease have also been identified. P-glycoprotein belongs to the adenosine triphosphate binding cassette transporter superfamily and its structure comprises a chain of approximately 1280 aminoacid residues with an N-C terminal structure, arranged as 2 homologous halves, each of which has 6 transmembrane segments, with a total of 12 segments with 2 cytoplasmic nucleotide binding domains. Many cytokines like interleukin 2 and tumor necrosis factor alpha increase Pgp expression and activity. Pgp functions as an efflux pump for a variety of toxins in order to protect particular organs and tissues as the central nervous system. Pgp transports a variety of substrates including glucocorticoids while other drugs such as tacrolimus and cyclosporine A act as modulators of this protein. The most widely used method to measure Pgp activity is flow cytometry using naturally fluorescent substrates such as anthracyclines or rhodamine 123. The study of drug resistance and its association to Pgp began with the study of resistance to chemotherapy in the treatment of cancer and antiretroviral therapy for human immunodeficiency virus; however, the role of Pgp in the treatment of systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis has been a focus of study lately and has emerged as an important mechanism by which treatment failure occurs. The present review analyzes the role of Pgp in these autoimmune diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast

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Doublier Sophie

2012-01-01

Full Text Available Abstract Background Invasive micropapillary carcinoma (IMPC of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1 activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters. Methods HIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay. Results In MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl-1-benzylindazole (a widely used HIF-1α inhibitor or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids. Conclusions MCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

pH-Responsive therapeutic solid lipid nanoparticles for reducing P-glycoprotein-mediated drug efflux of multidrug resistant cancer cells

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Chen HH

2015-08-01

Full Text Available Hsin-Hung Chen,1 Wen-Chia Huang,2 Wen-Hsuan Chiang,2 Te-I Liu,2 Ming-Yin Shen,2,3 Yuan-Hung Hsu,4 Sung-Chyr Lin,1 Hsin-Cheng Chiu2 1Department of Chemical Engineering, National Chung Hsing University, Taichung, 2Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 3Department of Surgery, National Taiwan University Hospital-Hsinchu Branch, 4Pharmaceutical Optimization Technology Division, Biomedical Technology and Device Research Laboratory, Industrial Technology Research Institute, Hsinchu, Taiwan Abstract: In this study, a novel pH-responsive cholesterol-PEG adduct-coated solid lipid nanoparticles (C-PEG-SLNs carrying doxorubicin (DOX capable of overcoming multidrug resistance (MDR breast cancer cells is presented. The DOX-loaded SLNs have a mean hydrodynamic diameter of ~100 nm and a low polydispersity index (under 0.20 with a high drug-loading efficiency ranging from 80.8% to 90.6%. The in vitro drug release profiles show that the DOX-loaded SLNs exhibit a pH-controlled drug release behavior with the maximum and minimum unloading percentages of 63.4% at pH 4.7 and 25.2% at pH 7.4, respectively. The DOX-loaded C-PEG-SLNs displayed a superior ability in inhibiting the proliferation of MCF-7/MDR cells. At a DOX concentration of 80 µM, the cell viabilities treated with C-PEG-SLNs were approximately one-third of the group treated with free DOX. The inhibition activity of C-PEG-SLNs could be attributed to the transport of C-PEG to cell membrane, leading to the change of the composition of the cell membrane and thus the inhibition of permeability glycoprotein activity. This hypothesis is supported by the confocal images showing the accumulation of DOX in the nuclei of cancer cells and the localization of C-PEG on the cell membranes. The results of in vivo study further demonstrated that the DOX delivered by the SLNs accumulates predominantly in tumor via enhanced permeability and retention effect, the

P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

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He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

2010-08-27

P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

The guinea-pig expresses functional CYP2C and P-glycoprotein: further validation of its usefulness in drug biotransformation/transport studies.

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Hasibu, Ibrahim; Patoine, Dany; Pilote, Sylvie; Drolet, Benoit; Simard, Chantale

2015-04-01

The guinea-pig is an excellent animal model for studying cardiopulmonary physiology/pharmacology. Interestingly, it also possesses a number of drug-metabolizing enzymes found in humans, such as CYP1A, CYP2D and CYP3A. To evaluate the hypothesis that the guinea-pig also expresses a functional CYP2C drug-metabolizing enzyme and the P-glycoprotein (P-gp) drug transporter in various tissues. cDNAs encoding CYP2C and P-gp were obtained from guinea-pig liver or small intestine and sequenced. Western blotting was performed to confirm the expression of CYP2C and P-gp. The functional enzymatic activity of guinea-pig CYP2C was evaluated with microsomal preparations using diclofenac and tolbutamide as specific drug substrates in HPLC analyses. To further study both P-gp and CYP2C functional activities, the guinea-pig ABCB1/MDR1 and CYP2C genes were cloned. The recombinant plasmids were then transfected in HEK293 (human embryonic kidney) cells and either calcein-acetoxymethyl ester (AM) accumulation assays or 14,15-EET/DHET formation experiments were performed to evaluate either P-gp transport activity or CYP2C epoxygenase activity, respectively. The guinea-pig tissue distribution of P-gp was studied by Western blotting. Functional expression of CYP2C was demonstrated in guinea-pig liver microsomal preparations. CYP2C-mediated biotransformation of diclofenac and tolbutamide were shown. Expression of P-gp protein was detected in guinea-pig liver and small intestine. Functional activity of guinea-pig P-gp was demonstrated in ABCB1/MDR1-transfected cells. GP-CYP2C-transfected cells also showed functional epoxygenase activity. The guinea-pig expresses functional CYP2C and P-gp, thus suggesting its usefulness for further validating data obtained with other animal models in drug biotransformation/transport studies. Copyright © 2015 John Wiley & Sons, Ltd.

Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus

NARCIS (Netherlands)

Arceci, R. J.; Baas, F.; Raponi, R.; Horwitz, S. B.; Housman, D.; Croop, J. M.

1990-01-01

The multidrug resistance (mdr) gene family has been shown to encode a membrane glycoprotein, termed the P-glycoprotein, which functions as a drug efflux pump with broad substrate specificity. This multigene family is expressed in a tissue-specific fashion in a wide variety of normal and neoplastic

P-Glycoprotein/MDR1 Regulates Pokemon Gene Transcription Through p53 Expression in Human Breast Cancer Cells

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Wei Xu

2010-08-01

Full Text Available P-glycoprotein (Pgp, encoded by the multidrug resistance 1 (MDR1 gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

Effects of Zuccagnia punctata extracts and their flavonoids on the function and expression of ABCB1/P-glycoprotein multidrug transporter.

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Chieli, Elisabetta; Romiti, Nadia; Catiana Zampini, Iris; Garrido, Gabino; Inés Isla, María

2012-12-18

Zuccagnia punctata extracts (ZpE) are used in ethnomedicine as antimicrobial and anti-inflammatory drugs. The pharmacological properties of ZpE and their polyphenolic components suggest that they may be used as potential modulators on the P-glycoprotein (P-gp) multidrug transporter. P-gp is well known for its role in the acquired drug resistance by tumors following chemotherapy, causing a low drug bioavailability by extruding them out of the cells. To evaluate the effects of ZpE and three of their phenolic components: 7-hydroxyflavanone (HF), 3,7-dihydroxyflavone (DHF) and 2',4'-dihydroxychalcone (DHC) on P-gp activity and expression. The effects of natural products on ABCB1/P-gp function and expression were evaluated by R-123 accumulation assay and western blot analysis using HK-2 cells as experimental model. The ABCB1 mRNA content was determined by SQRT-PCR. The accumulation of R-123 in HK-2 cells was significantly increased by ZpE and DHF, and to a lesser extent by DHC, indicating their roles on the efflux transporter activity. However, HF did not show any effect. HK-2 cells maintained in the presence of ZpE or DHF for 72 h, showed an increase in P-gp expression whereas activity was unchanged or decreased. No changes were observed in ABCB1 mRNA content. Furthermore, in these assay conditions, more sensibility of HK-2 cells to the cytotoxic action of cyclosporine A (P-gp substrate) was observed. These results may suggest an impact of Zuccagnia punctata and some of its components on the pharmacokinetics of drugs that are P-gp substrates, as well as a potential role on multidrug resistance modulation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Expression of P-gp, MRP, LRP, GST-π and TopoIIα and intrinsic resistance in human lung cancer cell lines.

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Wang, Jiarui; Zhang, Jinhui; Zhang, Lichuan; Zhao, Long; Fan, Sufang; Yang, Zhonghai; Gao, Fei; Kong, Ying; Xiao, Gary Guishan; Wang, Qi

2011-11-01

This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST‑π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.

Anaplasia and drug selection-independent overexpression of the multidrug resistance gene, MDR1, in Wilms' tumor.

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Re, G G; Willingham, M C; el Bahtimi, R; Brownlee, N A; Hazen-Martin, D J; Garvin, A J

1997-02-01

One reason for the failure of chemotherapy is the overexpression of the multidrug resistance gene, MDR1. The product of this gene is the multidrug transporter P-glycoprotein, an ATP-dependent pump that extrudes drugs from the cytoplasm. Some tumors inherently express P-glycoprotein, whereas others acquire the ability to do so after exposure to certain chemotherapeutic agents, often by the mechanism of gene amplification. Classical Wilms' tumors (nephroblastoma) typically respond to therapy and have a good prognosis. On the contrary, anaplastic Wilms' tumors are generally refractory to chemotherapy. These anaplastic variants are rare (4.5% of all Wilms' tumors reported in the United States), aggressive, and often fatal forms of tumor, which are commonly thought to result from the progression of classical Wilms' tumors. To investigate the basis for this differential response to therapy, we examined a number of classical and anaplastic Wilms' tumors for the expression of the MDR1 gene by immunohistochemical and mRNA analysis. Classical Wilms' tumors consistently did not express P-glycoprotein except in areas of tubular differentiation, as in normal kidney. Similarly, two of three anaplastic tumors failed to show P-glycoprotein expression. In contrast, cultured cells derived from a third anaplastic tumor, W4, exhibited strong P-glycoprotein expression and were drug resistant in vitro. Southern analysis revealed that W4 cells contained a single copy of the MDR1 gene per haploid genome similar to normal cells, demonstrating that the overexpression of MDR1 was not caused by gene amplification. Transcriptional activation of the MDR1 gene would be in keeping with the concept that p53 might act as a transcriptional repressor of the MDR1 gene.

A new in vivo method to study P-glycoprotein transport in tumors and the blood-brain barrier

NARCIS (Netherlands)

Hendrikse, NH; de Vries, EGE; Eriks-Fluks, L; van der Graaf, WTA; Hospers, GAP; Willemsen, ATM; Vaalburg, W; Franssen, EJF

1999-01-01

Drug resistance is a major cause of chemotherapy failure in cancer treatment, One reason is the overexpression of the drug efflux pump P-glycoprotein (P-gp), involved in multidrug resistance (MDR), In vivo pharmacokinetic analysis of P-gp transport might identify the capacity of modulation by P-gp

Disinfectant-susceptibility of multi-drug-resistant Mycobacterium tuberculosis isolated in Japan

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Noriko Shinoda

2016-02-01

Full Text Available Abstract Background Multi-drug-resistant Mycobacterium tuberculosis has been an important problem in public health around the world. However, limited information about disinfectant-susceptibility of multi-drug-resistant strain of M. tuberculosis was available. Findings We studied susceptibility of several Japanese isolates of multi-drug-resistant M. tuberculosis against disinfectants, which are commonly used in clinical and research laboratories. We selected a laboratory reference strain (H37Rv and eight Japanese isolates, containing five drug-susceptible strains and three multi-drug-resistant strains, and determined profiles of susceptibility against eight disinfectants. The M. tuberculosis strains were distinguished into two groups by the susceptibility profile. There was no relationship between multi-drug-resistance and disinfectant-susceptibility in the M. tuberculosis strains. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance. Conclusions Disinfectant-resistance is independent from multi-drug-resistance in M. tuberculosis. Cresol soap and oxydol were effective against all strains we tested, regardless of drug resistance.

A review on the relation between the brain-serum concentration ratio of drugs and the influence of P-glycoprotein

DEFF Research Database (Denmark)

Ejsing, Thomas Broeng; Morling, Niels; Linnet, Kristian

2007-01-01

This overview on the brain-serum relationship for drugs illustrates the importance of the drug transporter P-glycoprotein at the blood-brain barrier. Generally, an inverse relationship exists between the magnitude of the brain-serum ratio and the influence of P-glycoprotein. Concerning the pharma...... the pharmacogenomics of P-glycoprotein, no clear effect of single nucleotide polymorphisms (SNPs) has been demonstrated in humans....

Evodiamine synergizes with doxorubicin in the treatment of chemoresistant human breast cancer without inhibiting P-glycoprotein.

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Shengpeng Wang

Full Text Available Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO, a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose polymerase (PARP, caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs. Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp. Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.

vPARP Adjusts MVP Expression in Drug-resistant Cell Lines in Conjunction with MDR Proteins.

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Wojtowicz, Karolina; Januchowski, Radoslaw; Nowicki, Michal; Zabel, Maciej

2017-06-01

The definition of vault (ribonucleoprotein particles) function remains highly complex. Vaults may cooperate with multidrug resistance (MDR) proteins, supporting their role in drug resistance. This topic is the main theme of this publication. The cell viability was determined by an MTT assay. The protein expression was detected by western blot analysis. The proteins were knocked-down using siRNA. No major vault protein (MVP) in the LoVo/Dx and W1PR cell lines after tunicamycin treatment was shown. In W1PR cells with knocked-down MVP, a statistically significant decrease in cell viability was noted. In LoVo/Dx, W1TR and A2780TR cells were vault poly-ADP-ribose polymerase (vPARP) was knockdown, a decrease in cell viability was shown. Also, MVP silencing induced an increase in glycoprotein P (Pgp) expression in LoVo/Dx cells. MVP is important for the drug resistance of cancer cells, but it probably requires the presence of vPARP for full activation. Some correlations between MDR proteins and vaults exist. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Expression of multidrug resistance gene and P-glycoprotein in nasopharyngealcarcinoma cells after irradiation

International Nuclear Information System (INIS)

Wang Ruoyu; Wang Hui; Fan Kai; Lv Shen

2007-01-01

Objective: To mimick a clinical fractionated protocol of exposure to X-radiation in vitro in order to investigate the changes in the function of MDR1 and P-gp in nasopharyngeal carcinoma (NPC) CNE cell before and after irradiation to determine the sequential order of radiotherapy and chemotherapy or the time of chemotherapy after radiotherapy in the treatment of NPC. Methods: Exponentially growing CNE cells were treated with fractionated X-radiation with total dose of 10 Gy (2 Gy per day for 5 days consecutively) in vitro. The expression of MDR1 gene was examined in CNE cells before irradiation and on days 4,8,13,17 and 21 after irradiation by RT-PCR, and its protein P-gp were detected by immunocytochemistry. The function of multidrug resistance protein P-gp was examined by MTT method. Results: Expression of MDR1 gene was below the level of detection before irradiation. Irradiation induced an overexpression of MDR1 gene on day 4, expression of MDR1 was decreased from day 8 to day 21. The overall expression of MDR1 was significantly more than that before irradiation (P<0.05) Expression of P-gp was below the level of detection before irradiation, which demonstrated that irradiation induced an overexpression of P-gp. This overexpression was increased from day 8 to day 21. The overpression of MDR1 gene was maintained dining a short period, however, the emergence of overpression of protein P-gp was later than that of MDR1 gene. Resistance index was 1 for both pre-irradiation and on day 8, and up to 8,10,11.2 on days 13, 17 and 21, respectively. The change of resistance index was accordant with the condition of overexpression of P-gp . Conclusions: Expression of P-gp in nasopharyngeal carcinoma (NPC) CNE cell was below the level of detection before irradiation. Irradiation can induce an overexpression of MDR1 gene and its protein P-gp in CNE cells. The overexpression of MDR1 gene and its protein P-gp lasted a long term. (authors)

Molecular Analysis and Expression of bap Gene in Biofilm-Forming Multi-Drug-Resistant Acinetobacter baumannii

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Omid Azizi

2016-10-01

Full Text Available Background: Acinetobacter baumannii is commonly resistant to nearly all antibiotics due to presence of antibiotic resistance genes and biofilm formation. In this study we determined the presence of certain antibiotic-resistance genes associated with biofilm production and the influence of low iron concentration on expression of the biofilm-associated protein gene (bap in development of biofilm among multi-drug-resistant A. baumannii (MDRAB. Methods: Sixty-five MDRAB isolates from clinical samples were collected. Molecular typing was carried out by random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR. Biofilm formation was assayed by the microtiter method. Results: The sequence of bap was determined and deposited in the GenBank database (accession no. KR080550.1. Expression of bap in the presence of low iron was analyzed by relative quantitative real time PCR (rqRT-PCR. Nearly half of the isolates belonged to RAPD-types A and B remaining were either small clusters or singleton. The results of biofilm formation revealed that 23 (35.4%, 18 (27.7%, 13 (20%, and 11 (16.9% of the isolates had strong, moderate, weak, and no biofilm activities, respectively. ompA and csuE genes were detected in all, while bap and blaPER-1 were detected in 43 (66% and 42 (64% of the isolates that showed strong and moderate biofilm activities (p ≤ 0.05, respectively. Analysis of bap expression by rqRT-PCR revealed five isolates with four-fold bap overexpression in the presence of low iron concentration (20 μM. Conclusion: The results suggest that bap overexpression may influence biofilm formation in presence of low iron concentration.

Technetium-99m methoxyisobutylisonitrile imaging for parathyroid adenoma: relationship to P-glycoprotein or multidrug resistance-related protein expression

International Nuclear Information System (INIS)

Kao, Albert; Shiau, Yu-Chien; Tsai, Shih-Chuan; Wang, Jhi-Joung; Ho, Shung-Tai

2002-01-01

Gland size has been reported to have a major influence on localisation of parathyroid adenomas by technetium-99m methoxyisobutylisonitrile ( 99m Tc-MIBI) imaging. It has also been suggested that P-glycoprotein (Pgp) expression in parathyroid adenomas may influence localisation because false negative studies have been reported with large tumours and true positives with very small tumours. Therefore, the purpose of this study was to retrospectively evaluate the relationship between 99m Tc-MIBI parathyroid imaging results and Pgp or multidrug resistance-related protein (MRP) expression in parathyroid adenomas. Before surgery, 47 patients with large parathyroid adenomas (larger than 1.5 g) underwent early and delayed parathyroid imaging, 10 min and 2 h after intravenous injection of 99m Tc-MIBI. Immunohistochemical analyses (IHA) were performed, using multiple non-consecutive sections of the operative specimens, to detect Pgp or MRP expression. According to the results of IHA, the 34 parathyroid adenomas were separated into four groups: (1) three adenomas positive for both Pgp and MRP expression, (2) one adenoma positive for Pgp but negative for MRP expression, (3) four adenomas negative for Pgp but positive for MRP expression and (4) 39 adenomas with negative for both Pgp and MRP expression. All 39 adenomas in group 4 could be detected by 99m Tc-MIBI parathyroid imaging. None of the eight adenomas in groups 1-3 could be detected by 99m Tc-MIBI parathyroid imaging (P 99m Tc-MIBI imaging in localising parathyroid adenomas preoperatively. (orig.)

The study of relationship between scintimammography of breast cancer and the expression of P-glycoprotein and GST-π

International Nuclear Information System (INIS)

Cheng Bing; Liu Baoping; Han Xingmin

2003-01-01

Objective: To study the relationship of 99 Tc m -MIBI uptake and washout in untreated breast cancer with immunohistochemically determined glutathione-S-transferase π(GST-π) and P-glycoprotein (P-gp) expression. Methods: Thirty-six patients with untreated breast cancer were studied prospectively. 99 Tc m -MIBI scintigraphy and immunohistochemical analyses of P-gp and GST-π expression were used to evaluate the expected tumor tissues after surgical operations. Anterior planar images were acquired 10 and 180 min after intravenous injection of 740 MBq 99 Tc m -MIBI. The tumor-to-normal breast ratio (T/N) and washout index (WI) were calculated. Results: The early T/N ratios were significantly lower in 9 patients with negative P-gp expression when compared with that in 27 patients with positive P-gp expression (main scores were 8.33 vs 21.89 and Z=-3.32, P=0.002). The WI was significantly different between the two groups (t=3.59, P=0.001). On the other hand there was no significant relationship between negative and positive GST-π expression when calculated the early T/N ratio and WI. Significant relationship between GST-π and P-gp expression was found in these patients. Conclusions: The coexpression of P-gp and GST-π is one of the major characteristics of drug resistance in untreated breast cancer. Double-phase scintimammography and WI of 99 Tc m -MIBI can be used as a simple functional test for in vivo imaging of tumoral P-gp expression in patients with untreated breast cancer

Relevance of P-glycoprotein on CXCR4+ B cells to organ manifestation in highly active rheumatoid arthritis.

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Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Kawabe, Akio; Tanaka, Yoshiya

2018-03-01

In rheumatoid arthritis (RA), P-glycoprotein (P-gp) expression on activated B cells is associated with active efflux of intracellular drugs, resulting in drug resistance. CXCR4 is associated with migration of B cells. This study was designed to elucidate the relevance of P-gp expression on CXCR4 + B cells to clinical manifestations in refractory RA. CD19 + B cells were analyzed using flow cytometry and immunohistochemistry. P-gp was highly expressed especially on CXCR4 + CD19 + B cells in RA. The proportion of P-gp-expressing CXCR4 + B cells correlated with disease activity, estimated by Simplified Disease Activity Index (SDAI), and showed marked expansion in RA patients with high SDAI and extra-articular involvement. In highly active RA, massive infiltration of P-gp + CXCR4 + CD19 + B cells was noted in CXCL12-expressing inflammatory lesions of RA synovitis and RA-associated interstitial pneumonitis. In RA patient with active extra-articular involvement, intracellular dexamethasone level (IDL) in lymphocytes diminished with expansion of P-gp + CXCR4 + CD19 + B cells. Adalimumab reduced P-gp + CXCR4 + CD19 + B cells, increased IDL in lymphocytes, and improved the clinical manifestation and allowed tapering of concomitant medications. Expansion of P-gp + CXCR4 + B cells seems to be associated with drug resistance, disease activity and progressive destructive arthritis with extra-articular involvement in RA.

Pyramidatine (Z88) Sensitizes Vincristine-Resistant Human Oral Cancer (KB/VCR) Cells to Chemotherapeutic Agents by Inhibition of P-glycoprotein.

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Liu, Zulong; Zhu, Hengrui; Qu, Shijin; Tang, Lisha; Cao, Lihuan; Yu, Wenbo; Yang, Xianmei; Jiang, Songmin; Zhu, Dayuan; Tan, Changheng; Yu, Long

2018-01-01

Multi-drug resistance (MDR) remains a major impediment in cancer therapy. A major goal for scientists is to discover more effective compounds that are able to circumvent MDR and simultaneously have minimal adverse side effects. In the present study, we aim to determine the anti-MDR effects of pyramidatine (Z88), a cinnamic acid-derived bisamide compound isolated from the leaves of Aglaia perviridis, on KB/VCR (vincristineresistant human oral cancer cells) and MCF-7/ADR (adriamycin-resistant human breast adenocarcinoma) cells. Cell viability and average resistant fold (RF) of Z88 were examined by Cell Counting Kit-8 (CCK-8) assay. Flow cytometry, western blot, RT-PCR, Rhodamine 123 accumulation assay and P-glycoprotein (P-gp) ATPase assay were used to demonstrate the anti-MDR activity and mechanism of Z88. The average RF of Z88 is 0.09 and 0.51 in KB/VCR and MCF-7/ADR cells. A CCK-8 assay showed that Z88 could enhance the cytotoxicity of VCR toward KB/VCR cells. A FACS analysis revealed that Z88 could enhance the VCR-induced apoptosis as well as G2/M arrest in a dose-dependent manner in KB/VCR cells. Western blot results showed that the expression levels of PARP, Bax, and cyclin B1 all increased after treatment with 0.2 µmol/L (µM) of VCR combined with 10 µM of Z88 for 24 h in KB/VCR cells. Z88 also could enhance the accumulation of rhodamine 123. Further studies showed that Z88 could inhibit the verapamil stimulated Pgp ATPase activity. Additionally, qPCR detection and western blot assays revealed that Z88 could decrease the expression of P-gp at both RNA and protein level. Z88 exerted potent anti-MDR activity in vitro and its mechanisms are associated with dualinhibition of the function and expression of P-gp. These findings encourage efforts to develop more effective reversal agents to circumvent MDR based on Z88. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Comparison of constitutive and thiabendazole-induced expression of five cytochrome P450 genes in fourth-stage larvae of Haemonchus contortus isolates with different drug susceptibility identifies one gene with high constitutive expression in a multi-resistant isolate

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Esra Yilmaz

2017-12-01

Full Text Available Benzimidazoles (BZs remain amongst the most widely used anthelmintic drug classes against gastro-intestinal nematode infections, although their efficacy is increasingly compromised by resistance. The primary underlying mechanisms for BZ resistance are single-nucleotide polymorphisms (SNPs in the isotype 1 β-tubulin gene causing the substitutions F167Y, E198A or F200Y. However, resistance is believed to be multi-genic and previous studies have shown that isolates carrying 90–100% F200Y can vary considerably in their resistance level in the egg hatch assay (EHA. Cytochrome P450 monooxygenases (CYPs are associated with drug resistance in mammals and arthropods and have been considered as mediators of anthelmintic resistance. In Caenorhabditis elegans, several members of the CYP34/35 and CYP31 families are BZ and/or xenobiotic inducible and thiabendazole (TBZ is metabolised by CYP35D1. Here, expression of all 5 CYPs closely related to the C. elegans CYP34/35 and CYP31 families was investigated in fourth-stage larvae of two susceptible and three BZ-resistant Haemonchus contortus isolates following in vitro exposure to TBZ for 3 and 6 h using real-time RT-PCR. The resistance status of all isolates was determined using EHAs and quantification of resistance-associated β-tubulin SNPs using pyrosequencing. While none of the CYPs was TBZ inducible, constitutive expression of CYP34/35 family member HCOI100383400 was significantly 2.4–3.7-fold higher in the multi-drug resistant WR isolate with the strongest BZ resistance phenotype compared to susceptible and intermediate-level BZ-resistant isolates. Although this increase is only moderate, HCOI100383400 might still be involved in high-level BZ resistance by further decreasing susceptibility in isolates already carrying 100% of a β-tubulin SNP causing BZ resistance. Lower transcript levels were observed for all CYPs in the intermediately resistant IRE isolate in comparison to the susceptible Hc

Detecting parathyroid adenoma using technetium-99m tetrofosmin: comparison with P-glycoprotein and multidrug resistance related protein expression--a preliminary report

International Nuclear Information System (INIS)

Shiau, Y.C.; Tsai, S.C.; Wang, J.J.; Ho, S.T.; Kao, A.

2002-01-01

The aim of this study was to investigate the relationships among technetium-99m tetrofosmin (Tc-TF) accumulation in parathyroid adenoma and the expression of P-glycoprotein (Pgp) or multidrug resistance related protein (MRP). Before operation, 33 patients with parathyroid adenomas (larger than 1.5 gm) were studied with parathyroid scintigraphy 10 minutes and 2 hours after intravenous injection of Tc-TF before operation. Immunohistochemical analyses (IHA) were performed on multiple nonconsecutive sections of operative parathyroid specimens to detect Pgp or MRP expression. According to the results of IHA, the 33 parathyroid adenomas were separated into four groups: (1) 2 adenomas with both positive Pgp and positive MRP expression, (2) 1 adenomas with positive Pgp but negative MRP expression, (3) 2 adenomas with negative Pgp but positive MRP expression, and (4) 28 adenomas with both negative Pgp and negative MRP expression. All of 28 adenomas in the group 4 could be detected by Tc-TF parathyroid imaging. All of 5 adenomas in the groups 1 to 3 could not be detected by TcTF parathyroid imaging (p < 0.05). Not only the size of parathyroid adenomas, but also significant Pgp or MRP expression limited the sensitivity of Tc-TF parathyroid imaging to localize parathyroid adenomas before operation

Technetium-99m methoxyisobutylisonitrile imaging for parathyroid adenoma: relationship to P-glycoprotein or multidrug resistance-related protein expression

Energy Technology Data Exchange (ETDEWEB)

Kao, Albert [Departments of Nuclear Medicine and Medical Research, China Medical College Hospital, No. 2, Yuh-Der Road, Taichung 404 (Taiwan); Shiau, Yu-Chien [Department of Nuclear Medicine, Far Eastern Memorial Hospital, Institute of Biomedical Engineering, College of Electrical Engineering, National Taiwan University, Taipei (Taiwan); Tsai, Shih-Chuan [Department of Nuclear Medicine, Show-Chwan Memorial Hospital, Chunghua (Taiwan); Wang, Jhi-Joung [Department of Medical Research, Chi-Mei Medical Center, Tainan (Taiwan); Ho, Shung-Tai [School of Medicine, National Defense Medical Center, Taipe (Taiwan)

2002-08-01

Gland size has been reported to have a major influence on localisation of parathyroid adenomas by technetium-99m methoxyisobutylisonitrile ({sup 99m}Tc-MIBI) imaging. It has also been suggested that P-glycoprotein (Pgp) expression in parathyroid adenomas may influence localisation because false negative studies have been reported with large tumours and true positives with very small tumours. Therefore, the purpose of this study was to retrospectively evaluate the relationship between {sup 99m}Tc-MIBI parathyroid imaging results and Pgp or multidrug resistance-related protein (MRP) expression in parathyroid adenomas. Before surgery, 47 patients with large parathyroid adenomas (larger than 1.5 g) underwent early and delayed parathyroid imaging, 10 min and 2 h after intravenous injection of {sup 99m}Tc-MIBI. Immunohistochemical analyses (IHA) were performed, using multiple non-consecutive sections of the operative specimens, to detect Pgp or MRP expression. According to the results of IHA, the 34 parathyroid adenomas were separated into four groups: (1) three adenomas positive for both Pgp and MRP expression, (2) one adenoma positive for Pgp but negative for MRP expression, (3) four adenomas negative for Pgp but positive for MRP expression and (4) 39 adenomas with negative for both Pgp and MRP expression. All 39 adenomas in group 4 could be detected by {sup 99m}Tc-MIBI parathyroid imaging. None of the eight adenomas in groups 1-3 could be detected by {sup 99m}Tc-MIBI parathyroid imaging (P<0.05). It is concluded that not only the size of parathyroid adenomas but also significant Pgp or MRP expression limits the sensitivity of {sup 99m}Tc-MIBI imaging in localising parathyroid adenomas preoperatively. (orig.)

Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein.

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Hongping Min

Full Text Available Development of multidrug resistance (MDR is a continuous clinical challenge partially due to the overexpression of P-glycoprotein (P-gp for chronic myelogenous leukemia (CML patients. Herein, we evaluated the inhibitory potency of emodin, a natural anthraquinone derivative isolated from Rheum palmatum L, on P-gp in P-gp positive K562/ADM cells. Competition experiments combined with molecular docking analysis were utilized to investigate the binding modes between emodin and binding sites of P-gp. Emodin reversed adriamycin resistance in K562/ADM cells accompanied with the decrease of P-gp protein expression, further increasing the uptake of rhodamine123 in both K562/ADM and Caco-2 cells, indicating the inhibition of P-gp efflux function. Moreover, when incubated with emodin under different conditions where P-gp was inhibited, K562/ADM cells displayed increasing intracellular uptake of emodin, suggesting that emodin may be the potential substrate of P-gp. Importantly, rhodamine 123 could increase the Kintrinsic (Ki value of emodin linearly, whereas, verapamil could not, implying that emodin competitively bound to the R site of P-gp and noncompetition existed between emodin and verapamil at the M site, in a good accordance with the results of molecular docking that emodin bound to the R site of P-gp with higher affinity. Based on our results, we suggest that emodin might be used to modulate P-gp function and expression.

Increased platelet expression of glycoprotein IIIa following aspirin treatment in aspirin-resistant but not aspirin-sensitive subjects

Science.gov (United States)

Floyd, Christopher N; Goodman, Timothy; Becker, Silke; Chen, Nan; Mustafa, Agnesa; Schofield, Emma; Campbell, James; Ward, Malcolm; Sharma, Pankaj; Ferro, Albert

2014-01-01

Aims Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. Methods Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B2, and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. Results In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. Conclusions In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker. PMID:25099258

Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction

International Nuclear Information System (INIS)

Noonan, K.E.; Beck, C.; Holzmayer, T.A.; Chin, J.E.; Roninson, I.B.; Wunder, J.S.; Andrulis, I.L.; Gazdar, A.F.; Willman, C.L.; Griffith, B.; Von Hoff, D.D.

1990-01-01

The resistance of tumor cells ot chemotheraprutic drugs is a major obstacle to successful cancer chemotherapy. In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR). The levels of MDR in cell lines selected in bitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein. In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays. MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refactory tumors untreated with chemotherapeutic drugs. The authors have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clincal samples, based on the polymerase chain reaction. They have used this assay to measure MDR1 gene expression in MDR cell lines and >300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays. Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested. The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were ost common among tumor types known to be relatively responsive to chemotherapy

P-glycoprotein expression and DNA topoisomerase I and II activity in benign tumors of the ovary and in malignant tumors of the ovary, before and after platinum/cyclophosphamide chemotherapy

NARCIS (Netherlands)

van der Zee, A G; Hollema, H; de Jong, S; Boonstra, H; Gouw, A; Willemse, P H; Zijlstra, J G; de Vries, E G; de Jong, Steven

1991-01-01

P-glycoprotein (P-gp) expression and DNA topoisomerase (Topo) II are important variables in multidrug resistant tumor cell lines. The aim of this study was to evaluate P-gp expression and Topo I and II activity in benign and malignant epithelial ovarian tumors. P-gp expression was analyzed

Multi-clonal evolution of multi-drug-resistant/extensively drug-resistant Mycobacterium tuberculosis in a high-prevalence setting of Papua New Guinea for over three decades

Science.gov (United States)

Bainomugisa, Arnold; Lavu, Evelyn; Hiashiri, Stenard; Majumdar, Suman; Honjepari, Alice; Moke, Rendi; Dakulala, Paison; Hill-Cawthorne, Grant A.; Pandey, Sushil; Marais, Ben J.; Coulter, Chris; Coin, Lachlan

2018-01-01

An outbreak of multi-drug resistant (MDR) tuberculosis (TB) has been reported on Daru Island, Papua New Guinea. Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. Whole genome sequencing of 100 of 165 clinical isolates referred from Daru General Hospital to the Supranational reference laboratory, Brisbane, during 2012–2015 revealed that 95 belonged to a single modern Beijing sub-lineage strain. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with potentially enhanced virulence mediated by an mycP1 mutation. The Beijing sub-lineage strain demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2 %) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multi-drug resistance, observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance, and evidence of local transmission of extensively drug resistant (XDR) strains from 2009. These findings underline the importance of whole genome sequencing in informing an effective public health response to MDR/XDR TB. PMID:29310751

Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?

Science.gov (United States)

Abdullahi, Wazir; Davis, Thomas P; Ronaldson, Patrick T

2017-07-01

Drug delivery to the central nervous system (CNS) is greatly limited by the blood-brain barrier (BBB). Physical and biochemical properties of the BBB have rendered treatment of CNS diseases, including those with a hypoxia/reoxygenation (H/R) component, extremely difficult. Targeting endogenous BBB transporters from the ATP-binding cassette (ABC) superfamily (i.e., P-glycoprotein (P-gp)) or from the solute carrier (SLC) family (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents)) has been suggested as a strategy that can improve delivery of drugs to the brain. With respect to P-gp, direct pharmacological inhibition using small molecules or selective regulation by targeting intracellular signaling pathways has been explored. These approaches have been largely unsuccessful due to toxicity issues and unpredictable pharmacokinetics. Therefore, our laboratory has proposed that optimization of CNS drug delivery, particularly for treatment of diseases with an H/R component, can be achieved by targeting Oatp isoforms at the BBB. As the major drug transporting Oatp isoform, Oatp1a4 has demonstrated blood-to-brain transport of substrate drugs with neuroprotective properties. Furthermore, our laboratory has shown that targeting Oatp1a4 regulation (i.e., TGF-β signaling mediated via the ALK-1 and ALK-5 transmembrane receptors) represents an opportunity to control Oatp1a4 functional expression for the purpose of delivering therapeutics to the CNS. In this review, we will discuss limitations of targeting P-gp-mediated transport activity and the advantages of targeting Oatp-mediated transport. Through this discussion, we will also provide critical information on novel approaches to improve CNS drug delivery by targeting endogenous uptake transporters expressed at the BBB.

Comparison of the multi-drug resistant human hepatocellular carcinoma cell line Bel-7402/ADM model established by three methods

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Zhong Xingguo

2010-08-01

Full Text Available Abstract Background To compare the biological characteristics of three types of human hepatocellular carcinoma multi-drug resistant cell sub-lines Bel-7402/ADM models established by three methods. Methods Established human hepatocellular carcinoma adriamycin (ADM multi-drug resistant cell sub-lines models Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS by three methods of in vitro concentration gradient increased induction, nude mice liver-implanted induction and subcutaneous-implanted induction respectively. Phase contrast microscopy was used to observe the cells and the MTT (methyl thiazolyl tetrazolium method was used to detect drug resistance of the three different sub-lines of cells. Results The three groups of drug resistant cells, Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS generated cross-resistance to ADM and CDDP (cis-Diaminedichloroplatinum, but showed a significant difference in resistance to Bel-7402 IC50 value (P V, 46 h (Bel-7402/ADML, and 45 h (Bel-7402/ADMS. The excretion rates of ADM were significantly increased compared with the parent cell (34.14% line and were 81.06% (Bel-7402/ADMV, 66.56% (Bel-7402/ADML and 61.56% (Bel-7402/ADMS. Expression of P-gp and MRP in the three groups of resistant cells was significantly enhanced (P P > 0.05. Conclusions Stable resistance was involved in the resistant cell line model established by the above three methods. Liver implantation was a good simulation of human hepatocellular and proved to be an ideal model with characteristics similar to human hepatocellular biology and the pharmacokinetics of anticancer drugs.

A mechanism for overcoming P-glycoprotein-mediated drug resistance: novel combination therapy that releases stored doxorubicin from lysosomes via lysosomal permeabilization using Dp44mT or DpC.

Science.gov (United States)

Seebacher, Nicole A; Richardson, Des R; Jansson, Patric J

2016-12-01

The intracellular distribution of a drug can cause significant variability in both activity and selectivity. Herein, we investigate the mechanism by which the anti-cancer agents, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and the clinically trialed, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), re-instate the efficacy of doxorubicin (DOX), in drug-resistant P-glycoprotein (Pgp)-expressing cells. Both Dp44mT and DpC potently target and kill Pgp-expressing tumors, while DOX effectively kills non-Pgp-expressing cancers. Thus, the combination of these agents should be considered as an effective rationalized therapy for potently treating advanced and resistant tumors that are often heterogeneous in terms of Pgp-expression. These studies demonstrate that both Dp44mT and DpC are transported into lysosomes via Pgp transport activity, where they induce lysosomal-membrane permeabilization to release DOX trapped within lysosomes. This novel strategy of loading lysosomes with DOX, followed by permeabilization with Dp44mT or DpC, results in the relocalization of stored DOX from its lysosomal 'safe house' to its nuclear targets, markedly enhancing cellular toxicity against resistant tumor cells. Notably, the combination of Dp44mT or DpC with DOX showed a very high level of synergism in multiple Pgp-expressing cell types, for example, cervical, breast and colorectal cancer cells. These studies revealed that the level of drug synergy was proportional to Pgp activity. Interestingly, synergism was ablated by inhibiting Pgp using the pharmacological inhibitor, Elacridar, or by inhibiting Pgp-expression using Pgp-silencing, demonstrating the importance of Pgp in the synergistic interaction. Furthermore, lysosomal-membrane stabilization inhibited the relocalization of DOX from lysosomes to the nucleus upon combination with Dp44mT or DpC, preventing synergism. This latter observation demonstrated the importance of lysosomal

Effect of expression of P-glycoprotein on technetium-99m methoxyisobutylisonitrile single photon emission computed tomography of brain tumors

Energy Technology Data Exchange (ETDEWEB)

Shibata, Yasushi; Matsumura, Akira; Nose, Tadao [Tsukuba Univ., Ibaraki (Japan). Inst. of Clinical Medicine

2002-08-01

The expression of P-glycoprotein was investigated imunohistochemically in 26 brain tumor tissues and compared with the findings of technetium-99m methoxyisobutylisonitrile single photon emission computed tomography ({sup 99m}Tc-MIBI SPECT) to clarify the effect of P-glycoprotein on the diagnostic accuracy. P-glycoprotein labeling index of both tumor cells and vascular endothelial cells showed no clear relationship with the findings of {sup 99m}Tc-MIBI SPECT imaging. Expression of P-glycoprotein has no effect on the diagnostic accuracy of {sup 99m}Tc-MIBI SPECT. (author)

P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia

NARCIS (Netherlands)

de Vries, EGE; van Putten, WLJ; Verdonck, LF; Ossenkoppele, GJ; Verhoef, GEG; Vellenga, E

Despite treatment with intensive chemotherapy, a considerable number of patients with acute myeloid leukemia (AML) die from their disease due to the occurrence of resistance. Overexpression of the transporter proteins P-glycoprotein (P-gp) and multidrug resistance protein (MRP) 1 has been identified

Partial circumvention of P-glycoprotein-mediated multidrug resistance by doxorubicin-14-O-hemiadipate.

Science.gov (United States)

Leontieva, Olga V; Preobrazhenskaya, Maria N; Bernacki, Ralph J

2002-02-01

Previously, we have reported partial circumvention of P-glycoprotein (Pgp)-associated resistance to doxorubicin (Dox) in MCF7/R human breast carcinoma and P388/R murine leukemia cell lines by doxorubicin-14-O-hemiadipate (H-Dox) [Povarov L.S. et al. (1995) Russian J. Bioorganic Chemistry 21: 797-803]. We felt that these changes were due to alterations in the cellular pharmacokinetics of the analog in multidrug (MDR) resistant cells, as compared to that of Dox. To address this hypothesis, we performed comparative studies of the accumulation, retention and intracellular localization of H-Dox and Dox in Dox-sensitive murine leukemia cell line P388/S and its Dox-selected. Pgp-positive drug resistant P388/R subline. These studies were performed in the presence or absence of cyclosporin A (CsA), a competitive inhibitor of Pgp. Flow cytometric analysis revealed significant differences in Dox and H-Dox accumulation in P388/R cells when compared to P388/S cells. In P388/R versus P388/S cells, there was a 38-fold decrease in Dox accumulation, but only a 5-fold decrease in H-Dox accumulation, indicating over a 7-fold increase in H-Dox buildup in resistant cells. CsA did not affect uptake or retention of either drug by sensitive cells. However, coincubation with CsA resulted in a 54-fold increase in Dox accumulation and only a 5-fold increase in H-Dox uptake in P388/R cells, restoring anthracycline levels in P388/R to 100% of that found in P388/S cells. Once internalized by the resistant cells, H-Dox was retained better than Dox regardless of presence or absence of CsA. Confocal microscopic analysis revealed the presence of H-Dox but no Dox in cellular nuclei of P388/R cells. Thus, increased activity of H-Dox toward P388/R cells was correlated with its enhanced ability to enter and be retained in these cells, and also with redistribution of H-Dox into the nuclei of the resistant cells as compared to Dox. Overall, our findings support our initial hypothesis and provide evidence

P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected against ischemic renal injury

NARCIS (Netherlands)

Huls, M.; Kramers, C.; Levtchenko, E.N.; Wilmer, M.J.G.; Dijkman, H.B.P.M.; Kluijtmans, L.A.J.; Hoorn, J.W.A. van der; Russel, F.G.M.; Masereeuw, R.

2007-01-01

The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful

Identification of multi-drug resistant Pseudomonas aeruginosa clinical isolates that are highly disruptive to the intestinal epithelial barrier

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Shevchenko Olga

2006-06-01

Full Text Available Abstract Background Multi-drug resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. In this study, we focused on the virulence of multi-drug resistant clinical strains P. aeruginosa against the intestinal epithelial barrier, since P. aeruginosa can cause lethal sepsis from within the intestinal tract of critically ill and immuno-compromised patients via mechanisms involving disruption of epithelial barrier function. Methods We screened consecutively isolated multi-drug resistant P. aeruginosa clinical strains for their ability to disrupt the integrity of human cultured intestinal epithelial cells (Caco-2 and correlated these finding to related virulence phenotypes such as adhesiveness, motility, biofilm formation, and cytotoxicity. Results Results demonstrated that the majority of the multi-drug resistant P. aeruginosa clinical strains were attenuated in their ability to disrupt the barrier function of cultured intestinal epithelial cells. Three distinct genotypes were found that displayed an extreme epithelial barrier-disrupting phenotype. These strains were characterized and found to harbor the exoU gene and to display high swimming motility and adhesiveness. Conclusion These data suggest that detailed phenotypic analysis of the behavior of multi-drug resistant P. aeruginosa against the intestinal epithelium has the potential to identify strains most likely to place patients at risk for lethal gut-derived sepsis. Surveillance of colonizing strains of P. aeruginosa in critically ill patients beyond antibiotic sensitivity is warranted.

Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs

Science.gov (United States)

Regula, Gertraud; Petrini, Orlando; Zinsstag, Jakob; Schelling, Esther

2013-01-01

We investigated the distribution of commensal staphylococcal species and determined the prevalence of multi-drug resistance in healthy cats and dogs. Risk factors associated with the carriage of multi-drug resistant strains were explored. Isolates from 256 dogs and 277 cats were identified at the species level using matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The diversity of coagulase-negative Staphylococci (CNS) was high, with 22 species in dogs and 24 in cats. Multi-drug resistance was frequent (17%) and not always associated with the presence of the mecA gene. A stay in a veterinary clinic in the last year was associated with an increased risk of colonisation by multi-drug resistant Staphylococci (OR = 2.4, 95% CI: 1.1~5.2, p value LRT = 0.04). When identifying efficient control strategies against antibiotic resistance, the presence of mechanisms other than methicillin resistance and the possible role of CNS in the spread of resistance determinants should be considered. PMID:23820161

Effect of dietary fat on hepatic liver X receptor expression in P-glycoprotein deficient mice: implications for cholesterol metabolism

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Lee Stephen D

2008-06-01

Full Text Available Abstract Pgp (P-glycoprotein, MDR1, ABCB1 is an energy-dependent drug efflux pump that is a member of the ATP-binding cassette (ABC family of proteins. Preliminary studies have reported that nonspecific inhibitors of Pgp affect synthesis and esterification of cholesterol, putatively by blocking trafficking of cholesterol from the plasma membrane to the endoplasmic reticulum, and that relative increases in Pgp within a given cell type are associated with increased accumulation of cholesterol. Several key efflux proteins involved in the cholesterol metabolic pathway are transcriptionally regulated by the nuclear hormone liver X receptor (LXR. Therefore, to examine the interplay between P-glycoprotein and the cholesterol metabolic pathway, we utilized a high fat, normal cholesterol diet to upregulate LXRα without affecting dietary cholesterol. Our research has demonstrated that mice lacking in P-glycoprotein do not exhibit alterations in hepatic total cholesterol storage, circulating plasma total cholesterol levels, or total cholesterol concentration in the bile when compared to control animals on either a normal (25% calories from dietary fat or high fat (45% calories from dietary fat diet. However, p-glycoprotein deficient mice (Mdr1a-/-/1b-/- exhibit increased hepatic LXRα protein expression and an elevation in fecal cholesterol concentration when compared to controls.

Modulation of P-glycoprotein activity by novel synthetic curcumin derivatives in sensitive and multidrug-resistant T-cell acute lymphoblastic leukemia cell lines

International Nuclear Information System (INIS)

Ooko, Edna; Alsalim, Tahseen; Saeed, Bahjat; Saeed, Mohamed E.M.; Kadioglu, Onat; Abbo, Hanna S.; Titinchi, Salam J.J.; Efferth, Thomas

2016-01-01

Background: Multidrug resistance (MDR) and drug transporter P-glycoprotein (P-gp) represent major obstacles in cancer chemotherapy. We investigated 19 synthetic curcumin derivatives in drug-sensitive acute lymphoblastic CCRF–CEM leukemia cells and their multidrug-resistant P-gp-overexpressing subline, CEM/ADR5000. Material and methods: Cytotoxicity was tested by resazurin assays. Doxorubicin uptake was assessed by flow cytometry. Binding modes of compounds to P-gp were analyzed by molecular docking. Chemical features responsible for bioactivity were studied by quantitative structure activity relationship (QSAR) analyses. A 7-descriptor QSAR model was correlated with doxorubicin uptake values, IC 50 values and binding energies. Results: The compounds displayed IC 50 values between 0.7 ± 0.03 and 20.2 ± 0.25 μM. CEM/ADR5000 cells exhibited cross-resistance to 10 compounds, collateral sensitivity to three compounds and regular sensitivity to the remaining six curcumins. Molecular docking studies at the intra-channel transmembrane domain of human P-gp resulted in lowest binding energies ranging from − 9.00 ± 0.10 to − 6.20 ± 0.02 kcal/mol and pKi values from 0.24 ± 0.04 to 29.17 ± 0.88 μM. At the ATP-binding site of P-gp, lowest binding energies ranged from − 9.78 ± 0.17 to − 6.79 ± 0.01 kcal/mol and pKi values from 0.07 ± 0.02 to 0.03 ± 0.03 μM. CEM/ADR5000 cells accumulated approximately 4-fold less doxorubicin than CCRF–CEM cells. The control P-gp inhibitor, verapamil, partially increased doxorubicin uptake in CEM/ADR5000 cells. Six curcumins increased doxorubicin uptake in resistant cells or even exceeded uptake levels compared to sensitive one. QSAR yielded good activity prediction (R = 0.797 and R = 0.794 for training and test sets). Conclusion: Selected derivatives may serve to guide future design of novel P-gp inhibitors and collateral sensitive drugs to combat MDR. - Highlights: • Novel derivatives of curcumin in reversing multidrug

Modulation of P-glycoprotein activity by novel synthetic curcumin derivatives in sensitive and multidrug-resistant T-cell acute lymphoblastic leukemia cell lines

Energy Technology Data Exchange (ETDEWEB)

Ooko, Edna [Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Alsalim, Tahseen; Saeed, Bahjat [Department of Chemistry, College of Education for Pure Sciences, University of Basrah, P.O. Box 49 Basrah, Al Basrah (Iraq); Saeed, Mohamed E.M.; Kadioglu, Onat [Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Abbo, Hanna S. [Department of Chemistry, University of the Western Cape, P/B X17, Bellville, 7535 Cape Town (South Africa); Titinchi, Salam J.J., E-mail: stitinchi@uwc.ac.za [Department of Chemistry, University of the Western Cape, P/B X17, Bellville, 7535 Cape Town (South Africa); Efferth, Thomas, E-mail: efferth@uni-mainz.de [Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany)

2016-08-15

Background: Multidrug resistance (MDR) and drug transporter P-glycoprotein (P-gp) represent major obstacles in cancer chemotherapy. We investigated 19 synthetic curcumin derivatives in drug-sensitive acute lymphoblastic CCRF–CEM leukemia cells and their multidrug-resistant P-gp-overexpressing subline, CEM/ADR5000. Material and methods: Cytotoxicity was tested by resazurin assays. Doxorubicin uptake was assessed by flow cytometry. Binding modes of compounds to P-gp were analyzed by molecular docking. Chemical features responsible for bioactivity were studied by quantitative structure activity relationship (QSAR) analyses. A 7-descriptor QSAR model was correlated with doxorubicin uptake values, IC{sub 50} values and binding energies. Results: The compounds displayed IC{sub 50} values between 0.7 ± 0.03 and 20.2 ± 0.25 μM. CEM/ADR5000 cells exhibited cross-resistance to 10 compounds, collateral sensitivity to three compounds and regular sensitivity to the remaining six curcumins. Molecular docking studies at the intra-channel transmembrane domain of human P-gp resulted in lowest binding energies ranging from − 9.00 ± 0.10 to − 6.20 ± 0.02 kcal/mol and pKi values from 0.24 ± 0.04 to 29.17 ± 0.88 μM. At the ATP-binding site of P-gp, lowest binding energies ranged from − 9.78 ± 0.17 to − 6.79 ± 0.01 kcal/mol and pKi values from 0.07 ± 0.02 to 0.03 ± 0.03 μM. CEM/ADR5000 cells accumulated approximately 4-fold less doxorubicin than CCRF–CEM cells. The control P-gp inhibitor, verapamil, partially increased doxorubicin uptake in CEM/ADR5000 cells. Six curcumins increased doxorubicin uptake in resistant cells or even exceeded uptake levels compared to sensitive one. QSAR yielded good activity prediction (R = 0.797 and R = 0.794 for training and test sets). Conclusion: Selected derivatives may serve to guide future design of novel P-gp inhibitors and collateral sensitive drugs to combat MDR. - Highlights: • Novel derivatives of curcumin in reversing

Interaction of coenzyme Q10 with the intestinal drug transporter P-glycoprotein.

Science.gov (United States)

Itagaki, Shirou; Ochiai, Akiko; Kobayashi, Masaki; Sugawara, Mitsuru; Hirano, Takeshi; Iseki, Ken

2008-08-27

In clinical trials, patients usually take many kinds of drugs at the same time. Thus, drug-drug interactions can often directly affect the therapeutic safety and efficacy of many drugs. Oral delivery is the most desirable means of drug administration. Changes in the activity of drug transporters may substantially influence the absorption of administered drugs from the intestine. However, there have been a few studies on food-drug interactions involving transporters. It is important to be aware of the potential of food-drug interactions and to act in order to prevent undesirable and harmful clinical consequences. Coenzyme Q10 (CoQ10) is very widely consumed by humans as a food supplement because of its recognition by the public as an important nutrient in supporting human health. Since intestinal efflux transporter P-glycoprotein (P-gp) is one of the major factors in drug-drug interactions, we focused on this transporter. We report here for the first time that CoQ10, which is widely used as a food supplement, affects the transport activity of P-gp.

Alteration of gene expression and DNA methylation in drug-resistant gastric cancer.

Science.gov (United States)

Maeda, Osamu; Ando, Takafumi; Ohmiya, Naoki; Ishiguro, Kazuhiro; Watanabe, Osamu; Miyahara, Ryoji; Hibi, Yoko; Nagai, Taku; Yamada, Kiyofumi; Goto, Hidemi

2014-04-01

The mechanisms of drug resistance in cancer are not fully elucidated. To study the drug resistance of gastric cancer, we analyzed gene expression and DNA methylation profiles of 5-fluorouracil (5-FU)- and cisplatin (CDDP)-resistant gastric cancer cells and biopsy specimens. Drug-resistant gastric cancer cells were established with culture for >10 months in a medium containing 5-FU or CDDP. Endoscopic biopsy specimens were obtained from gastric cancer patients who underwent chemotherapy with oral fluoropyrimidine S-1 and CDDP. Gene expression and DNA methylation analyses were performed using microarray, and validated using real-time PCR and pyrosequencing, respectively. Out of 17,933 genes, 541 genes commonly increased and 569 genes decreased in both 5-FU- and CDDP-resistant AGS cells. Genes with expression changed by drugs were related to GO term 'extracellular region' and 'p53 signaling pathway' in both 5-FU- and CDDP-treated cells. Expression of 15 genes including KLK13 increased and 12 genes including ETV7 decreased, in both drug-resistant cells and biopsy specimens of two patients after chemotherapy. Out of 10,365 genes evaluated with both expression microarray and methylation microarray, 74 genes were hypermethylated and downregulated, or hypomethylated and upregulated in either 5-FU-resistant or CDDP-resistant cells. Of these genes, expression of 21 genes including FSCN1, CPT1C and NOTCH3, increased from treatment with a demethylating agent. There are alterations of gene expression and DNA methylation in drug-resistant gastric cancer; they may be related to mechanisms of drug resistance and may be useful as biomarkers of gastric cancer drug sensitivity.

Simple strategy to assess linezolid exposure in patients with multi-drug-resistant and extensively-drug-resistant tuberculosis

NARCIS (Netherlands)

Kamp, Jasper; Bolhuis, Mathieu S.; Tiberi, Simon; Akkerman, Onno W.; Centis, Rosella; de lange, Wiel C.; Kosterink, Jos G.; van der Werf, Tjip S.; Migliori, Giovanni B.; Alffenaar, Jan-Willem C.

Linezolid is used increasingly for the treatment of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis (TB). However, linezolid can cause severe adverse events, such as peripheral and optical neuropathy or thrombocytopenia related to higher drug exposure. This study aimed

Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide

DEFF Research Database (Denmark)

Nielsen, D; Maare, C; Eriksen, J

2000-01-01

-extractable immunoreactive topoisomerase IIalpha and beta in EHR2/VP16 was reduced by 30-40% relative to that in EHR2. The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expression of P-glycoprotein was unchanged. In EHR2/VP16, the steady......M. ATPase activity was slightly stimulated by daunorubicin, whereas vinblastine, verapamil, and cyclosporin A had no effect. In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20......-fold). MRP displays several points of resemblance to P-glycoprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistance to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in the cells and this decrease is abolished by omission of energy; and 3) MRP increases efflux...

DNA origami/gold nanorod hybrid nanostructures for the circumvention of drug resistance.

Science.gov (United States)

Song, Linlin; Jiang, Qiao; Liu, Jianbing; Li, Na; Liu, Qing; Dai, Luru; Gao, Yuan; Liu, Weili; Liu, Dongsheng; Ding, Baoquan

2017-06-14

We herein demonstrate that DNA origami can work as a multifunctional platform integrating a chemotherapeutic drug (doxorubicin), gold nanorods and a tumour-specific aptamer MUC-1, to realize the effective circumvention of drug resistance. Doxorubicin (DOX) was loaded efficiently onto DNA origami through base pair intercalation and surface-modified gold nanorods (AuNRs) were assembled onto the DNA origami through DNA hybridization. Due to the active targeting effect of the assembled aptamers, the multifunctional nanostructures achieved increased cellular internalization of DOX and AuNRs. Upon near-infrared (NIR) laser irradiation, the P-glycoprotein (multidrug resistance pump) expression of multidrug resistant MCF-7 (MCF-7/ADR) cells was down-regulated, achieving the synergistically chemotherapeutic (DOX) and photothermal (AuNRs) effects.

Development of Classification Models for Identifying “True” P-glycoprotein (P-gp Inhibitors Through Inhibition, ATPase Activation and Monolayer Efflux Assays

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Anna Maria Bianucci

2012-06-01

Full Text Available P-glycoprotein (P-gp is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as “true” P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

Small-molecule synthetic compound norcantharidin reverses multi-drug resistance by regulating Sonic hedgehog signaling in human breast cancer cells.

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Yu-Jen Chen

Full Text Available Multi-drug resistance (MDR, an unfavorable factor compromising treatment efficacy of anticancer drugs, involves upregulated ATP binding cassette (ABC transporters and activated Sonic hedgehog (Shh signaling. By preparing human breast cancer MCF-7 cells resistant to doxorubicin (DOX, we examined the effect and mechanism of norcantharidin (NCTD, a small-molecule synthetic compound, on reversing multidrug resistance. The DOX-prepared MCF-7R cells also possessed resistance to vinorelbine, characteristic of MDR. At suboptimal concentration, NCTD significantly inhibited the viability of DOX-sensitive (MCF-7S and DOX-resistant (MCF-7R cells and reversed the resistance to DOX and vinorelbine. NCTD increased the intracellular accumulation of DOX in MCF-7R cells and suppressed the upregulated the mdr-1 mRNA, P-gp and BCRP protein expression, but not the MRP-1. The role of P-gp was strengthened by partial reversal of the DOX and vinorelbine resistance by cyclosporine A. NCTD treatment suppressed the upregulation of Shh expression and nuclear translocation of Gli-1, a hallmark of Shh signaling activation in the resistant clone. Furthermore, the Shh ligand upregulated the expression of P-gp and attenuated the growth inhibitory effect of NCTD. The knockdown of mdr-1 mRNA had not altered the expression of Shh and Smoothened in both MCF-7S and MCF-7R cells. This indicates that the role of Shh signaling in MDR might be upstream to mdr-1/P-gp, and similar effect was shown in breast cancer MDA-MB-231 and BT-474 cells. This study demonstrated that NCTD may overcome multidrug resistance through inhibiting Shh signaling and expression of its downstream mdr-1/P-gp expression in human breast cancer cells.

P-glycoprotein Modulates Morphine Uptake into the CNS: A Role for the Non-steroidal Anti-inflammatory Drug Diclofenac

Science.gov (United States)

Sanchez-Covarrubias, Lucy; Slosky, Lauren M.; Thompson, Brandon J.; Zhang, Yifeng; Laracuente, Mei-Li; DeMarco, Kristin M.; Ronaldson, Patrick T.; Davis, Thomas P.

2014-01-01

Our laboratory has previously demonstrated that peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). The result of increased P-gp functional expression was a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. A major concern in the treatment of acute pain/inflammation is the potential for drug-drug interactions resulting from P-gp induction by therapeutic agents co-administered with opioids. Such effects on P-gp activity can profoundly modulate CNS distribution of opioid analgesics and alter analgesic efficacy. In this study, we examined the ability of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) that is commonly administered in conjunction with the opioids during pain therapy, to alter BBB transport of morphine via P-gp and whether such changes in P-gp morphine transport could alter morphine analgesic efficacy. Administration of diclofenac reduced paw edema and thermal hyperalgesia in rats subjected to PIP, which is consistent with the known mechanism of action of this NSAID. Western blot analysis demonstrated an increase in P-gp expression in rat brain microvessels not only following PIP induction but also after diclofenac treatment alone. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased morphine brain uptake. Critically, morphine analgesia was significantly reduced in animals pretreated with diclofenac (3 h), as compared to animals administered diclofenac and morphine concurrently. These novel findings suggest that administration of diclofenac and P-gp substrate opioids during pain pharmacotherapy may result in a clinically significant drug-drug interaction. PMID:24520393

P-glycoprotein modulates morphine uptake into the CNS: a role for the non-steroidal anti-inflammatory drug diclofenac.

Science.gov (United States)

Sanchez-Covarrubias, Lucy; Slosky, Lauren M; Thompson, Brandon J; Zhang, Yifeng; Laracuente, Mei-Li; DeMarco, Kristin M; Ronaldson, Patrick T; Davis, Thomas P

2014-01-01

Our laboratory has previously demonstrated that peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). The result of increased P-gp functional expression was a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. A major concern in the treatment of acute pain/inflammation is the potential for drug-drug interactions resulting from P-gp induction by therapeutic agents co-administered with opioids. Such effects on P-gp activity can profoundly modulate CNS distribution of opioid analgesics and alter analgesic efficacy. In this study, we examined the ability of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) that is commonly administered in conjunction with the opioids during pain therapy, to alter BBB transport of morphine via P-gp and whether such changes in P-gp morphine transport could alter morphine analgesic efficacy. Administration of diclofenac reduced paw edema and thermal hyperalgesia in rats subjected to PIP, which is consistent with the known mechanism of action of this NSAID. Western blot analysis demonstrated an increase in P-gp expression in rat brain microvessels not only following PIP induction but also after diclofenac treatment alone. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased morphine brain uptake. Critically, morphine analgesia was significantly reduced in animals pretreated with diclofenac (3 h), as compared to animals administered diclofenac and morphine concurrently. These novel findings suggest that administration of diclofenac and P-gp substrate opioids during pain pharmacotherapy may result in a clinically significant drug-drug interaction.

P-glycoprotein modulates morphine uptake into the CNS: a role for the non-steroidal anti-inflammatory drug diclofenac.

Directory of Open Access Journals (Sweden)

Lucy Sanchez-Covarrubias

Full Text Available Our laboratory has previously demonstrated that peripheral inflammatory pain (PIP, induced by subcutaneous plantar injection of λ-carrageenan, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp that is endogenously expressed at the blood-brain barrier (BBB. The result of increased P-gp functional expression was a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. A major concern in the treatment of acute pain/inflammation is the potential for drug-drug interactions resulting from P-gp induction by therapeutic agents co-administered with opioids. Such effects on P-gp activity can profoundly modulate CNS distribution of opioid analgesics and alter analgesic efficacy. In this study, we examined the ability of diclofenac, a non-steroidal anti-inflammatory drug (NSAID that is commonly administered in conjunction with the opioids during pain therapy, to alter BBB transport of morphine via P-gp and whether such changes in P-gp morphine transport could alter morphine analgesic efficacy. Administration of diclofenac reduced paw edema and thermal hyperalgesia in rats subjected to PIP, which is consistent with the known mechanism of action of this NSAID. Western blot analysis demonstrated an increase in P-gp expression in rat brain microvessels not only following PIP induction but also after diclofenac treatment alone. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased morphine brain uptake. Critically, morphine analgesia was significantly reduced in animals pretreated with diclofenac (3 h, as compared to animals administered diclofenac and morphine concurrently. These novel findings suggest that administration of diclofenac and P-gp substrate opioids during pain pharmacotherapy may result in a clinically significant drug-drug interaction.

Overcoming multidrug resistance via simultaneous delivery of cytostatic drug and P-glycoprotein inhibitor to cancer cells by HPMA copolymer conjugate

Czech Academy of Sciences Publication Activity Database

Sivák, Ladislav; Šubr, Vladimír; Tomala, Jakub; Říhová, Blanka; Strohalm, Jiří; Etrych, Tomáš; Kovář, Marek

2017-01-01

Roč. 115, JAN 2017 (2017), s. 65-80 ISSN 0142-9612 R&D Projects: GA ČR(CZ) GAP301/12/1254; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 ; RVO:61389013 Keywords : Multidrug resistance * P-glycoprotein * Doxorubicin Subject RIV: EE - Microbiology, Virology; CD - Macromolecular Chemistry (UMCH-V) OBOR OECD: Microbiology; Polymer sci ence (UMCH-V) Impact factor: 8.402, year: 2016

Inhibition of ABCB1 (MDR1 expression by an siRNA nanoparticulate delivery system to overcome drug resistance in osteosarcoma.

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Michiro Susa

2010-05-01

Full Text Available The use of neo-adjuvant chemotherapy in treating osteosarcoma has improved patients' average 5 year survival rate from 20% to 70% in the past 30 years. However, for patients who progress after chemotherapy, its effectiveness diminishes due to the emergence of multi-drug resistance (MDR after prolonged therapy.In order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure resulting from MDR, we designed and evaluated a novel drug delivery system for MDR1 siRNA delivery. Novel biocompatible, lipid-modified dextran-based polymeric nanoparticles were used as the platform for MDR1 siRNA delivery; and the efficacy of combination therapy with this system was evaluated. In this study, multi-drug resistant osteosarcoma cell lines (KHOS(R2 and U-2OS(R2 were treated with the MDR1 siRNA nanocarriers and MDR1 protein (P-gp expression, drug retention, and immunofluoresence were analyzed. Combination therapy of the MDR1 siRNA loaded nanocarriers with increasing concentrations of doxorubicin was also analyzed. We observed that MDR1 siRNA loaded dextran nanoparticles efficiently suppresses P-gp expression in the drug resistant osteosarcoma cell lines. The results also demonstrated that this approach may be capable of reversing drug resistance by increasing the amount of drug accumulation in MDR cell lines.Lipid-modified dextran-based polymeric nanoparticles are a promising platform for siRNA delivery. Nanocarriers loaded with MDR1 siRNA are a potential treatment strategy for reversing MDR in osteosarcoma.

Molecular Cloning and Characterization of a P-Glycoprotein from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

Science.gov (United States)

Tian, Lixia; Yang, Jiaqiang; Hou, Wenjie; Xu, Baoyun; Xie, Wen; Wang, Shaoli; Zhang, Youjun; Zhou, Xuguo; Wu, Qingjun

2013-01-01

Macrocyclic lactones such as abamectin and ivermectin constitute an important class of broad-spectrum insecticides. Widespread resistance to synthetic insecticides, including abamectin and ivermectin, poses a serious threat to the management of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a major pest of cruciferous plants worldwide. P-glycoprotein (Pgp), a member of the ABC transporter superfamily, plays a crucial role in the removal of amphiphilic xenobiotics, suggesting a mechanism for drug resistance in target organisms. In this study, PxPgp1, a putative Pgp gene from P. xylostella, was cloned and characterized. The open reading frame (ORF) of PxPgp1 consists of 3774 nucleotides, which encodes a 1257-amino acid peptide. The deduced PxPgp1 protein possesses structural characteristics of a typical Pgp, and clusters within the insect ABCB1. PxPgp1 was expressed throughout all developmental stages, and showed the highest expression level in adult males. PxPgp1 was highly expressed in midgut, malpighian tubules and testes. Elevated expression of PxPgp1 was observed in P. xylostella strains after they were exposed to the abamectin treatment. In addition, the constitutive expressions of PxPgp1 were significantly higher in laboratory-selected and field-collected resistant strains in comparison to their susceptible counterpart. PMID:24264038

Multi-drug resistant tuberculosis in Tanzania: Initial description of ...

African Journals Online (AJOL)

Background: Drug resistant Tuberculosis is well documented worldwide and is associated with increasing morbidity and mortality complicating Tuberculosis control with increasing costs of managing the disease. Broad. Objective: To describe clinical and laboratory characteristics of multi-drug resistant Tuberculosis ...

Towards Comprehension of the ABCB1/P-Glycoprotein Role in Chronic Myeloid Leukemia

Directory of Open Access Journals (Sweden)

Raquel C. Maia

2018-01-01

Full Text Available Abstract: The introduction of imatinib (IM, a BCR-ABL1 tyrosine kinase inhibitor (TKI, has represented a significant advance in the first-line treatment of chronic myeloid leukemia (CML. However, approximately 30% of patients need to discontinue IM due to resistance or intolerance to this drug. Both resistance and intolerance have also been observed in treatment with the second-generation TKIs—dasatinib, nilotinib, and bosutinib—and the third-generation TKI—ponatinib. The mechanisms of resistance to TKIs may be BCR-ABL1-dependent and/or BCR-ABL1-independent. Although the role of efflux pump P-glycoprotein (Pgp, codified by the ABCB1 gene, is unquestionable in drug resistance of many neoplasms, a longstanding question exists about whether Pgp has a firm implication in TKI resistance in the clinical scenario. The goal of this review is to offer an overview of ABCB1/Pgp expression/activity/polymorphisms in CML. Understanding how interactions, associations, or cooperation between Pgp and other molecules—such as inhibitor apoptosis proteins, microRNAs, or microvesicles—impact IM resistance risk may be critical in evaluating the response to TKIs in CML patients. In addition, new non-TKI compounds may be necessary in order to overcome the resistance mediated by Pgp in CML.

Comparative uptake of Tc-99m sestamibi and Tc-99m tetrofosmin in cancer cells and tissue expressing P-Glycoprotein or multidrug resistance associated protein

International Nuclear Information System (INIS)

Cho, Jung Ah; Lee, Jae Tae; Yoo, Jung Ah

2005-01-01

99m Tc-sestamibi(MIBI) and 99m Tc-tetrofosmin have been used as substrates for P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP), which are closely associated with multidrug resistance of the tumors. To understand different handling of radiotracers in cancer cell lines expressing Pgp and MRP, we compared cellular uptakes of 99m Tc-MIBI and 99m Tc-tetrofosmin. The effects of cyclosporin A (CsA), well-known multidrug resistant reversing agent, on the uptake of both tracers were also compared. HCT15/CL02 human colorectal cancer cells for Pgp expressing cells, and human non-small cell lung cancer A549 cells for MRP expressing cells, were used for in vitro and in vivo studies. RT-PCR, western blot analysis and immunohistochemistry were used for detection of Pgp and MRP. MDR-reversal effect with CsA was evaluated at different drug concentrations after incubation with MIBI or tetrofosmin. Radioactivities of supernatant and pellet were measured with gamma well counter. Tumoral uptake of the tracers were measured from tumor bearing nude mice treated with or without CsA. RT-PCR, western blot analysis of the cells and immunochemical staining revealed selective expression of Pgp and MRP for HCT15/CL02 and A549 cells, respectively. There were no significant difference in cellular uptakes of both tracers in HCT15/CL02 cells, but MIBI uptake was slightly higher than that of tetrofosmin in A549 cells. Co-incubation with CsA resulted in a increase in cellular uptakes of MIBI and tetrofosmin. Uptake of MIBI or tetrofosmin in HCT15/CL02 cells was increased by 10-and 2.4-fold, and by 7.5 and 6.3-fold in A549 cells, respectively. Percentage increase of MIBI was higher than that of tetrofosmin with CsA for both cells (ρ < 0.05). In vivo biodistribution study showed that MIBI (114% at 10 min, 257% at 60 min, 396% at 24C min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively increased by the time, up to 240 min with CsA. But

Comparative uptake of Tc-99m sestamibi and Tc-99m tetrofosmin in cancer cells and tissue expressing P-Glycoprotein or multidrug resistance associated protein

Energy Technology Data Exchange (ETDEWEB)

Cho, Jung Ah; Lee, Jae Tae; Yoo, Jung Ah [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)] (and others)

2005-02-15

{sup 99m}Tc-sestamibi(MIBI) and {sup 99m}Tc-tetrofosmin have been used as substrates for P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP), which are closely associated with multidrug resistance of the tumors. To understand different handling of radiotracers in cancer cell lines expressing Pgp and MRP, we compared cellular uptakes of {sup 99m}Tc-MIBI and {sup 99m}Tc-tetrofosmin. The effects of cyclosporin A (CsA), well-known multidrug resistant reversing agent, on the uptake of both tracers were also compared. HCT15/CL02 human colorectal cancer cells for Pgp expressing cells, and human non-small cell lung cancer A549 cells for MRP expressing cells, were used for in vitro and in vivo studies. RT-PCR, western blot analysis and immunohistochemistry were used for detection of Pgp and MRP. MDR-reversal effect with CsA was evaluated at different drug concentrations after incubation with MIBI or tetrofosmin. Radioactivities of supernatant and pellet were measured with gamma well counter. Tumoral uptake of the tracers were measured from tumor bearing nude mice treated with or without CsA. RT-PCR, western blot analysis of the cells and immunochemical staining revealed selective expression of Pgp and MRP for HCT15/CL02 and A549 cells, respectively. There were no significant difference in cellular uptakes of both tracers in HCT15/CL02 cells, but MIBI uptake was slightly higher than that of tetrofosmin in A549 cells. Co-incubation with CsA resulted in a increase in cellular uptakes of MIBI and tetrofosmin. Uptake of MIBI or tetrofosmin in HCT15/CL02 cells was increased by 10-and 2.4-fold, and by 7.5 and 6.3-fold in A549 cells, respectively. Percentage increase of MIBI was higher than that of tetrofosmin with CsA for both cells ({rho} < 0.05). In vivo biodistribution study showed that MIBI (114% at 10 min, 257% at 60 min, 396% at 24C min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively increased by the time, up to

The co-delivery of a low-dose P-glycoprotein inhibitor with doxorubicin sterically stabilized liposomes against breast cancer with low P-glycoprotein expression

Directory of Open Access Journals (Sweden)

Gao W

2014-07-01

Full Text Available Wei Gao,1 Zhiqiang Lin,1 Meiwan Chen,2 Xiucong Yang,1 Zheng Cui,1 Xiaofei Zhang,1 Lan Yuan,3 Qiang Zhang11State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 2State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, 3Medical and Healthy Analytical Center, Peking University, Beijing, People's Republic of China Introduction: P-glycoprotein (P-gp inhibitors are usually used to treat tumors that overexpress P-gps. However, most common types of breast cancers, such as Luminal A, are low-P-gp expressing, at least during the initial phases of treatment. Therefore, it would be interesting to know if P-gp inhibitors are still useful in treating low-P-gp-expressing tumors. Methods: In the study reported here, the human breast-cancer cell line MCF-7, chosen as a model of Luminal A, was found to be low-P-gp expressing. We designed a novel doxorubicin (DOX sterically stabilized liposome system co-loaded with the low-dose P-gp inhibitor cyclosporine A (CsA (DOX/CsA/SSL. Results: The co-delivery system showed good size uniformity, high encapsulation efficiency, and a desirable release profile. The cell-uptake and cytotoxicity studies demonstrated that CsA could significantly enhance the intracellular accumulation and toxicity of free DOX and the liposomal DOX in MCF-7 cells. The confocal microscopy and in vivo imaging study confirmed the intracellular and in vivo targeting effect of DOX/CsA/SSL, respectively. Finally, the in vivo study proved that DOX/CsA/SSL could achieve significantly better antitumor effect against MCF-7 tumor than controls, without inducing obvious systemic toxicity. Conclusion: This study demonstrated that the co-delivery of a low-dose P-gp inhibitor and liposomal DOX could improve the therapy of low-P-gp-expressing cancer, which is of significance in clinical tumor therapy. Keywords: liposomes, low-P-gp-expressing

pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells

DEFF Research Database (Denmark)

Litman, Thomas; Pedersen, S F; Kramhøft, B

1998-01-01

Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3. Steady-state pHi was similar in cells expressing...

Effect of methylxanthines derived from pentoxifylline on P-glycoprotein mediated multidrug resistance

International Nuclear Information System (INIS)

Kupsakova, I.; Drobna, Z.; Breier, A.

2001-01-01

In this paper study of multidrug resistance (MDR) antitumor agents - P-glycoprotein (PGP) is presented. The ability of pentoxifylline (PTX) to depress resistance mediated by overexpression of PGP in mouse leukemic cell line L 121 ONCR resistant to vincristine (VCR) was described earlier. PTX depressed the resistance of these cells in a dose and time dependent manner. This effect was accompanied by increased level of [ 3 H]-vincristine accumulation by these cells. The methylxanthines with different length of this aliphatic side chain were synthesized and their capability to depress MDR was tested. The results indicated that the position of carbonyl group plays a crucial role for the ability of the derivative to depress MDR of L 121 ONCR cells. (authors)

Synthesis and Preclinical Evaluation of Novel PET Probes for P-Glycoprotein Function and Expression

NARCIS (Netherlands)

van Waarde, Aren; Ramakrishnan, Nisha K.; Rybczynska, Anna A.; Elsinga, Philip H.; Berardi, Francesco; de Jong, Johan R.; Kwizera, Chantal; Perrone, Roberto; Cantore, Mariangela; Sijbesma, Jurgen W. A.; Dierckx, Rudi A.; Colabufo, Nicola A.

2009-01-01

P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. A P-gp substrate (7) and an inhibitor (6) were labeled with (11)C, resulting in potential tracers of P-gp function and expression. Methods: 6 and 7 were labeled using (11)CH(3)I. (11)C-verapamil was

Co-treatment with grapefruit juice inhibits while chronic administration activates intestinal P-glycoprotein-mediated drug efflux.

Science.gov (United States)

Panchagnula, R; Bansal, T; Varma, M V S; Kaul, C L

2005-12-01

P-Glycoprotein (P-gp) mediated efflux is recognized as a significant biochemical barrier affecting oral absorption for a number of drugs. Various conflicting reports have been published regarding the effects of grapefruit juice (GFJ) on P-gp-mediated drug efflux, in which GFJ has been shown both to inhibit and activate it. Hence, the present study adopted a two-way approach, involving both co-treatment and chronic administration. Bi-directional transport of paclitaxel (PCL) was carried out in the absence and presence of GFJ extract, in rat everted ileum sac. Further, the effect of chronic administration of GFJ to rats was characterized by permeability studies with indinavir (INDI). Co-treatment of GFJ extract at 100% concentration reduced the asymmetric transport of PCL (efflux ratio = 20.8) by increasing absorptive (A --> B) transport by 921% and reducing secretory (B --> A) transport by 41%. Further, GFJ showed a concentration dependent effect on PCL permeability. Imipramine, a passive permeability marker with absorptive permeability of 15.33 +/- 4.26 x 10(-6) cm/s showed no asymmetric transport and also no significant (P extract inhibited P-gp-mediated efflux in co-treatment, whereas chronic administration led to increased levels of P-gp expression, thus having a profound effect on intestinal absorption and GFJ-drug interactions in vivo.

Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

Directory of Open Access Journals (Sweden)

Balaguer Trinidad

2012-07-01

Full Text Available Abstract Background It has been reported that the histone deacetylase inhibitor (iHDAc trichostatin A (TSA induces an increase in MDR1 gene transcription (ABCB1. This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp. It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a translational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. Methods A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry has been used in this study. Results The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a translational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5′ end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5′ end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. Conclusions The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we demonstrate that TSA in fact, regulates

Multi drug resistance tuberculosis: pattern seen in last 13 years

International Nuclear Information System (INIS)

Iqbal, R.; Shabbir, I.; Munir, K.; Tabassum, M.N.; Khan, S.U.; Khan, M.Z.U.

2011-01-01

Background: Drug resistance in tuberculosis is a serious problem throughout the world especially, after the emergence of multi drug resistant TB strains. Objectives: To estimate drug resistance in TB patients and compare it with previous studies to see the changing trends. Materials and Methods: The PMRC Research Centre receives sputum samples from all the leading hospitals of Lahore. This retrospective analysis was done from 1996 to 2008 on the multi drug resistant TB strains that were seen during these years. Five first lines anti tuberculosis drugs were tested on Lowenstein Jensen medium using standard proportion method. Results: A total of 2661 confirmed isolates of Mycobacterium tuberculosis were seen over the past 13 years. Of the total, 2182 were pulmonary and 479 were extra pulmonary specimens. The patients comprised of those with and without history of previous treatment. These specimens were subjected to drug susceptibility testing. Almost half of the patient had some resistance; multiple drug resistance was seen in 12.3% and 23.0% cases without and with history of previous treatment respectively. Overall resistance to rifampicin was 26.4%, isoniazid 24.1% streptomycin 21.6% ethambutol 13.4% and pyrazinamide 28.4% respectively. Statistically significant difference was seen between primary and acquired resistance. When compared with the reports from previous studies from the same area, there was a trend of gradual increase of drug resistance. Conclusions Resistance to anti tuberculosis drugs is high. Policy message. TB Control Program should start 'DOTS Plus' schemes for which drug susceptibility testing facilities should be available for correctly managing the patients. (author)

Multi drug resistance tuberculosis: pattern seen in last 13 years

Energy Technology Data Exchange (ETDEWEB)

Iqbal, R; Shabbir, I; Munir, K [King Edward Medical University Hospital, Lahore (Pakistan). Dept. of Research Centre; Tabassum, M N; Khan, S U; Khan, M Z.U. [King Edward Medical University Hospital, Lahore (Pakistan). Dept. of Chest Medicine

2011-01-15

Background: Drug resistance in tuberculosis is a serious problem throughout the world especially, after the emergence of multi drug resistant TB strains. Objectives: To estimate drug resistance in TB patients and compare it with previous studies to see the changing trends. Materials and Methods: The PMRC Research Centre receives sputum samples from all the leading hospitals of Lahore. This retrospective analysis was done from 1996 to 2008 on the multi drug resistant TB strains that were seen during these years. Five first lines anti tuberculosis drugs were tested on Lowenstein Jensen medium using standard proportion method. Results: A total of 2661 confirmed isolates of Mycobacterium tuberculosis were seen over the past 13 years. Of the total, 2182 were pulmonary and 479 were extra pulmonary specimens. The patients comprised of those with and without history of previous treatment. These specimens were subjected to drug susceptibility testing. Almost half of the patient had some resistance; multiple drug resistance was seen in 12.3% and 23.0% cases without and with history of previous treatment respectively. Overall resistance to rifampicin was 26.4%, isoniazid 24.1% streptomycin 21.6% ethambutol 13.4% and pyrazinamide 28.4% respectively. Statistically significant difference was seen between primary and acquired resistance. When compared with the reports from previous studies from the same area, there was a trend of gradual increase of drug resistance. Conclusions Resistance to anti tuberculosis drugs is high. Policy message. TB Control Program should start 'DOTS Plus' schemes for which drug susceptibility testing facilities should be available for correctly managing the patients. (author)

Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

Science.gov (United States)

Wise, John G

2012-06-26

Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.

A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics

DEFF Research Database (Denmark)

Saaby, Lasse; Brodin, Birger

2017-01-01

Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties...... and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may...... mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate...

The influence of P-glycoprotein expression and its inhibitors on the distribution of doxorubicin in breast tumors

International Nuclear Information System (INIS)

Patel, Krupa J; Tannock, Ian F

2009-01-01

Anti-cancer drugs access solid tumors via blood vessels, and must penetrate tumor tissue to reach all cancer cells. Previous studies have demonstrated steep gradients of decreasing doxorubicin fluorescence with increasing distance from blood vessels, such that many tumor cells are not exposed to drug. Studies using multilayered cell cultures show that increased P-glycoprotein (PgP) is associated with better penetration of doxorubicin, while PgP inhibitors decrease drug penetration in tumor tissue. Here we evaluate the effect of PgP expression on doxorubicin distribution in vivo. Mice bearing tumor sublines with either high or low expression of PgP were treated with doxorubicin, with or without pre-treatment with the PgP inhibitors verapamil or PSC 833. The distribution of doxorubicin in relation to tumor blood vessels was quantified using immunofluorescence. Our results indicate greater uptake of doxorubicin by cells near blood vessels in wild type as compared to PgP-overexpressing tumors, and pre-treatment with verapamil or PSC 833 increased uptake in PgP-overexpressing tumors. However, there were steeper gradients of decreasing doxorubicin fluorescence in wild-type tumors compared to PgP overexpressing tumors, and treatment of PgP overexpressing tumors with PgP inhibitors led to steeper gradients and greater heterogeneity in the distribution of doxorubicin. PgP inhibitors increase uptake of doxorubicin in cells close to blood vessels, have little effect on drug uptake into cells at intermediate distances, and might have a paradoxical effect to decrease doxorubicin uptake into distal cells. This effect probably contributes to the limited success of PgP inhibitors in clinical trials

Sea lice population and sex differences in P-glycoprotein expression and emamectin benzoate resistance on salmon farms in the Bay of Fundy, New Brunswick, Canada.

Science.gov (United States)

Igboeli, Okechukwu O; Burka, John F; Fast, Mark D

2014-06-01

Parasitic sea lice are a major challenge for salmon aquaculture. This is especially due to the recent development of resistance to emamectin benzoate (EMB) in the parasite. We investigated: (1) whether EMB treatment success in Grand Manan, Bay of Fundy, NB, Canada can be explained through EMB bioassay and P-glycoprotein (P-gp) mRNA expression studies; (2) if other populations of sea lice not under EMB selective pressure possess similar EMB sensitivity as Grand Manan sea lice populations; and (3) the heritability of EMB resistance in Lepeophtheirus salmonis. EMB bioassay results indicated population, species, sex and temporal differences in EMB EC50 values. RT-qPCR analyses revealed population and sex differences in P-gp mRNA levels, correlating with the bioassay results. Laboratory-reared sea lice maintained their EMB sensitivity status up to the F3 generation. Caligus elongatus, collected from Grand Manan showed more than twofold lower EMB EC50 values compared with L. salmonis collected from the same site. Concurrent exposure to EMB and verapamil yielded no increase in C. elongatus sensitivity to the parasiticide. Sea lice bioassay and P-gp mRNA studies can be used to track EMB resistance and sex differences in EMB sensitivity and P-gp mRNA levels exist in the parasite. © 2013 Society of Chemical Industry.

Effect of pentoxifylline on P-glycoprotein mediated vincristine resistance of L1210 mouse leukemic cell line

International Nuclear Information System (INIS)

Breier, A.; Uhrik, B.; Barancik, M.; Stefankova, Z.; Tribulova, N.

1994-01-01

Effect of pentoxifylline (PTX) on vincristine (VCR) resistance of multidrug resistant L1210/VCR mouse leukemic cell line was studied. Reversal effect of PTX (in concentration 50-150 mg dm -3 ) on vincristine resistance, i.e. potentiation of vincristine cytotoxicity on L1210/VCR cells by PTX was found. PTX alone in the above concentration did not exert any significant effect on sensitive or resistant cell lines in the absence of vincristine. Resistance of L1210/VCR cell line was found previously to be accompanied with overexpression of drug transporting P-glycoprotein. Indeed, lower level of 3 H-vincristine accumulation by resistant L1210/VCR cell line in comparison with sensitive L1210 cell line was observed. Accumulation of 3 H-vincristine by L1210/VCR cell line was significantly increased in the presence of PTX. PTX in the same condition did not exert any considerable effect on accumulation of 3 H-vincristine by nonresistant L1210 cells. Observable morphological damage was observed in 1210/VCR cells cultivated in medium containing vincristine (0.2 mg dm -3 ) and pentoxifylline (100 mg dm -3 ) in comparison with the non-damaged cells in the presence of vincristine or pentoxifylline alone. The results obtained indicate that pentoxifylline may be considered as a reversal agent in multidrug resistance. (author)

HSA-based multi-target combination therapy: regulating drugs' release from HSA and overcoming single drug resistance in a breast cancer model.

Science.gov (United States)

Gou, Yi; Zhang, Zhenlei; Li, Dongyang; Zhao, Lei; Cai, Meiling; Sun, Zhewen; Li, Yongping; Zhang, Yao; Khan, Hamid; Sun, Hongbing; Wang, Tao; Liang, Hong; Yang, Feng

2018-11-01

Multi-drug delivery systems, which may be promising solution to overcome obstacles, have limited the clinical success of multi-drug combination therapies to treat cancer. To this end, we used three different anticancer agents, Cu(BpT)Br, NAMI-A, and doxorubicin (DOX), to build human serum albumin (HSA)-based multi-drug delivery systems in a breast cancer model to investigate the therapeutic efficacy of overcoming single drug (DOX) resistance to cancer cells in vivo, and to regulate the drugs' release from HSA. The HSA complex structure revealed that NAMI-A and Cu(BpT)Br bind to the IB and IIA sub-domain of HSA by N-donor residue replacing a leaving group and coordinating to their metal centers, respectively. The MALDI-TOF mass spectra demonstrated that one DOX molecule is conjugated with lysine of HSA by a pH-sensitive linker. Furthermore, the release behavior of three agents form HSA can be regulated at different pH levels. Importantly, in vivo results revealed that the HSA-NAMI-A-Cu(BpT)Br-DOX complex not only increases the targeting ability compared with a combination of the three agents (the NAMI-A/Cu(BpT)Br/DOX mixture), but it also overcomes DOX resistance to drug-resistant breast cancer cell lines.

Single photon emission computed tomography imaging using 99Tcm-methoxyisobutylisonitrile predict the multi-drug resistance and chemotherapy efficacy of lung cancer

International Nuclear Information System (INIS)

Zhang Yiqiu; Shi Hongcheng

2008-01-01

Chemotherapy is one of the main comprehensive treatments for lung cancer, especially for non-small cell lung cancer (NSCIC) Multi-drug resistance of lung cancer plays an important role in the failure of chemotherapy. Early detection of multi-drug resistance (MDR) is essential for choosing a suitable chemotherapy regimen for the patients of lung cancer. In recent years lots of literature reports that MDR of lung cancer is related to many kinds of multi-drug resistance protein (MRP) expression in lung cancer. Some lipophilic chemotherapy drugs and 99 Tc m -methoxyisobutylisonitrile( 99 Tc m -MIBI)may be the same substrate for some MRP. These MRP can transport them out of the tumor cells, then the chemotherapy is invalid or non-radioactive concentration. The retention of 99 Tc m -MIBI in tumor cells is correlated with the expression of MRP, thus the prediction of the MRP expression before chemotherapy or monitoring MRP expression changes in the process of chemotherapy by using the noninvasive 99 Tc m -MIBI single photon emission computed tomography imaging is helpful to predict the MDR and chemotherapy efficacy of lung cancer. (authors)

P-glycoprotein-9 and macrocyclic lactone resistance status in selected strains of the ovine gastrointestinal nematode, Teladorsagia circumcincta.

Science.gov (United States)

Turnbull, Frank; Jonsson, Nicholas N; Kenyon, Fiona; Skuce, Philip J; Bisset, Stewart A

2018-04-01

The Teladorsagia circumcincta P-glycoprotein-9 (Tci-pgp-9) gene has previously been implicated in multiple-anthelmintic resistance in this parasite. Here we further characterise genetic diversity in Tci-pgp-9 and its possible role in ivermectin (IVM) and multi-drug resistance using two UK field isolates of T. circumcincta, one susceptible to anthelmintics (MTci2) and the other resistant to most available anthelmintics including IVM (MTci5). A comparison of full-length Tci-pgp-9 cDNA transcripts from the MTci2 and MTci5 isolates (∼3.8 kb in both cases) indicated that they shared 95.6% and 99.5% identity at the nucleotide and amino acid levels, respectively. Nine non-synonymous SNPs were found in the MTci5 sequences relative to their MTci2 counterparts. Twelve genomic sequence variants of the first internucleotide binding domain of Tci-pgp-9 were identified and up to 10 of these were present in some individual worms, strongly supporting previous evidence that amplification of this gene has occurred in T. circumcincta. On average, fewer distinct sequence variants of Tci-pgp-9 were present in individual worms of the MTci5 isolate than in those of the MTci2 isolate. A further reduction in the number of sequence variants was observed in individuals derived from an IVM-treated sub-population of MTci5. These findings suggest that Tci-pgp-9 was under purifying selection in the face of IVM treatment in T. circumcincta, with some sequence variants being selected against. Copyright © 2018. Published by Elsevier Ltd.

New-Onset Psychosis in a Multi-Drug Resistant Tuberculosis Patient ...

African Journals Online (AJOL)

Drug-resistant tuberculosis poses a serious challenge to global control of TB. These forms of TB do not respond to the standard six-month treatment; it can take two years or more to treat with category IV drugs that are less potent, more toxic and much more expensive. Treatment of multi-drug resistant tuberculosis is still ...

Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding.

Science.gov (United States)

Rosenberg, Mark F; Kamis, Alhaji Bukar; Callaghan, Richard; Higgins, Christopher F; Ford, Robert C

2003-03-07

P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.

HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein

Directory of Open Access Journals (Sweden)

Cara Andrea

2007-03-01

Full Text Available Abstract Background The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN (IN inhibitors, IINs has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA derivatives active on the HIV-1 IN strand transfer (ST step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR reversing ability. Results The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells. Conclusion To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.

The process behind the expression of mdr-1/P-gp and mrp/MRP in human leukemia/lymphoma.

Science.gov (United States)

Hirose, Masao

2009-04-01

There is a controversy over the link between phenotypes of multidrug resistance (MDR) and clinical outcome in leukemia/lymphoma patients. This may be because the process behind the induction and loss of expression of genotypes and phenotypes by which MDR develops and the role of MDR in fresh cells of human leukemia/lymphoma are not clearly defined. P-glycoprotein (P-gp) increased and decreased along with mdr-1 expression in three cell lines out of five vincristine (VCR)-resistant cell lines. MRP appeared with increased mrp expression in the other two cell lines. After the drug was removed from the culture system, mdr-1/P-gp changed in parallel with the level of VCR resistance, although mrp and MRP did not. It was concluded that P-gp is directly derived from mdr-1 and that mdr-1/P-gp supports the VCR-resistance but mrp/MRP is not directly linked to the VCR-resistance. These results should contribute to a better understanding of MDR phenomenon in cancer.

Involvement of CUL4A in Regulation of Multidrug Resistance to P-gp Substrate Drugs in Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Yunshan Wang

2013-12-01

Full Text Available CUL4A encodes a core component of a cullin-based E3 ubiquitin ligase complex that regulates many critical processes such as cell cycle progression, DNA replication, DNA repair and chromatin remodeling by targeting a variety of proteins for ubiquitination and degradation. In the research described in this report we aimed to clarify whether CUL4A participates in multiple drug resistance (MDR in breast cancer cells. We first transfected vectors carrying CUL4A and specific shCUL4A into breast cancer cells and corresponding Adr cells respectively. Using reverse transcription polymerase chain reactions and western blots, we found that overexpression of CUL4A in MCF7 and MDA-MB-468 cells up-regulated MDR1/P-gp expression on both the transcription and protein levels, which conferred multidrug resistance to P-gp substrate drugs, as determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assays. On the other hand, silencing CUL4A in MCF7/Adr and MDA-MB-468/Adr cells led to the opposite effect. Moreover, ERK1/2 in CUL4A-overexpressing cells was highly activated and after treatment with PD98059, an ERK1/2-specific inhibitor, CUL4A-induced expression of MDR1/P-gp was decreased significantly. Lastly, immunohistochemistry in breast cancer tissues showed that P-gp expression had a positive correlation with the expression of CUL4A and ERK1/2. Thus, these results implied that CUL4A and ERK1/2 participated in multi-drug resistance in breast cancer through regulation of MDR1/P-gp expression.

Multi drug resistant tuberculosis: a challenge in the management of ...

African Journals Online (AJOL)

Multi drug resistant tuberculosis (MDR-TB) will not usually respond to short course chemotherapy. Unless the individual infected with this bug is treated appropriately, they can continue spreading resistant strains in the community and further fuel the tuberculosis epidemic. Diagnosis requires drug sensitivity testing and the ...

Epigenetic modulation of the biophysical properties of drug-resistant cell lipids to restore drug transport and endocytic functions.

Science.gov (United States)

Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Lu, Shan; Labhasetwar, Vinod

2012-09-04

In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane's biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug 5-aza-2'-deoxycytidine (decitabine) significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells

International Nuclear Information System (INIS)

Zhang, Hai-chang; Zhang, Fei; Wu, Bing; Han, Jing-hua; Ji, Wei; Zhou, Yan; Niu, Rui-fang

2012-01-01

To explore the interaction of Anxa2 with P-Glycoprotein (P-gp) in the migration and invasion of the multidrug-resistant (MDR) human breast cancer cell line MCF-7/ADR. A pair of short hairpin RNA (shRNA) targeting P-gp was transfected into MCF-7/ADR cells, and monoclonal cell strains were screened. The expression of P-gp was detected by Western blot. Transwell chambers were used to observe the cell migration capacity and invasion ability. The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses. P-gp expression was significantly knocked down, and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells (P<0.05). There was a close interaction between Anxa2 and P-gp. MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities. The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells. The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells

Direct interaction between verapamil and doxorubicin causes the lack of reversal effect of verapamil on P-glycoprotein mediated resistance to doxorubicin in vitro using L1210/VCR cells

International Nuclear Information System (INIS)

Breier, A.; Drobna, Z.; Barancik, M.

1998-01-01

Mouse leukemic cell sub-line L 1210/VCR exerts expressive multidrug resistance (MDR) that is mediated by P-glycoprotein. Cells originally adapted to vincristine are also extremely resistant to doxorubicin. Resistance to both vincristine and doxorubicin is connected with depression of drug uptake. While resistance of L 121 O cells to vincristine could be reversed by verapamil as chemo-sensitizer, resistance of cells to doxorubicin was insensitive to verapamil. Action of verapamil (well-known inhibitor of PGP activity) on multidrug resistance was often used as evidence that MDR is mediated by PGP. From this point it may be possible that the resistance of L1210/VCR cells to vincristine is mediated by PGP and the resistance to doxorubicin is mediated by other PGP-independent system. Another and more probable explanation of different effect of verapamil on resistance of L1210/VCR cells to vincristine and doxorubicin may be deduced from the following fact: Using UV spectroscopy we found that doxorubicin dissolved in water buffered medium interacts effectively with verapamil. This interaction may be responsible for the decrease of concentration of both drugs in free effective form and consequently for higher survival of cells. In contrast to doxorubicin vincristine does not give any interaction with verapamil that is measurable by UV spectroscopy and resistance of L1210/VCR cells to vincristine may be fully reversed by verapamil. (authors)

Prognostic significance of multidrug-resistance protein (MDR-1 in renal clear cell carcinomas: A five year follow-up analysis

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Strazzullo Viviana

2006-12-01

Full Text Available Abstract Background A large number of renal cancer patients shows poor or partial response to chemotherapy and the mechanisms have not been still understood. Multi-drug resistance is the principal mechanism by which many cancers develop resistance to chemotherapic drugs. The role of the multi-drug resistant transporter (MDR-1/P-glycoprotein, the gene product of MDR-1, and that one of the so-called multi-drug resistance associated protein (MRP, two energy-dependent efflux pumps, are commonly known to confer drug resistance. We studied MDR-1 expression in selected cases of renal cell carcinoma (RCC, clear cell type, with long-term follow-up, in order to establish its prognostic role and its possible contribution in the choice of post-surgical therapy. Methods MDR-1 has been studied by standard LSAB-HRP immunohistochemical technique, in paraffin embedded RCC samples. Protein expression has been compared to clinical and histopathological data and to disease specific survival of RCC patients, by Kaplan-Meier curve and Cox multivariate regression analyses. Results Two groups of RCCs were obtained by esteeming MDR-1 expression and disease specific survival (obtained with Kaplan-Meier curve and Cox multivariate regression analyses: the first one presents low or absent MDR-1 expression and good survival; the second one is characterized by high MDR-1 expression and significant poor outcome (p p p p Conclusion In our opinion, the results of this study well prove the relationship between MDR-1 expression and worse clinical prognosis in RCC, because MDR-1 over-expressing RCCs can be considered a group of tumours with a more aggressive behavior. This finding outlines a possible role of MDR-1 as prognostic factor, dependent and independent of multidrug resistance. These results could be useful to predict cancer evolution and to choose the appropriate treatment: this is another step that can stimulate further promising and interesting investigations on broader

Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

Energy Technology Data Exchange (ETDEWEB)

Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

2016-07-27

<p>P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.>

Differential effects of the enantiomers of tamsulosin and tolterodine on P-glycoprotein and cytochrome P450 3A4.

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Doricakova, Aneta; Theile, Dirk; Weiss, Johanna; Vrzal, Radim

2017-01-01

The pregnane X receptor (PXR) is a transcription factor regulating P-glycoprotein (P-gp; ABCB1)-mediated transport and cytochrome P450 3A4 (CYP3A4)-mediated metabolism of xenobiotics thereby affecting the pharmacokinetics of many drugs and potentially modulating clinical efficacy. Thus, pharmacokinetic drug-drug interactions can arise from PXR activation. Here, we examined whether the selective α1-adrenoreceptor blocker tamsulosin or the antagonist of muscarinic receptors tolterodine affect PXR-mediated regulation of CYP3A4 and of P-gp at the messenger RNA (mRNA) and protein level in an enantiomer-specific way. In addition, the effect of tamsulosin and tolterodine on P-gp activity was evaluated. We used quantitative real-time PCR, gene reporter assay, western blotting, rhodamine efflux assay, and calcein assay for determination of expression, activity, and inhibition of P-glycoprotein. The studied compounds significantly and concentration-dependently increased PXR activity in the ABCB1-driven luciferase-based reporter gene assay. We observed much stronger induction of ABCB1 mRNA by S-tamsulosin as compared to the R or racemic form. R or racemic form of tolterodine and R-tamsulosin concentration-dependently increased P-gp protein expression; the latter also enhanced P-gp efflux function in a rhodamine-based efflux assay. R-tamsulosin and all forms of tolderodine slightly inhibited P-gp. The effect on CYP3A4 expression followed the same pattern but was much weaker. Taken together, tamsulosin and tolterodine are demonstrated to interfere with P-gp and CYP3A4 regulation in an enantiomer-specific way.

Signaling to P-glycoprotein-A new therapeutic target to treat drug-resistant epilepsy?

NARCIS (Netherlands)

Hartz, A.M.; Notenboom, S.; Bauer, B.

2009-01-01

Epilepsy affects more than 60 million people worldwide. While most patients can be treated with antiepileptic drugs, up to 40% of patients respond poorly to pharmacotherapy. This drug resistance is not well understood and presents a major clinical problem. In this short review we provide background

Copper bis(diphosphine) complexes: radiopharmaceuticals for the detection of multi-drug resistance in tumours by PET

International Nuclear Information System (INIS)

Lewis, J.S.; Dearling, J.L.S.; Blower, P.J.; Sosabowski, J.K.; Zweit, J.; Carnochan, P.; Kelland, L.R.; Coley, H.M.

2000-01-01

Experience with imaging of the multi-drug resistance (MDR) phenotype in tumours using technetium-99m sestamibi, a substrate of the P-glycoprotein (Pgp) transporter, suggests that better quantification of images and separation of MDR from other variables affecting tracer uptake in tumours are required. One approach to these problems is the development of short half-life positron-emitting tracers which are substrates of Pgp. Several lipophilic cationic copper(I) bis(diphosphine) complexes labelled with copper-64 have been synthesised and evaluated in vitro as substrates for Pgp. The synthesis is rapid and efficient with no need for purification steps. The chemistry is suitable for use with very short half-life radionuclides such as copper-62 (9.7 min) and copper-60 (23.7 min). Incubation of the complexes with human serum in vitro showed that they are sufficiently stable in serum to support clinical imaging, and the more lipophilic members of the series are taken up rapidly by cells (Chinese hamster ovary and human ovarian carcinoma) in vitro with great avidity. Uptake in human ovarian carcinoma cells is significantly reduced after several months of conditioning in the presence of doxorubicin, which induces increased Pgp expression. Uptake in hooded rat sarcoma (HSN) cells, which express Pgp, is significantly increased in the presence of the MDR modulator cyclosporin A. Biodistribution studies in hooded rats show rapid blood clearance, excretion through both kidneys and liver, and low uptake in other tissues. The one complex investigated in HSN tumour-bearing rats showed uptake in tumour increasing up to 30 min p.i. while it was decreasing in other tissues. We conclude that diphosphine ligands offer a good basis for development of radiopharmaceuticals containing copper radionuclides, and that this series of complexes should undergo further evaluation in vivo as positron emission tomography imaging agents for MDR. (orig.)

Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone

DEFF Research Database (Denmark)

Nielsen, D; Eriksen, J; Maare, C

2000-01-01

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cel...... to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA....

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents

NARCIS (Netherlands)

Michaelis, M.; Rothweiler, F.; Agha, B.; Barth, S.; Voges, Y.; Loeschmann, N.; von Deimling, A.; Breitling, R.; Doerr, H. Wilhelm; Roedel, F.; Speidel, D.; Cinatl, J.; Cinatl Jr., J.; Stephanou, A.

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3,

An ETP model (exclusion-tolerance-progression for multi drug resistance

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Kannan Subburaj

2005-04-01

Full Text Available Abstract Background It is known that sensitivity or resistance of tumor cells to a given chemotherapeutic agent is an acquired characteristic(s, depending on the heterogeneity of the tumor mass subjected to the treatment. The clinical success of a chemotherapeutic regimen depends on the ratio of sensitive to resistant cell populations. Results Based on findings from clinical and experimental studies, a unifying model is proposed to delineate the potential mechanism by which tumor cells progress towards multi drug resistance, resulting in failure of chemotherapy. Conclusion It is suggested that the evolution of multi drug resistance is a developmentally orchestrated event. Identifying stage-specific time windows during this process would help to identify valid therapeutic targets for the effective elimination of malignancy.

In silico screening for inhibitors of p-glycoprotein that target the nucleotide binding domains.

Science.gov (United States)

Brewer, Frances K; Follit, Courtney A; Vogel, Pia D; Wise, John G

2014-12-01

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Multi-drug resistant Ewingella Americana

International Nuclear Information System (INIS)

Bukhari, Syed Z.; Ashshi, Ahmad M.; Hussain, Waleed M.; Fatani, Mohammad I.

2008-01-01

We report a case of pneumonia due to multi-drug resistant Ewingella Americana in a young patient admitted in the Intensive Care Unit of Hera General Hospital, Makkah, Saudi Arabia with severe head injury in a road traffic accident. He was an Indonesian pilgrim who had traveled to the Kingdom of Saudi Arabia to perform Hajj in December 2007. Ewingella Americana was identified to be the pathogen of pneumonia with clinical signs and symptoms along with positive radiological findings. (author)

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

Science.gov (United States)

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

Science.gov (United States)

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y.; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

Multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP and lung resistance protein (LRP gene expression in childhood acute lymphoblastic leukemia

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Elvis Terci Valera

Full Text Available CONTEXT: Despite the advances in the cure rate for acute lymphoblastic leukemia, approximately 25% of affected children suffer relapses. Expression of genes for the multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP, and lung resistance protein (LRP may confer the phenotype of resistance to the treatment of neoplasias. OBJECTIVE: To analyze the expression of the MDR-1, MRP and LRP genes in children with a diagnosis of acute lymphoblastic leukemia via the semiquantitative reverse transcription polymerase chain reaction (RT-PCR, and to determine the correlation between expression and event-free survival and clinical and laboratory variables. DESIGN: A retrospective clinical study. SETTING: Laboratory of Pediatric Oncology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. METHODS: Bone marrow aspirates from 30 children with a diagnosis of acute lymphoblastic leukemia were assessed for the expression of messenger RNA for the MDR-1, MRP and LRP genes by semi-quantitative RT-PCR. RESULTS: In the three groups studied, only the increased expression of LRP was related to worsened event-free survival (p = 0.005. The presence of the common acute lymphoblastic leukemia antigen (CALLA was correlated with increased LRP expression (p = 0.009 and increased risk of relapse or death (p = 0.05. The relative risk of relapse or death was six times higher among children with high LRP expression upon diagnosis (p = 0.05, as confirmed by multivariate analysis of the three genes studied (p = 0.035. DISCUSSION: Cell resistance to drugs is a determinant of the response to chemotherapy and its detection via RT-PCR may be of clinical importance. CONCLUSIONS: Evaluation of the expression of genes for resistance to antineoplastic drugs in childhood acute lymphoblastic leukemia upon diagnosis, and particularly the expression of the LRP gene, may be of clinical relevance, and should be the

Gene expression analysis of two extensively drug-resistant tuberculosis isolates show that two-component response systems enhance drug resistance.

Science.gov (United States)

Yu, Guohua; Cui, Zhenling; Sun, Xian; Peng, Jinfu; Jiang, Jun; Wu, Wei; Huang, Wenhua; Chu, Kaili; Zhang, Lu; Ge, Baoxue; Li, Yao

2015-05-01

Global analysis of expression profiles using DNA microarrays was performed between a reference strain H37Rv and two clinical extensively drug-resistant isolates in response to three anti-tuberculosis drug exposures (isoniazid, capreomycin, and rifampicin). A deep analysis was then conducted using a combination of genome sequences of the resistant isolates, resistance information, and related public microarray data. Certain known resistance-associated gene sets were significantly overrepresented in upregulated genes in the resistant isolates relative to that observed in H37Rv, which suggested a link between resistance and expression levels of particular genes. In addition, isoniazid and capreomycin response genes, but not rifampicin, either obtained from published works or our data, were highly consistent with the differentially expressed genes of resistant isolates compared to those of H37Rv, indicating a strong association between drug resistance of the isolates and genes differentially regulated by isoniazid and capreomycin exposures. Based on these results, 92 genes of the studied isolates were identified as candidate resistance genes, 10 of which are known resistance-related genes. Regulatory network analysis of candidate resistance genes using published networks and literature mining showed that three two-component regulatory systems and regulator CRP play significant roles in the resistance of the isolates by mediating the production of essential envelope components. Finally, drug sensitivity testing indicated strong correlations between expression levels of these regulatory genes and sensitivity to multiple anti-tuberculosis drugs in Mycobacterium tuberculosis. These findings may provide novel insights into the mechanism underlying the emergence and development of drug resistance in resistant tuberculosis isolates and useful clues for further studies on this issue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Monoclonal antibody to an external epitope of the human mdr1 P-glycoprotein

NARCIS (Netherlands)

Arceci, R. J.; Stieglitz, K.; Bras, J.; Schinkel, A.; Baas, F.; Croop, J.

1993-01-01

A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents. P-glycoprotein, encoded in humans by the mdr1 gene, functions as an energy-dependent efflux pump to exclude these

Molecular Genetics of Drug-resistance in Epilepsies

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Kurupath Radhakrishnan

2015-06-01

Full Text Available Nearly one-third of newly diagnosed patients with epilepsy remain unresponsive to antiepileptic drugs (AEDs, etiopathogenesis of which is poorly understood. The genes encoding the proteins that regulate the pharmacokinetics such as P-glycoprotein [ABCBI], major vault protein [MVP gene] and drug metabolizing enzymes [ABCB1, ABCG2, MVP, CYP2C9, CYP2C19, CYP3A4, CYP3A5, EPHX1, UGT1A1, UGT2B7], and pharmacodynamics such as sodium channels [SCN1A, SCN2A] and GABA receptors [GABRA1, GABRA6, GABRB2, GABRG2] of AEDs are under intense investigation to unravel the mysteries of AED-resistance. However, till today, a consistent and reliable result that could help the clinician either to predict drug resistance or to overcome it has not been forthcoming. The discrepant results may be related to variations in the definition of drug-resistance, heterogeneous patient populations, ethnic variations in the frequency distribution of single nucleotide polymorphisms (SNPs and the selection of SNPs. Understanding of these limitations of existing studies, hopefully, will help in designing better studies. Nearly one-third of newly diagnosed patients with epilepsy remain unresponsive toantiepileptic drugs (AEDs, etiopathogenesis of which is poorly understood. The genesencoding the proteins that regulate the pharmacokinetics such as P-glycoprotein[ABCBI], major vault protein [MVP gene] and drug metabolizing enzymes [ABCB1,ABCG2, MVP, CYP2C9, CYP2C19, CYP3A4, CYP3A5, EPHX1, UGT1A1, UGT2B7],and pharmacodynamics such as sodium channels [SCN1A, SCN2A] and GABAreceptors [GABRA1, GABRA6, GABRB2, GABRG2] of AEDs are under intenseinvestigation to unravel the mysteries of AED-resistance. However, till today, aconsistent and reliable result that could help the clinician either to predict drugresistanceor to overcome it has not been forthcoming. The discrepant results may berelated to variations in the definition of drug-resistance, heterogeneous patientpopulations, ethnic

Prognostic implications of the nuclear localization of Y-box-binding protein-1 and CXCR4 expression in ovarian cancer: their correlation with activated Akt, LRP/MVP and P-glycoprotein expression.

Science.gov (United States)

Oda, Yoshinao; Ohishi, Yoshihiro; Basaki, Yuji; Kobayashi, Hiroaki; Hirakawa, Toshio; Wake, Norio; Ono, Mayumi; Nishio, Kazuto; Kuwano, Michihiko; Tsuneyoshi, Masazumi

2007-07-01

The nuclear localization of Y-box-binding protein-1 (YB-1) is known to be a poor prognostic factor in several human malignancies, including ovarian carcinoma. Following on from our basic study dealing with microarray analyses of YB-1-associated gene expression in ovarian cancer cells, we examined whether nuclear localization of YB-1 is associated with the expression of CXCR4, a vault protein named lung resistance-related vault protein (LRP/MVP), phosphorylated Akt (p-Akt) or P-glycoprotein (P-gp) in human ovarian carcinoma. Fifty-three surgically resected ovarian carcinomas treated with paclitaxel and carboplatin were examined immunohistochemically for nuclear YB-1 expression and intrinsic expression of p-Akt, P-gp, LRP/MVP and CXCR4. Nuclear expression of YB-1 demonstrated significant correlation with p-Akt, P-gp and LRP expression, but no relationship with CXCR4 expression. By multivariate analysis, only YB-1 nuclear expression and CXCR4 expression were independent prognostic factors with regard to overall survival. These results indicate that YB-1 nuclear expression and CXCR4 expression are important prognostic factors in ovarian carcinoma.

The Role of ABC Proteins in Drug Resistant Breast Cancer Cells

Science.gov (United States)

2008-04-01

called the Plasmodium falciparum Chloroquine Transporter (PfCRT). While PfCRT is known to be the main molecular determinant of chloroquine resistance...proteins (such as human P-glycoprotein) and labeled PfCRT with a photoaffinity drug analogue . A manuscript is currently in preparation detailing my results...directly responsible for drug response, the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) (Fidock et al 2000). While not a member of

A novel curcumin derivative which inhibits P-glycoprotein, arrests cell cycle and induces apoptosis in multidrug resistance cells.

Science.gov (United States)

Lopes-Rodrigues, Vanessa; Oliveira, Ana; Correia-da-Silva, Marta; Pinto, Madalena; Lima, Raquel T; Sousa, Emília; Vasconcelos, M Helena

2017-01-15

Cancer multidrug resistance (MDR) is a major limitation to the success of cancer treatment and is highly associated with the overexpression of drug efflux pumps such as P-glycoprotein (P-gp). In order to achieve more effective chemotherapeutic treatments, it is important to develop P-gp inhibitors to block/decrease its activity. Curcumin (1) is a secondary metabolite isolated from the turmeric of Curcuma longa L.. Diverse biological activities have been identified for this compound, particularly, MDR modulation in various cancer cell models. However, curcumin (1) has low chemical stability, which severely limits its application. In order to improve stability and P-gp inhibitory effect, two potential more stable curcumin derivatives were synthesized as building blocks, followed by several curcumin derivatives. These compounds were then analyzed in terms of antitumor and anti-P-gp activity, in two MDR and sensitive tumor lines (from chronic myeloid leukemia and non-small cell lung cancer). We identified from a series of curcumin derivatives a novel curcumin derivative (1,7-bis(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)hepta-1,6-diene-3,5-dione, 10) with more potent antitumor and anti-P-gp activity than curcumin (1). This compound (10) was shown to promote cell cycle arrest (at the G2/M phase) and induce apoptosis in the MDR chronic myeloid leukemia cell line. Therefore it is a really interesting P-gp inhibitor due to its ability to inhibit both P-gp function and expression. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of S1P receptor signaling pathway on the survival and drug resistance in multiple myeloma cells.

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Fu, Di; Li, Yingchun; Li, Jia; Shi, Xiaoyan; Yang, Ronghui; Zhong, Yuan; Wang, Huihan; Liao, Aijun

2017-01-01

Multiple myeloma (MM) remains incurable by conventional chemotherapy. Sphingosine-1-phosphate (S1P) receptor-mediated signaling has been recently demonstrated to have critical roles in cell survival and drug resistance in a number of hematological malignancies. To dissect the roles of S1P receptor pathway in MM, we systematically examined cell viability and protein expression associated with cell survival and drug resistance in MM cell lines upon treatment with either pathway activator (S1P) or inhibitor (FTY720). Our results reveal that FTY720 inhibits cell proliferation by downregulating expression of target genes, while S1P has an opposite effect. Knocking down of S1P receptor S1P5R results in a reduction of cell survival-related gene expression; however, it does not have impacts on expression of drug resistance genes. These results suggest that S1P signaling plays a role in cell proliferation and drug resistance in MM, and targeting this pathway will provide a new therapeutic direction for MM management.

Tumor suppressor WWOX and p53 alterations and drug resistance in glioblastomas

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Ming-Fu eChiang

2013-03-01

Full Text Available Tumor suppressor p53 are frequently mutated in glioblastomas (GBMs and appears to contribute, in part, to resistance to temozolomide and therapeutic drugs. WW domain-containing oxidoreductase WWOX (FOR or WOX1 is a proapoptotic protein and is considered as a tumor suppressor. Loss of WWOX gene expression is frequently seen in malignant cancer cells due to promoter hypermethylation, genetic alterations, and translational blockade. Intriguingly, ectopic expression of wild type WWOX preferentially induces apoptosis in human glioblastoma cells harboring mutant p53. WWOX is known to physically bind and stabilize wild type p53. Here, we provide an overview for the updated knowledge in p53 and WWOX, and postulate a potential scenarios that wild type and mutant p53, or isoforms, modulate the apoptotic function of WWOX. We propose that triggering WWOX activation by therapeutic drugs under p53 functional deficiency is needed to overcome TMZ resistance and induce GBM cell death.

Expression and functional activity of P-glycoprotein in passaged primary human nasal epithelial cell monolayers cultured by the air-liquid interface method for nasal drug transport study.

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Cho, Hyun-Jong; Choi, Min-Koo; Lin, Hongxia; Kim, Jung Sun; Chung, Suk-Jae; Shim, Chang-Koo; Kim, Dae-Duk

2011-03-01

P-glycoprotein (P-gp) is an efflux transporter encoded by the multidrug resistance gene (MDR1), which is also known as the human ABCB1 gene (ATP-binding cassette, subfamily-B). The objectives of this study were to investigate the expression of P-gp in passaged primary human nasal epithelial (HNE) cell monolayer, cultured by the air-liquid interface (ALI) method, and to evaluate its feasibility as an in-vitro model for cellular uptake and transport studies of P-gp substrates. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the expression of the MDR1 gene. Transport and cellular uptake studies with P-gp substrate (rhodamine123) and P-gp inhibitors (verapamil and cyclosporin A) were conducted to assess the functional activity of P-gp in HNE cell monolayers cultured by the ALI method. MDR1 gene expression in primary HNE cell monolayers cultured by ALI method was confirmed by RT-PCR. The apparent permeability coefficient (P(app) ) of the P-gp substrate (rhodamine123) in the basolateral to apical (B to A) direction was 6.9 times higher than that in the apical to basolateral (A to B) direction. B to A transport was saturated at high rhodamine123 concentration, and the treatment of P-gp inhibitors increased cellular uptake of rhodamine123 in a time- and concentration-dependent manner. These results support the MDR1 gene expression and the functional activity of P-gp in primary HNE cell monolayers cultured by the ALI method. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

The Consequence of Drug-Drug Interactions Influencing the Interplay between P-Glycoprotein and Cytochrome P450 3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices.

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Li, Ming; de Graaf, Inge A M; Siissalo, Sanna; de Jager, Marina H; van Dam, Annie; Groothuis, Geny M M

2016-05-01

P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites. Copyright © 2016 by The American Society for Pharmacology and Experimental

Multi drug resistance and β-lactamase production by Klebsiella ...

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SERVER

2007-08-06

Aug 6, 2007 ... *Corresponding author. E-mail: gnsimha123@rediffmail.com. (Rice, 1999). plasmid that can be easily spread from one organisms to another (Sirot, 1995) these enzymes are capable of inactivating a variety of β-lactam drugs (Rice,. 1999). The ESBL producing organisms often show multi- drug resistant as ...

Modulation of human multidrug-resistance MDR-1 gene by natural curcuminoids

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Buddhasukh Duang

2004-04-01

Full Text Available Abstract Background Multidrug resistance (MDR is a phenomenon that is often associated with decreased intracellular drug accumulation in patient's tumor cells resulting from enhanced drug efflux. It is related to the overexpression of a membrane protein, P-glycoprotein (Pgp-170, thereby reducing drug cytotoxicity. A variety of studies have tried to find MDR modulators which increase drug accumulation in cancer cells. Methods In this study, natural curcuminoids, pure curcumin, demethoxycurcumin and bisdemethoxycurcumin, isolated from turmeric (Curcuma longa Linn, were compared for their potential ability to modulate the human MDR-1 gene expression in multidrug resistant human cervical carcinoma cell line, KB-V1 by Western blot analysis and RT-PCR. Results Western blot analysis and RT-PCR showed that all the three curcuminoids inhibited MDR-1 gene expression, and bisdemethoxycurcumin produced maximum effect. In additional studies we found that commercial grade curcuminoid (approximately 77% curcumin, 17% demethoxycurcumin and 3% bisdemthoxycurcumin decreased MDR-1 gene expression in a dose dependent manner and had about the same potent inhibitory effect on MDR-1 gene expression as our natural curcuminoid mixtures. Conclusion These results indicate that bisdemethoxycurcumin is the most active of the curcuminoids present in turmeric for modulation of MDR-1 gene. Treatment of drug resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with a decreased MDR-1 gene product, a P-glycoprotein, on the cell plasma membrane. Although many drugs that prevent the P-glycoprotein function have been reported, this report describes the inhibition of MDR-1 expression by a phytochemical. The modulation of MDR-1 expression may be an attractive target for new chemosensitizing agents.

Modulation of human multidrug-resistance MDR-1 gene by natural curcuminoids

International Nuclear Information System (INIS)

Limtrakul, Pornngarm; Anuchapreeda, Songyot; Buddhasukh, Duang

2004-01-01

Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in patient's tumor cells resulting from enhanced drug efflux. It is related to the overexpression of a membrane protein, P-glycoprotein (Pgp-170), thereby reducing drug cytotoxicity. A variety of studies have tried to find MDR modulators which increase drug accumulation in cancer cells. In this study, natural curcuminoids, pure curcumin, demethoxycurcumin and bisdemethoxycurcumin, isolated from turmeric (Curcuma longa Linn), were compared for their potential ability to modulate the human MDR-1 gene expression in multidrug resistant human cervical carcinoma cell line, KB-V1 by Western blot analysis and RT-PCR. Western blot analysis and RT-PCR showed that all the three curcuminoids inhibited MDR-1 gene expression, and bisdemethoxycurcumin produced maximum effect. In additional studies we found that commercial grade curcuminoid (approximately 77% curcumin, 17% demethoxycurcumin and 3% bisdemthoxycurcumin) decreased MDR-1 gene expression in a dose dependent manner and had about the same potent inhibitory effect on MDR-1 gene expression as our natural curcuminoid mixtures. These results indicate that bisdemethoxycurcumin is the most active of the curcuminoids present in turmeric for modulation of MDR-1 gene. Treatment of drug resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with a decreased MDR-1 gene product, a P-glycoprotein, on the cell plasma membrane. Although many drugs that prevent the P-glycoprotein function have been reported, this report describes the inhibition of MDR-1 expression by a phytochemical. The modulation of MDR-1 expression may be an attractive target for new chemosensitizing agents

PPAR-α, a lipid-sensing transcription factor, regulates blood-brain barrier efflux transporter expression.

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More, Vijay R; Campos, Christopher R; Evans, Rebecca A; Oliver, Keith D; Chan, Gary Ny; Miller, David S; Cannon, Ronald E

2017-04-01

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR- α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood-brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR- α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR- α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood-brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain.

P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery.

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Davis, Thomas P; Sanchez-Covarubias, Lucy; Tome, Margaret E

2014-01-01

The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription. © 2014 Elsevier Inc. All rights reserved.

Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2

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Sanja Perkovic

2012-09-01

Full Text Available Aggressive natural killer-cell leukaemia (ANKL is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV and P-glycoprotein (P-gp expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2 MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

Microdose study of a P-glycoprotein substrate, fexofenadine, using a non-radioisotope-labelled drug and LC/MS/MS.

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Yamazaki, A; Kumagai, Y; Yamane, N; Tozuka, Z; Sugiyama, Y; Fujita, T; Yokota, S; Maeda, M

2010-04-01

Fexofenadine is a P-glycoprotein substrate of low bioavailability. It is primarily excreted into faeces as a parent drug via biliary excretion. The predictability from microdose data for the drug absorbed via transporters such as P-glycoprotein is not known. Therefore, this study assessed the predictability of therapeutic-dose pharmacokinetics of fexofenadine from microdosing data using non-radioisotope-labelled drug and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). In a single dose, randomized, two-way crossover study, eight subjects received a microdose (100 microg) or a therapeutic dose (60 mg) of fexofenadine. Blood samples were collected until 12 h after dosing, and assayed using LC/MS/MS. Plasma concentration-time curves of fexofenadine between microdose and therapeutic dose were similar. The mean +/- SD of C(max) normalized to 60 mg dose after microdose and therapeutic dose were 379 +/- 147 and 275 +/- 145 ng/mL respectively. The mean AUC(last) normalized to 60 mg dose after microdose and therapeutic dose were 1914 +/- 738 and 1431 +/- 432 ng/h/mL respectively. The mean dose-adjusted C(max) and AUC(last) after microdose were higher compared with those after therapeutic dose. Individual plots of C(max) and AUC(last) normalized to 60 mg dose, were similar for microdose and therapeutic dose. None of the pharmacokinetic parameters were statistically different using anova. Overall, the microdose pharmacokinetics profile was similar to, and hence predictive of, that of the therapeutic dose. For the P-glycoprotein substrate fexofenadine, the predictability of therapeutic-dose pharmacokinetics from microdose data was good. A microdose study using a non-radioisotope-labelled drug and LC/MS/MS is convenient, and has the potential to aid the early selection of drug candidates.

Quercetin and doxorubicin co-encapsulated biotin receptor-targeting nanoparticles for minimizing drug resistance in breast cancer.

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Lv, Li; Liu, Chunxia; Chen, Chuxiong; Yu, Xiaoxia; Chen, Guanghui; Shi, Yonghui; Qin, Fengchao; Ou, Jiebin; Qiu, Kaifeng; Li, Guocheng

2016-05-31

The combination of a chemotherapeutic drug with a chemosensitizer has emerged as a promising strategy for cancers showing multidrug resistance (MDR). Herein we describe the simultaneous targeted delivery of two drugs to tumor cells by using biotin-decorated poly(ethylene glycol)-b-poly(ε-caprolactone) nanoparticles encapsulating the chemotherapeutic drug doxorubicin and the chemosensitizer quercetin (BNDQ). Next, the potential ability of BNDQ to reverse MDR in vitro and in vivo was investigated. Studies demonstrated that BNDQ was more effectively taken up with less efflux by doxorubicin-resistant MCF-7 breast cancer cells (MCF-7/ADR cells) than by the cells treated with the free drugs, single-drug-loaded nanoparticles, or non-biotin-decorated nanoparticles. BNDQ exhibited clear inhibition of both the activity and expression of P-glycoprotein in MCF-7/ADR cells. More importantly, it caused a significant reduction in doxorubicin resistance in MCF-7/ADR breast cancer cells both in vitro and in vivo, among all the groups. Overall, this study suggests that BNDQ has a potential role in the treatment of drug-resistant breast cancer.

Overview of P-glycoprotein inhibitors: a rational outlook

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Kale Mohana Raghava Srivalli

2012-09-01

Full Text Available P-glycoprotein (P-gp, a transmembrane permeability glycoprotein, is a member of ATP binding cassette (ABC super family that functions specifically as a carrier mediated primary active efflux transporter. It is widely distributed throughout the body and has a diverse range of substrates. Several vital therapeutic agents are substrates to P-gp and their bioavailability is lowered or a resistance is induced because of the protein efflux. Hence P-gp inhibitors were explored for overcoming multidrug resistance and poor bioavailability problems of the therapeutic P-gp substrates. The sensitivity of drug moieties to P-gp and vice versa can be established by various experimental models in silico, in vitro and in vivo. Ever since the discovery of P-gp, the research plethora identified several chemical structures as P-gp inhibitors. The aim of this review was to emphasize on the discovery and development of newer, inert, non-toxic, and more efficient, specifically targeting P-gp inhibitors, like those among the natural herb extracts, pharmaceutical excipients and formulations, and other rational drug moieties. The applications of cellular and molecular biology knowledge, in silico designed structural databases, molecular modeling studies and quantitative structure-activity relationship (QSAR analyses in the development of novel rational P-gp inhibitors have also been mentioned.Glicoproteína-p (P-gp, uma glicoproteína de transmembrana permeável, é um membro da superfamília (ABC de cassete de gene de ligação de ATP que funciona especificamente como um carreador mediado pelo transportador de efluxo ativo primário. É amplamente distribuído por todo o corpo e apresenta uma gama diversificada de substratos. Diversos agentes terapêuticos vitais são substratos para P-gp e sua biodisponibilidade é reduzida ou a resistência é induzida devido ao efluxo de proteínas. Portanto, os inibidores da P-gp foram explorados para a superação da resistência a

The application of 99Tcm-MIBI scintimammography to diagnose multidrug resistance of breast cancer

International Nuclear Information System (INIS)

Cheng Bing

2002-01-01

The author discussed the main mechanism of multidrug resistance of breast cancer tissues, and the correlation between technetium-99m sestamibi ( 99 Tc m -MIBI) breast imaging results, with the expression of drug resistance proteins P-glycoprotein and glutathione-S-transferase-π in human breast cancer. Through not all the results reported before matched each other, as a kind of a noninvasive simple functional test imaging technology in vitro, SPECT can be used to diagnose P-glycoprotein expression in breast cancer, and can be used to predict chemotherapy response

The reversal effects of 3-bromopyruvate on multidrug resistance in vitro and in vivo derived from human breast MCF-7/ADR cells.

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Long Wu

Full Text Available P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound.The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer's instructions.3-Bromopyruvate treatment led to marked decreases in the IC50 values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds, paclitaxel (85 folds, daunorubicin (201 folds, and epirubicin (171 folds] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied.We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the

The reversal effects of 3-bromopyruvate on multidrug resistance in vitro and in vivo derived from human breast MCF-7/ADR cells.

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Wu, Long; Xu, Jun; Yuan, Weiqi; Wu, Baojian; Wang, Hao; Liu, Guangquan; Wang, Xiaoxiong; Du, Jun; Cai, Shaohui

2014-01-01

P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer's instructions. 3-Bromopyruvate treatment led to marked decreases in the IC50 values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied. We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular

Age-related P-glycoprotein expression in the intestine and affecting the pharmacokinetics of orally administered enrofloxacin in broilers.

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Guo, Mengjie; Bughio, Shamsuddin; Sun, Yong; Zhang, Yu; Dong, Lingling; Dai, Xiaohua; Wang, Liping

2013-01-01

Bioavailability is the most important factor for the efficacy of any drug and it is determined by P- glycoprotein (P-gp) expression. Confirmation of P-gp expression during ontogeny is needed for understanding the differences in therapeutic efficacy of any drug in juvenile and adult animals. In this study, Abcb1 mRNA levels in the liver and intestine of broilers during ontogeny were analysed by RT qPCR. Cellular distribution of P-gp was detected by immunohistochemstry. Age-related differences of enrofloxacin pharmacokinetics were also studied. It was found that broilers aged 4 week-old expressed significantly (P0.05) age-related difference in the duodenum. Furthermore, the highest and lowest levels of Abcb1 mRNA expression were observed in the jejunum, and duodenum, respectively. P-gp immunoreactivity was detected on the apical surface of the enterocytes and in the bile canalicular membranes of the hepatocytes. Pharmacokinetic analysis revealed that the 8 week-old broilers, when orally administrated enrofloxacin, exhibited significantly higher Cmax (1.97 vs. 0.98 μg • ml(-1), P=0.009), AUC(14.54 vs. 9.35 μg • ml(-1) • h, P=0.005) and Ka (1.38 vs. 0.43 h(-1), P=0.032), as well as lower Tpeak (1.78 vs. 3.28 h, P=0.048) and T1/2 ka (0.6 vs. 1.64 h, P=0.012) than the 4 week-old broilers. The bioavailability of enrofloxacin in 8 week-old broilers was increased by 15.9%, compared with that in 4 week-old birds. Interestingly, combining verapamil, a P-gp modulator, significantly improved pharmacokinetic behaviour of enrofloxacin in all birds. The results indicate juvenile broilers had a higher expression of P-gp in the intestine, affecting the pharmacokinetics and reducing the bioavailability of oral enrofloxacin in broilers. On the basis of our results, it is recommended that alternative dose regimes are necessary for different ages of broilers for effective therapy.

Design, synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance inhibitors.

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Wu, Yuxiang; Pan, Miaobo; Dai, Yuxuan; Liu, Baomin; Cui, Jian; Shi, Wei; Qiu, Qianqian; Huang, Wenlong; Qian, Hai

2016-05-15

A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity toward K562 cells (IC50>100μM). Compared with VRP, compound 4 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123 (Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising candidate for developing P-gp-mediated MDR inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

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Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

2016-01-01

Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p Cordia verbenacea (-47%, p activity (-48%, p active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

Drug accumulation in the presence of the multidrug resistance pump

DEFF Research Database (Denmark)

Ayesh, S; Litman, Thomas; Stein, W D

1997-01-01

We studied the interaction between the multidrug transporter, P-glycoprotein, and two compounds that interact with it: vinblastine, a classical substrate of the pump, and verapamil, a classical reverser. Steady-state levels of accumulation of these two drugs were determined in a multidrug resistant...... P388 leukemia cell line, P388/ADR. The time course of accumulation of these drugs, and the effect of energy starvation and the presence of chloroquine on the level of their steady-state accumulation were quite disparate. Vinblastine inhibited the accumulation of verapamil whereas it enhanced...

Expression of multi-drug resistance-related genes MDR3 and MRP as prognostic factors in clinical liver cancer patients.

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Yu, Zheng; Peng, Sun; Hong-Ming, Pan; Kai-Feng, Wang

2012-01-01

To investigate the expression of multi-drug resistance-related genes, MDR3 and MRP, in clinical specimens of primary liver cancer and their potential as prognostic factors in liver cancer patients. A total of 26 patients with primary liver cancer were enrolled. The expression of MDR3 and MRP genes was measured by real-time PCR and the association between gene expression and the prognosis of patients was analyzed by the Kaplan-Meier method and COX regression model. This study showed that increases in MDR3 gene expression were identified in cholangiocellular carcinoma, cirrhosis and HBsAg-positive patients, while MRP expression increased in hepatocellular carcinoma, non-cirrhosis and HBsAg-negative patients. Moreover, conjugated bilirubin and total bile acid in the serum were significantly reduced in patients with high MRP expression compared to patients with low expression. The overall survival tended to be longer in patients with high MDR3 and MRP expression compared to the control group. MRP might be an independent prognostic factor in patients with liver cancer by COX regression analysis. MDR3 and MRP may play important roles in liver cancer patients as prognostic factors and their underlying mechanisms in liver cancer are worthy of further investigation.

Prediction of promiscuous p-glycoprotein inhibition using a novel machine learning scheme.

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Max K Leong

Full Text Available BACKGROUND: P-glycoprotein (P-gp is an ATP-dependent membrane transporter that plays a pivotal role in eliminating xenobiotics by active extrusion of xenobiotics from the cell. Multidrug resistance (MDR is highly associated with the over-expression of P-gp by cells, resulting in increased efflux of chemotherapeutical agents and reduction of intracellular drug accumulation. It is of clinical importance to develop a P-gp inhibition predictive model in the process of drug discovery and development. METHODOLOGY/PRINCIPAL FINDINGS: An in silico model was derived to predict the inhibition of P-gp using the newly invented pharmacophore ensemble/support vector machine (PhE/SVM scheme based on the data compiled from the literature. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those structurally diverse molecules in the training set (n = 31, r(2 = 0.89, q(2 = 0.86, RMSE = 0.40, s = 0.28, the test set (n = 88, r(2 = 0.87, RMSE = 0.39, s = 0.25 and the outlier set (n = 11, r(2 = 0.96, RMSE = 0.10, s = 0.05. The generated PhE/SVM model also showed high accuracy when subjected to those validation criteria generally adopted to gauge the predictivity of a theoretical model. CONCLUSIONS/SIGNIFICANCE: This accurate, fast and robust PhE/SVM model that can take into account the promiscuous nature of P-gp can be applied to predict the P-gp inhibition of structurally diverse compounds that otherwise cannot be done by any other methods in a high-throughput fashion to facilitate drug discovery and development by designing drug candidates with better metabolism profile.

Determinants of foamy virus envelope glycoprotein mediated resistance to superinfection

International Nuclear Information System (INIS)

Berg, Angelika; Pietschmann, Thomas; Rethwilm, Axel; Lindemann, Dirk

2003-01-01

Little is known about the nature of foamy virus (FV) receptor molecules on target cells and their interaction with the viral glycoproteins. Similar to other viruses, cellular expression of the FV Env protein is sufficient to induce resistance to exogenous FV, a phenomenon called superinfection resistance (SIR). In this study we define determinants of the FV Env protein essential for mediating SIR. FV Env requires the extracellular domains of the SU and the TM subunits as well as membrane anchorage, efficient cell surface transport, and most probably correct subunit processing. This is in contrast to murine leukemia virus where secreted proteins comprising the receptor-binding domain in SU are sufficient to induce SIR. Furthermore, we demonstrate that cellular expression of the prototype FV envelope proteins induces SIR against pseudotypes with glycoproteins of other FV species, including of simian, feline, bovine, and equine origin. This implies that all of them use the same receptor molecules for viral entry

Withanolide D Exhibits Similar Cytostatic Effect in Drug-Resistant and Drug-Sensitive Multiple Myeloma Cells

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Mark E. Issa

2017-09-01

Full Text Available In spite of recent therapeutic advances, multiple myeloma (MM remains a malignancy with very low curability. This has been partly attributed to the existence of a drug-resistant subpopulation known as cancer stem cells (CSCs. MM-CSCs are equipped with the necessary tools that render them highly resistant to virtually all conventional therapies. In this study, the growth inhibitory effects of withanolide D (WND, a steroidal lactone isolated from Withania somnifera, on drug-sensitive tumoral plasma cells and drug-resistant MM cells have been investigated. In MTT/XTT assays, WND exhibited similar cytostatic effects between drug-resistant and drug-sensitive cell lines in the nM range. WND also induced cell death and apoptosis in MM-CSCs and RPMI 8226 cells, as examined by the calcein/ethidium homodimer and annexin V/propidium iodide stainings, respectively. To determine whether P-glycoprotein (P-gp efflux affected the cytostatic activity of WND, P-gp was inhibited with verapamil and results indicated that the WND cytostatic effect in MM-CSCs was independent of P-gp efflux. Furthermore, WND did not increase the accumulation of the fluorescent P-gp substrate rhodamine 123 in MM-CSCs, suggesting that WND may not inhibit P-gp at the tested relevant doses. Therefore, the WND-induced cytostatic effect may be independent of P-gp efflux. These findings warrant further investigation of WND in MM-CSC animal models.

Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China.

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Zhao, Ji-Li; Liu, Wei; Xie, Wan-Ying; Cao, Xu-Dong; Yuan, Li

2018-01-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is one of the most common chronic infectious amphixenotic diseases worldwide. Prevention and control of TB are greatly difficult, due to the increase in drug-resistant TB, particularly multidrug-resistant TB. We speculated that there were some differences between drug-sensitive and drug-resistant MTB strains and that mazEF 3,6,9 toxin-antitoxin systems (TASs) were involved in MTB viability. This study aimed to investigate differences in viability, biofilm formation, and MazEF expression between drug-sensitive and drug-resistant MTB strains circulating in Xinjiang, China, and whether mazEF 3,6,9 TASs contribute to MTB viability under stress conditions. Growth profiles and biofilm-formation abilities of drug-sensitive, drug-resistant MTB strains and the control strain H37Rv were monitored. Using molecular biology experiments, the mRNA expression of the mazF 3, 6, and 9 toxin genes, the mazE 3, 6, and 9 antitoxin genes, and expression of the MazF9 protein were detected in the different MTB strains, H37RvΔ mazEF 3,6,9 mutants from the H37Rv parent strain were generated, and mutant viability was tested. Ex vivo culture analyses demonstrated that drug-resistant MTB strains exhibit higher survival rates than drug-sensitive strains and the control strain H37Rv. However, there was no statistical difference in biofilm-formation ability in the drug-sensitive, drug-resistant, and H37Rv strains. mazE 3,6 mRNA-expression levels were relatively reduced in the drug-sensitive and drug-resistant strains compared to H37Rv. Conversely, mazE 3,9 expression was increased in drug-sensitive strains compared to drug-resistant strains. Furthermore, compared with the H37Rv strain, mazF 3,6 expression was increased in drug-resistant strains, mazF 9 expression was increased in drug-sensitive strains, and mazF 9 exhibited reduced expression in drug-resistant strains compared with drug-sensitive strains. Protein expression of mazF9

Multi-targeted therapy for leprosy: insilico strategy to overcome multi drug resistance and to improve therapeutic efficacy.

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Anusuya, Shanmugam; Natarajan, Jeyakumar

2012-12-01

Leprosy remains a major public health problem, since single and multi-drug resistance has been reported worldwide over the last two decades. In the present study, we report the novel multi-targeted therapy for leprosy to overcome multi drug resistance and to improve therapeutic efficacy. If multiple enzymes of an essential metabolic pathway of a bacterium were targeted, then the therapy would become more effective and can prevent the occurrence of drug resistance. The MurC, MurD, MurE and MurF enzymes of peptidoglycan biosynthetic pathway were selected for multi targeted therapy. The conserved or class specific active site residues important for function or stability were predicted using evolutionary trace analysis and site directed mutagenesis studies. Ten such residues which were present in at least any three of the four Mur enzymes (MurC, MurD, MurE and MurF) were identified. Among the ten residues G125, K126, T127 and G293 (numbered based on their position in MurC) were found to be conserved in all the four Mur enzymes of the entire bacterial kingdom. In addition K143, T144, T166, G168, H234 and Y329 (numbered based on their position in MurE) were significant in binding substrates and/co-factors needed for the functional events in any three of the Mur enzymes. These are the probable residues for designing newer anti-leprosy drugs in an attempt to reduce drug resistance. Copyright © 2012 Elsevier B.V. All rights reserved.

Quercetin suppresses drug-resistant spheres via the p38 MAPK-Hsp27 apoptotic pathway in oral cancer cells.

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Su-Feng Chen

Full Text Available BACKGROUND: Treatment failure in oral squamous cell carcinoma (OSCC leading to local recurrence(s and metastases is mainly due to drug resistance. Cancer stem cells (CSCs are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive. METHODS: A drug-resistant sphere (DRSP model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial-mesenchymal transition (EMT-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo. RESULTS: Our data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27 via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu, suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC. CONCLUSIONS: The p38 MAPK-Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis

Relationship between the 99mTc-MIBI and expression of P-glycoprotein in lung cancer

International Nuclear Information System (INIS)

Meng Zili; Shen Zhenya

2002-01-01

Objective: To correlate the imaging of 99m Tc-MIBI with the expression of P-glycoprotein (PGP) in lung cancer. Methods: All patients, undergoing early (30 min) and delayed (3h) 99m Tc-MIBI imaging before initiation of chemo-or radiotherapy, were diagnosed pathologically. Immunohistochemical studies were performed using a monoclonal antibody, P-170, developed against the internal epitope of PGP. Normal tissue and tumor washout rate and tumor-to-background ratio were correlated with the level of PGP expression. Results: There was an inverse correlation between tumor-to-background ratio and the density of PGP (P 0.1). Conclusion: The reduced ability for the tumors to accumulate MIBI correlates well with the increased levels of PGP expression, tumor washout rate of MIBI does not correlate with the density of PGP in tumor tissues

Multi-drug-resistant tuberculosis in HIV positive patients in Eastern Europe.

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Post, Frank A; Grint, Daniel; Werlinrud, Anne Marie; Panteleev, Alexander; Riekstina, Vieja; Malashenkov, Evgeniy A; Skrahina, Alena; Duiculescu, Dan; Podlekareva, Daria; Karpov, Igor; Bondarenko, Vasiliy; Chentsova, Nelly; Lundgren, Jens; Mocroft, Amanda; Kirk, Ole; Miro, Jose M

2014-03-01

Observational data from Eastern Europe on the management and outcome of multi-drug-resistant tuberculosis (MDR TB) in HIV positive populations remain sparse in the English-language literature. We compared clinical characteristics and outcomes of 55 patients who were diagnosed with HIV and MDR TB in Eastern Europe between 2004 and 2006 to 89 patients whose Mycobacterium tuberculosis isolates were susceptible to isoniazid and rifampicin. Patients with HIV and MDR TB were young and predominantly male with high rates of intravenous drug use, imprisonment and hepatitis C co-infection. Eighty-four per cent of patients with MDR TB had no history of previous TB drug exposure suggesting that the majority of MDR TB resulted from transmission of drug-resistant M. tuberculosis. The use of non-standardized tuberculosis treatment was common, and the use of antiretroviral therapy infrequent. Compared to those with susceptible tuberculosis, patients with MDR TB were less likely to achieve cure or complete tuberculosis treatment (21.8% vs. 62.9%, p < 0.0001), and they were more likely to die (65.5% vs. 27.0%, p < 0.0001). Our study documents suboptimal management and poor outcomes in HIV positive patients with MDR TB. Implementation of WHO guidelines, rapid TB diagnostics and TB drug susceptibility testing for all patients remain a priority in this region. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance

International Nuclear Information System (INIS)

Yang, Ji Yeon; Ha, Seon-Ah; Yang, Yun-Sik; Kim, Jin Woo

2010-01-01

Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells. Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated. YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics. Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists

Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency.

Science.gov (United States)

Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V

2017-11-01

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (transport large (>1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. Published by Elsevier Inc.

Association of drug transporter expression with mortality and progression-free survival in stage IV head and neck squamous cell carcinoma.

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Rolf Warta

Full Text Available Drug transporters such as P-glycoprotein (ABCB1 have been associated with chemotherapy resistance and are considered unfavorable prognostic factors for survival of cancer patients. Analyzing mRNA expression levels of a subset of drug transporters by quantitative reverse transcription polymerase chain reaction (qRT-PCR or protein expression by tissue microarray (TMA in tumor samples of therapy naïve stage IV head and neck squamous cell carcinoma (HNSCC (qRT-PCR, n = 40; TMA, n = 61, this in situ study re-examined the significance of transporter expression for progression-free survival (PFS and overall survival (OS. Data from The Cancer Genome Atlas database was used to externally validate the respective findings (n = 317. In general, HNSCC tended to lower expression of drug transporters compared to normal epithelium. High ABCB1 mRNA tumor expression was associated with both favorable progression-free survival (PFS, p = 0.0357 and overall survival (OS, p = 0.0535. Similar results were obtained for the mRNA of ABCC1 (MRP1, multidrug resistance-associated protein 1; PFS, p = 0.0183; OS, p = 0.038. In contrast, protein expression of ATP7b (copper transporter ATP7b, mRNA expression of ABCG2 (BCRP, breast cancer resistance protein, ABCC2 (MRP2, and SLC31A1 (hCTR1, human copper transporter 1 did not correlate with survival. Cluster analysis however revealed that simultaneous high expression of SLC31A1, ABCC2, and ABCG2 indicates poor survival of HNSCC patients. In conclusion, this study militates against the intuitive dogma where high expression of drug efflux transporters indicates poor survival, but demonstrates that expression of single drug transporters might indicate even improved survival. Prospectively, combined analysis of the 'transportome' should rather be performed as it likely unravels meaningful data on the impact of drug transporters on survival of patients with HNSCC.

Increasing BMI is associated with reduced expression of P-glycoprotein (ABCB1 gene) in the human brain with a stronger association in African-Americans than Caucasians

DEFF Research Database (Denmark)

Nielsen, Julie Vendelbo; Olesen, Rasmus Hansen; Lauridsen, Jesper Krogh

2016-01-01

The efflux pump, p-glycoprotein, controls bioavailability and excretion of pharmaceutical compounds. In the blood-brain barrier, p-glycoprotein regulates the delivery of pharmaceutical substances to the brain, influencing efficacy and side effects for some drugs notably antipsychotics. Common sid...... online publication, 29 November 2016; doi:10.1038/tpj.2016.74....

Reduced intracellular drug accumulation in drug-resistant leukemia cells is not solely due to MDR-mediated efflux but also to decreased uptake

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Angela Oliveira Pisco

2014-10-01

Full Text Available Expression of ABC family transporter proteins that promote drug efflux from cancer cells is a widely observed mechanism of multi-drug resistance of cancer cells. Cell adaptation in long-term culture of HL60 leukemic cells in the presence of chemotherapy leads to induction and maintenance of the ABC transporters expression, preventing further accumulation of drugs. However, we found that decreased accumulation of drugs and fluorescent dyes was also contributed by a reduced uptake by the resistant cells. Confocal time-lapse microscopy and flow cytometry revealed that fluid-phase endocytosis was diminished in drug-resistant cells compared to drug-sensitive cells. Drug uptake was increased by insulin co-treatment when cells were grown in methylcellulose and monitored under the microscope, but not when cultured in suspension. We propose that multi-drug resistance is not solely achieved by enhanced efflux capacity but also by supressed intake of the drug offering an alternative target to overcome drug resistance or potentiate chemotherapy.

Venetoclax (ABT-199) Might Act as a Perpetrator in Pharmacokinetic Drug-Drug Interactions.

Science.gov (United States)

Weiss, Johanna; Gajek, Thomas; Köhler, Bruno Christian; Haefeli, Walter Emil

2016-02-24

Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug-drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug-drug interactions.

The effect of P-glycoprotein on methadone hydrochloride flux in equine intestinal mucosa.

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Linardi, R L; Stokes, A M; Andrews, F M

2013-02-01

Methadone is an effective analgesic opioid that may have a place for the treatment of pain in horses. However, its absorption seems to be impaired by the presence of a transmembrane protein, P-glycoprotein, present in different tissues including the small intestine in other species. This study aims to determine the effect of the P-glycoprotein on methadone flux in the equine intestinal mucosa, as an indicator of in vivo drug absorption. Jejunum tissues from five horses were placed into the Ussing chambers and exposed to methadone solution in the presence or absence of Rhodamine 123 or verapamil. Electrical measurements demonstrated tissue viability for 120 min, and the flux of methadone across the jejunal membrane (mucosal to submucosal direction) was calculated based on the relative drug concentration measured by ELISA. The flux of methadone was significantly higher only in the presence of verapamil. P-glycoprotein was immunolocalized in the apical membrane of the jejunal epithelial cells (enterocytes), mainly located in the tip of the villi compared to cells of the crypts. P-glycoprotein is present in the equine jejunum and may possibly mediate the intestinal transport of methadone. This study suggests that P-glycoprotein may play a role in the poor intestinal absorption of methadone in vivo. © 2012 Blackwell Publishing Ltd.

In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

Science.gov (United States)

Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

2016-01-01

Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

Enhanced brain disposition and effects of Δ9-tetrahydrocannabinol in P-glycoprotein and breast cancer resistance protein knockout mice.

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Adena S Spiro

Full Text Available The ABC transporters P-glycoprotein (P-gp, Abcb1 and breast cancer resistance protein (Bcrp, Abcg2 regulate the CNS disposition of many drugs. The main psychoactive constituent of cannabis Δ(9-tetrahydrocannabinol (THC has affinity for P-gp and Bcrp, however it is unknown whether these transporters modulate the brain accumulation of THC and its functional effects on the CNS. Here we aim to show that mice devoid of Abcb1 and Abcg2 retain higher brain THC levels and are more sensitive to cannabinoid-induced hypothermia than wild-type (WT mice. Abcb1a/b (-/-, Abcg2 (-/- and wild-type (WT mice were injected with THC before brain and blood were collected and THC concentrations determined. Another cohort of mice was examined for THC-induced hypothermia by measuring rectal body temperature. Brain THC concentrations were higher in both Abcb1a/b (-/- and Abcg2 (-/- mice than WT mice. ABC transporter knockout mice exhibited delayed elimination of THC from the brain with the effect being more prominent in Abcg2 (-/- mice. ABC transporter knockout mice were more sensitive to THC-induced hypothermia compared to WT mice. These results show P-gp and Bcrp prolong the brain disposition and hypothermic effects of THC and offer a novel mechanism for both genetic vulnerability to the psychoactive effects of cannabis and drug interactions between CNS therapies and cannabis.

The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria.

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Walsh, Fiona; Duffy, Brion

2013-01-01

Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.

The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria.

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Fiona Walsh

Full Text Available Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR resistance genes were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.

Cytochrome P450s--Their expression, regulation, and role in insecticide resistance.

Science.gov (United States)

Liu, Nannan; Li, Ming; Gong, Youhui; Liu, Feng; Li, Ting

2015-05-01

P450s are known to be critical for the detoxification and/or activation of xenobiotics such as drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In insects, P450s play an important role in detoxifying exogenous compounds such as insecticides and plant toxins and their overexpression can result in increased levels of P450 proteins and P450 activities. This has been associated with enhanced metabolic detoxification of insecticides and has been implicated in the development of insecticide resistance in insects. Multiple P450 genes have been found to be co-overexpressed in individual insect species via several constitutive overexpression and induction mechanisms, which in turn are co-responsible for high levels of insecticide resistance. Many studies have also demonstrated that the transcriptional overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of regulatory genes/factors and resistance genes. This chapter reviews our current understanding of how the molecular mechanisms of P450 interaction/gene regulation govern the development of insecticide resistance in insects and our progress along the road to a comprehensive characterization of P450 detoxification-mediated insecticide resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Reduced expression of p27 is a novel mechanism of docetaxel resistance in breast cancer cells

International Nuclear Information System (INIS)

Brown, Iain; Shalli, Kawan; McDonald, Sarah L; Moir, Susan E; Hutcheon, Andrew W; Heys, Steven D; Schofield, Andrew C

2004-01-01

Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Breast cancers can have an inherent or acquired resistance to docetaxel but the causes of this resistance remain unclear. However, apoptosis and cell cycle regulation are key mechanisms by which most chemotherapeutic agents exert their cytotoxic effects. We created two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) and performed cDNA microarray analysis to identify candidate genes associated with docetaxel resistance. Gene expression changes were validated at the RNA and protein levels by reverse transcription PCR and western analysis, respectively. Gene expression cDNA microarray analysis demonstrated reduced p27 expression in docetaxel-resistant breast cancer cells. Although p27 mRNA expression was found to be reduced only in MCF-7 docetaxel-resistant sublines (2.47-fold), reduced expression of p27 protein was noted in both MCF-7 and MDA-MB-231 docetaxel-resistant breast cancer cells (2.83-fold and 3.80-fold, respectively). This study demonstrates that reduced expression of p27 is associated with acquired resistance to docetaxel in breast cancer cells. An understanding of the genes that are involved in resistance to chemotherapy may allow further development in modulating drug resistance, and may permit selection of those patients who are most likely to benefit from such therapies

20(S)-Protopanaxadiol (PPD) analogues chemosensitize multidrug-resistant cancer cells to clinical anticancer drugs.

Science.gov (United States)

Liu, Junhua; Wang, Xu; Liu, Peng; Deng, Rongxin; Lei, Min; Chen, Wantao; Hu, Lihong

2013-07-15

Novel 20(S)-protopanoxadiol (PPD) analogues were designed, synthesized, and evaluated for the chemosensitizing activity against a multidrug resistant (MDR) cell line (KBvcr) overexpressing P-glycoprotein (P-gp). Structure-activity relationship analysis showed that aromatic substituted aliphatic amine at the 24-positions (groups V) effectively and significantly sensitized P-gp overexpressing multidrug resistant (MDR) cells to anticancer drugs, such as docetaxel (DOC), vincristine (VCR), and adriamycin (ADM). PPD derivatives 12 and 18 showed 1.3-2.6 times more effective reversal ability than verapamil (VER) for DOC and VCR. Importantly, no cytotoxicity was observed by the active PPD analogues (5μM) against both non-MDR and MDR cells, suggesting that PPD analogues serve as novel lead compounds toward a potent and safe resistance modulator. Moreover, a preliminary mechanism study demonstrated that the chemosensitizing activity of PPD analogues results from inhibition of P-glycoprotein (P-gp) overexpressed in MDR cancer cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effects of cadmium exposure on expression and activity of P-glycoprotein in eastern oysters, Crassostrea virginica Gmelin

Energy Technology Data Exchange (ETDEWEB)

Ivanina, Anna V. [Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 (United States); Sokolova, Inna M. [Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 (United States)], E-mail: isokolov@uncc.edu

2008-06-02

Heavy metal pollution is a worldwide problem, and cadmium (Cd) is one of the most noxious pollutants in aquatic environments. We studied P-glycoprotein (P-gp) expression and function in control and Cd exposed (50 {mu}g L{sup -1} Cd, 30-40 days) oysters Crassostrea virginica as a possible mechanism of cell protection against Cd. Our data show that P-gp is expressed on cell membrane and in mitochondria of oyster gills and hepatopancreas. Inhibitor studies with verapamil, cyclosporine A and JS-2190 suggest that in the gills, mitochondrial P-gp pumps substrates from cytosol into the mitochondria, while cell membrane P-gp pumps substrates from cytosol out of the cell. Cd exposure resulted in a 2-2.5-fold increase in P-gp protein expression in cell membranes and a 3.5-7-fold increase in transport activity measured as the inhibitor-sensitive rhodamine B extrusion rate. In contrast, p-gp mRNA levels were similar in control and Cd-exposed oysters. No difference in P-gp protein expression was observed between mitochondria of control and Cd-exposed oysters but the apparent transport activity was higher in mitochondria from Cd-exposed oysters. Overall, a stronger increase in substrate transport activity in Cd-exposed oysters compared to a relatively weaker change in P-gp protein levels suggests that P-gp activity is post-translationally regulated. Our data show that direct determination of P-gp transport activity may be the best measure of the xenobiotic-resistant phenotype, whereas p-gp mRNA levels are not a good marker due to the likely involvement of multiple post-transcriptional regulatory steps. Cd exposure resulted in a significantly elevated rate of oxygen consumption of isolated oyster gills by 46%. Specific inhibitors of ATPase function of P-gp (cyclosporine A and JS-2190) had no significant effect on tissue oxygen consumption indicating that P-gp contribution to energy budget is negligible and supporting indirect estimates based on the ATP stoichiometry of substrate

Effects of cadmium exposure on expression and activity of P-glycoprotein in eastern oysters, Crassostrea virginica Gmelin

International Nuclear Information System (INIS)

Ivanina, Anna V.; Sokolova, Inna M.

2008-01-01

Heavy metal pollution is a worldwide problem, and cadmium (Cd) is one of the most noxious pollutants in aquatic environments. We studied P-glycoprotein (P-gp) expression and function in control and Cd exposed (50 μg L -1 Cd, 30-40 days) oysters Crassostrea virginica as a possible mechanism of cell protection against Cd. Our data show that P-gp is expressed on cell membrane and in mitochondria of oyster gills and hepatopancreas. Inhibitor studies with verapamil, cyclosporine A and JS-2190 suggest that in the gills, mitochondrial P-gp pumps substrates from cytosol into the mitochondria, while cell membrane P-gp pumps substrates from cytosol out of the cell. Cd exposure resulted in a 2-2.5-fold increase in P-gp protein expression in cell membranes and a 3.5-7-fold increase in transport activity measured as the inhibitor-sensitive rhodamine B extrusion rate. In contrast, p-gp mRNA levels were similar in control and Cd-exposed oysters. No difference in P-gp protein expression was observed between mitochondria of control and Cd-exposed oysters but the apparent transport activity was higher in mitochondria from Cd-exposed oysters. Overall, a stronger increase in substrate transport activity in Cd-exposed oysters compared to a relatively weaker change in P-gp protein levels suggests that P-gp activity is post-translationally regulated. Our data show that direct determination of P-gp transport activity may be the best measure of the xenobiotic-resistant phenotype, whereas p-gp mRNA levels are not a good marker due to the likely involvement of multiple post-transcriptional regulatory steps. Cd exposure resulted in a significantly elevated rate of oxygen consumption of isolated oyster gills by 46%. Specific inhibitors of ATPase function of P-gp (cyclosporine A and JS-2190) had no significant effect on tissue oxygen consumption indicating that P-gp contribution to energy budget is negligible and supporting indirect estimates based on the ATP stoichiometry of substrate

A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics.

Science.gov (United States)

Saaby, Lasse; Brodin, Birger

2017-09-01

Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate kinetic parameters, such as K m and V max , for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC 50 ) and the disassociation constant for an inhibitor/P-gp complex (K i ) have been determined to estimate P-gp inhibition. This review addresses in vitro methods commonly used to study P-gp transport kinetics and aims at providing a critical evaluation of the application of steady-state Michaelis-Menten analysis of kinetic parameters for substrate/P-gp interactions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Menadione serves as a substrate for P-glycoprotein: implication in chemosensitizing activity.

Science.gov (United States)

Oh, Seok-Jeong; Han, Hyo-Kyung; Kang, Keon-Wook; Lee, Young-Joo; Lee, Moo-Yeol

2013-04-01

Based on its chemosensitizing effect, we questioned whether menadione is an inhibitor or a substrate of P-glycoprotein (P-gp). To test this hypothesis, we assessed the effect of menadione on P-gp activity and examined the P-gp-dependency of cellular accumulation and cytotoxicity of menadione as well. Treatment with menadione resulted in the concentration-dependent increase of rhodamine 123 (Rh123) accumulation in P-gp-overexpressing MDCKII/MDR1 and NCI/ADR-RES cells, suggesting that menadione inhibits Rh123 extrusion by P-gp. Compared with MDCKII or MCF-7, intracellular distribution of [(3)H]-menadione was significantly lower in MDCKII/MDR1 or NCI/ADR-RES cells, which could be restored by the P-gp inhibitors, verapamil and quinidine. Consistent with these results, MDCKII/MDR1 or NCI/ADR-RES cells were more resistant to the cytotoxicity of menadione than MDCKII or MCF-7 cells, respectively. Such resistance was abolished by the combined treatment of verapamil and quinidine in NCI/ADR-RES cells. Our study identified menadione as a substrate of P-gp, which presumably, acts as the mechanism for the chemosensitizing effect. Menadione may be a promising chemotherapeutic enhancer by its ability of circumventing drug resistance, in addition to its own anti-cancer activity.

Collateral sensitivity to cisplatin in KB-8-5-11 drug-resistant cancer cells.

LENUS (Irish Health Repository)

Doherty, Ben

2014-01-01

KB-8-5-11 cells are a drug-resistant cervical cell model that overexpresses ABCB1 (P-glycoprotein). KB-8-5-11 has become sensitive to non-ABCB1 substrate cisplatin. Understanding the mechanism of collateral sensitivity to cisplatin may lead to biomarker discovery for platinum sensitivity in patients with cancer.

Pattern of intensive phase treatment outcomes of multi-drug resistant ...

African Journals Online (AJOL)

Pattern of intensive phase treatment outcomes of multi-drug resistant tuberculosis in University of Port Harcourt Treatment Centre: a review of records from ... Data on patients' age, sex, HIV status, treatment outcomes were extracted from the hospital book records into a computer data sheet at the UPTH treatment centre.

Association between HIV/AIDS and multi-drug resistance tuberculosis: a systematic review and meta-analysis.

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Yonatan Moges Mesfin

Full Text Available BACKGROUND: Human immunodeficiency virus (HIV, multi-drug resistant tuberculosis (MDR is emerging as major challenge facing tuberculosis control programs worldwide particularly in Asia and Africa. Findings from different studies on associations of HIV co-infection and drug resistance among patients with TB have been contradictory (discordant. Some institution based studies found strongly increased risks for multi-drug resistant TB (MDR TB among patients co-infected with TB and HIV, whereas other studies found no increased risk (it remains less clear in community based studies. The aim was to conduct a systematic review and meta-analysis of the association between multi-drug resistant tuberculosis and HIV infection. METHODS AND FINDINGS: Systematic review of the published literature of observational studies was conducted. Original studies were identified using databases of Medline/Pubmed, Google Scholar and HINARI. The descriptions of original studies were made using frequency and forest plot. Publication bias was assessed using Funnel plot graphically and Egger weighted and Begg rank regression tests statistically. Heterogeneity across studies was checked using Cochrane Q test statistic and I(2. Pool risk estimates of MDR-TB and sub-grouping analysis were computed to analyze associations with HIV. Random effects of the meta-analysis of all 24 observational studies showed that HIV is associated with a marginal increased risk of multi-drug resistant tuberculosis (estimated Pooled OR 1.24; 95%, 1.04-1.43. Subgroup analyses showed that effect estimates were higher (Pooled OR 2.28; 95%, 1.52-3.04 for primary multi-drug resistance tuberculosis and moderate association between HIV/AIDS and MDR-TB among population based studies and no significant association in institution settings. CONCLUSIONS: This study demonstrated that there is association between MDR-TB and HIV. Capacity for diagnosis of MDR-TB and initiating and scale up of antiretroviral

Low-level quinolone-resistance in multi-drug resistant typhoid

Energy Technology Data Exchange (ETDEWEB)

Mirza, S H; Khan, M A [Armed Forces Inst. of Pathology, Rawalpindi (Pakistan). Dept. of Microbiolgy

2008-01-15

To find out the frequency of low-level quinolone-resistance in Multi-Drug Resistant (MDR) typhoid using nalidixic acid screening disc. Blood was obtained from suspected cases of typhoid fever and cultured in to BacT/ALERT. The positive blood cultures bottles were subcultured. The isolates were identified by colony morphology and biochemical tests using API-20E galleries. Susceptibility testing of isolates was done by modified Kirby-Bauer disc diffusion method on Muellar Hinton Agar. For the isolates, which were resistant to nalidixic acid by disc diffusion method, Minimal Inhibitory Concentrations (MICs) of ciprofloxacin and nalidixic acid were determined by using the E-test strips. Disc diffusion susceptibility tests and MICs were interpreted according to the guidelines provided by National Committee for Control Laboratory Standard (NCCLS). A total of 21(65.5%) out of 32 isolates of Salmonellae were nalidixic acid-resistant by disk diffusion method. All the nalidixic acid-resistant isolates by disc diffusion method were confirmed by MICs for both ciprofloxacin and nalidixic acid. All the nalidixic acid-resistant isolates had a ciprofloxacin MIC of 0.25-1 microg/ml (reduced susceptibility) and nalidixic acid MICs > 32 microg (resistant). Out of all Salmonella isolates, 24 (75%) were found to be MDR, and all were S. typbi. Low-level quinolone-resistance in typhoid was high in this small series. Screening for nalidixic acid resistance with a 30 microg nalidixic acid disk is a reliable and cost-effective method to detect low-level fluoroquinolone resistance, especially in the developing countries. (author)

Low-level quinolone-resistance in multi-drug resistant typhoid

International Nuclear Information System (INIS)

Mirza, S.H.; Khan, M.A.

2008-01-01

To find out the frequency of low-level quinolone-resistance in Multi-Drug Resistant (MDR) typhoid using nalidixic acid screening disc. Blood was obtained from suspected cases of typhoid fever and cultured in to BacT/ALERT. The positive blood cultures bottles were subcultured. The isolates were identified by colony morphology and biochemical tests using API-20E galleries. Susceptibility testing of isolates was done by modified Kirby-Bauer disc diffusion method on Muellar Hinton Agar. For the isolates, which were resistant to nalidixic acid by disc diffusion method, Minimal Inhibitory Concentrations (MICs) of ciprofloxacin and nalidixic acid were determined by using the E-test strips. Disc diffusion susceptibility tests and MICs were interpreted according to the guidelines provided by National Committee for Control Laboratory Standard (NCCLS). A total of 21(65.5%) out of 32 isolates of Salmonellae were nalidixic acid-resistant by disk diffusion method. All the nalidixic acid-resistant isolates by disc diffusion method were confirmed by MICs for both ciprofloxacin and nalidixic acid. All the nalidixic acid-resistant isolates had a ciprofloxacin MIC of 0.25-1 microg/ml (reduced susceptibility) and nalidixic acid MICs > 32 microg (resistant). Out of all Salmonella isolates, 24 (75%) were found to be MDR, and all were S. typbi. Low-level quinolone-resistance in typhoid was high in this small series. Screening for nalidixic acid resistance with a 30 microg nalidixic acid disk is a reliable and cost-effective method to detect low-level fluoroquinolone resistance, especially in the developing countries. (author)

Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells

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Elodie Jouan

2016-12-01

Full Text Available Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP, organic anion-transporting polypeptides (OATPs and organic cation transporter 1 (OCT1, and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP. Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2, OCT1 and bile salt export pump or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3 in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR- and nuclear factor erythroid 2-related factor 2 (Nrf2-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation.

Effects on enantiomeric drug disposition and open-field behavior after chronic treatment with venlafaxine in the P-glycoprotein knockout mice model.

Science.gov (United States)

Karlsson, Louise; Hiemke, Christoph; Carlsson, Björn; Josefsson, Martin; Ahlner, Johan; Bengtsson, Finn; Schmitt, Ulrich; Kugelberg, Fredrik C

2011-05-01

P-glycoprotein (P-gp) plays an important role in the efflux of drugs from the brain back into the bloodstream and can influence the pharmacokinetics and pharmacodynamics of drug molecules. To our knowledge, no studies have reported pharmacodynamic effects of any antidepressant drug in the P-gp knockout mice model. The aim of this study was to investigate the enantiomeric venlafaxine and metabolite concentrations in serum and brain of abcb1ab⁻/⁻ mice compared to wild-type mice upon chronic dosing, and to assess the effect of venlafaxine treatment on open-field behavior. P-gp knockout and wild-type mice received two daily intraperitoneal injections of venlafaxine (10 mg/kg) over ten consecutive days. Locomotor and rearing activities were assessed on days 7 and 9. After 10 days, drug and metabolite concentrations in brain and serum were determined using an enantioselective LC/MS/MS method. The brain concentrations of venlafaxine and its three demethylated metabolites were two to four times higher in abcb1ab⁻/⁻ mice compared to abcb1ab+/+ mice. The behavioral results indicated an impact on exploration-related behaviors in the open-field as center activity was increased, and rears were decreased by venlafaxine treatment. Our results show that P-gp at the blood-brain barrier plays an important role in limiting brain entry of the enantiomers of venlafaxine and its metabolites after chronic dosing. Taken together, the present pharmacokinetic and pharmacodynamic findings offer the possibility that the expression of P-gp in patients may be a contributing factor for limited treatment response.

Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model

DEFF Research Database (Denmark)

Andersson, O.; Badisco, L.; Hansen, A. H.

2014-01-01

In earlier studies insects were proposed as suitable models for vertebrate blood–brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain b...... has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery.......In earlier studies insects were proposed as suitable models for vertebrate blood–brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain...

Inhibition of cellular efflux pumps involved in multi xenobiotic resistance (MXR) in echinoid larvae as a possible mode of action for increased ecotoxicological risk of mixtures

NARCIS (Netherlands)

Drs Anselmo, H.M.R.; Berg, van den J.H.J.; Rietjens, I.; Murk, A.J.

2012-01-01

In marine organisms the multi xenobiotic resistance (MXR) mechanism via e.g. P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) is an important first line of defense against contaminants by pumping contaminants out of the cells. If compounds would impair the MXR mechanism, this

Significance of MDR1 and multiple drug resistance in refractory human epileptic brain

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Dini Gabriele

2004-10-01

Full Text Available Abstract Background The multiple drug resistance protein (MDR1/P-glycoprotein is overexpressed in glia and blood-brain barrier (BBB endothelium in drug refractory human epileptic tissue. Since various antiepileptic drugs (AEDs can act as substrates for MDR1, the enhanced expression/function of this protein may increase their active extrusion from the brain, resulting in decreased responsiveness to AEDs. Methods Human drug resistant epileptic brain tissues were collected after surgical resection. Astrocyte cell cultures were established from these tissues, and commercially available normal human astrocytes were used as controls. Uptake of fluorescent doxorubicin and radioactive-labeled Phenytoin was measured in the two cell populations, and the effect of MDR1 blockers was evaluated. Frozen human epileptic brain tissue slices were double immunostained to locate MDR1 in neurons and glia. Other slices were exposed to toxic concentrations of Phenytoin to study cell viability in the presence or absence of a specific MDR1 blocker. Results MDR1 was overexpressed in blood vessels, astrocytes and neurons in human epileptic drug-resistant brain. In addition, MDR1-mediated cellular drug extrusion was increased in human 'epileptic' astrocytes compared to 'normal' ones. Concomitantly, cell viability in the presence of cytotoxic compounds was increased. Conclusions Overexpression of MDR1 in different cell types in drug-resistant epileptic human brain leads to functional alterations, not all of which are linked to drug pharmacokinetics. In particular, the modulation of glioneuronal MDR1 function in epileptic brain in the presence of toxic concentrations of xenobiotics may constitute a novel cytoprotective mechanism.

Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance in vitro.

Science.gov (United States)

Khdair, Ayman; Handa, Hitesh; Mao, Guangzhao; Panyam, Jayanth

2009-02-01

Drug resistance limits the success of many anticancer drugs. Reduced accumulation of the drug at its intracellular site of action because of overexpression of efflux transporters such as P-glycoprotein (P-gp) is a major mechanism of drug resistance. In this study, we investigated whether photodynamic therapy (PDT) using methylene blue, also a P-gp inhibitor, can be used to enhance doxorubicin-induced cytotoxicity in drug-resistant tumor cells. Aerosol OT (AOT)-alginate nanoparticles were used as a carrier for the simultaneous cellular delivery of doxorubicin and methylene blue. Methylene blue was photoactivated using light of 665 nm wavelength. Induction of apoptosis and necrosis following treatment with combination chemotherapy and PDT was investigated in drug-resistant NCI/ADR-RES cells using flow cytometry and fluorescence microscopy. Effect of encapsulation in nanoparticles on the intracellular accumulation of doxorubicin and methylene blue was investigated qualitatively using fluorescence microscopy and was quantitated using HPLC. Encapsulation in AOT-alginate nanoparticles significantly enhanced the cytotoxicity of combination therapy in resistant tumor cells. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs. Further, nanoparticle-mediated combination therapy resulted in significantly elevated reactive oxygen species (ROS) production compared to single drug treatment. In conclusion, nanoparticle-mediated combination chemotherapy and PDT using doxorubicin and methylene blue was able to overcome resistance mechanisms and resulted in improved cytotoxicity in drug-resistant tumor cells.

P-glycoprotein mediated efflux limits the transport of the novel anti-Parkinson's disease candidate drug FLZ across the physiological and PD pathological in vitro BBB models.

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Qian Liu

Full Text Available FLZ, a novel anti-Parkinson's disease (PD candidate drug, has shown poor blood-brain barrier (BBB penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp and breast cancer resistance protein (BCRP are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER and low permeability for sodium fluorescein (NaF confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.

Asiatic Acid (AA) Sensitizes Multidrug-Resistant Human Lung Adenocarcinoma A549/DDP Cells to Cisplatin (DDP) via Downregulation of P-Glycoprotein (MDR1) and Its Targets.

Science.gov (United States)

Cheng, Qilai; Liao, Meixiang; Hu, Haibo; Li, Hongliang; Wu, Longhuo

2018-01-01

P-glycoprotein (P-gp, i.e., MDR1) is associated with the phenotype of multidrug resistance (MDR) and causes chemotherapy failure in the management of cancers. Searching for effective MDR modulators and combining them with anticancer drugs is a promising strategy against MDR. Asiatic acid (AA), a natural triterpene isolated from the plant Centella asiatica, may have an antitumor activity. The present study assessed the reversing effect of AA on MDR and possible molecular mechanisms of AA action in MDR1-overexpressing cisplatin (DDP)-resistant lung cancer cells, A549/DDP. Human lung adenocarcinoma A549/DDP cells were either exposed to different concentrations of AA or treated with DDP, and their viability was measured by the MTT assay. A Rhodamine 123 efflux assay, immunofluorescent staining, ATPase assay, reverse-transcription PCR (RT-PCR), and western blot analysis were conducted to elucidate the mechanisms of action of AA on MDR. Our results showed that AA significantly enhanced the cytotoxicity of DDP toward A549/DDP cells but not its parental A549 cells. Furthermore, AA strongly inhibited P-gp expression by blocking MDR1 gene transcription and increased the intracellular accumulation of the P-gp substrate Rhodamine 123 in A549/DDP cells. Nuclear factor (NF)-kB (p65) activity, IkB degradation, and NF-kB/p65 nuclear translocation were markedly inhibited by pretreatment with AA. Additionally, AA inhibited the MAPK-ERK pathway, as indicated by decreased phosphorylation of ERK1 and -2, AKT, p38, and JNK, thus resulting in reduced activity of the Y-box binding protein 1 (YB1) via blockage of its nuclear translocation. AA reversed P-gp-mediated MDR by inhibition of P-gp expression. This effect was likely related to downregulation of YB1, and this effect was mediated by the NF-kB and MAPK-ERK pathways. AA may be useful as an MDR reversal agent for combination therapy in clinical trials. © 2018 The Author(s). Published by S. Karger AG, Basel.

Molecular Genetic Analysis of Multi-drug Resistance in Indian Isolates of Mycobacterium tuberculosis

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Noman Siddiqi

1998-09-01

Full Text Available A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo b, gyr A, kat G were studied for mutation(s by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP technique. The SSCP positive samples were sequenced to characterize the mutations in rpo b, and gyr A loci. While previously reported mutations in the gyr A and rpo b loci were found to be present, several novel mutations were also scored in the rpo b locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis one from India, highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

In vitro effects of four native Brazilian medicinal plants in CYP3A4 mRNA gene expression, glutathione levels and P-glycoprotein activity.

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Andre Luis Dias Araujo Mazzari

2016-08-01

Full Text Available Erythrina mulungu Benth. (Fabaceae, Cordia verbenacea A. DC. (Boraginaceae, Solanum paniculatum L. (Solanaceae and Lippia sidoides Cham. (Verbenaceae are medicinal plants species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI. In this work we assess non-toxic concentrations (100μg/mL of their infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp activity in vincristine-resistant Caco-2 cells (Caco-2 VCR. Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (two-fold decrease, p<0.05, this being correlated with an antagonist effect upon hPXR (EC50 = 0.38mg/mL. Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p<0.001, Lippia sidoides (-12%, p<0.05 and Cordia verbenacea (-47%, p<0.001. The later plant extract was able to decrease GGT activity (-48%, p<0.01. In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum.

Decreasing prevalence of multi-drugs resistant Mycobacterium tuberculosis in Nashik City, India

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Arun P. More

2013-03-01

Full Text Available Objective: In India, increasing prevalence of multi-drug resistant tuberculosis (MDR has aggravated the control oftuberculosis problem. In many urban and semi-urban regions of India, no surveillance data of multidrug resistance inMycobacterium tuberculosisis available.Methods: A surveillance study on multidrug resistance was carried out in semi-urban and rural regions in and aroundNashik City of Maharashtra, India. The surveillance study was conducted in this region found that the prevalence ofcombined resistance to first and second-line anti-tuberculosis drugs is remarkably high. The isolates of M. tuberculosiswas identified and subjected to drug susceptibility testing. The patterns of drug susceptibility of isolates of M. tuberculosisduring the periods 2000 and 2004 were compared with drug susceptibility patterns of the organisms during theperiod 2008 to 2011.Results: The 260 isolates identified as M. tuberculosis show mean drug resistance prevalence of 45.6% for more than anytwo drugs and the MDR rate as 37% in the years 2000 to 2004 whereas 305 isolates of the organism show mean drugresistance prevalence of 30.2% and the MDR rate as 25% in the years 2008 to 2011.Conclusion: The researcher found that, though the prevalence of multidrug resistance to the drugs tested is remarkablyhigh, it has come down noticeably during the past seven years due to efforts of State Government and strict implementationof treatment guidelines of WHO by the physicians. J Microbiol Infect Dis 2013; 3(1: 12-17Key words: MDR-TB, XDR-TB, DOTS, drug-resistance prevalence rate.

Multi-drug-resistant tuberculosis in HIV positive patients in Eastern Europe

DEFF Research Database (Denmark)

Post, Frank A; Grint, Daniel; Efsen, Anne Marie Werlinrud

2014-01-01

Observational data from Eastern Europe on the management and outcome of multi-drug-resistant tuberculosis (MDR TB) in HIV positive populations remain sparse in the English-language literature.We compared clinical characteristics and outcomes of 55 patients who were diagnosed with HIV and MDR TB...... in Eastern Europe between 2004 and 2006 to 89 patients whose Mycobacterium tuberculosis isolates were susceptible to isoniazid and rifampicin.Patients with HIV and MDR TB were young and predominantly male with high rates of intravenous drug use, imprisonment and hepatitis C co-infection. Eighty-four per cent...... of patients with MDR TB had no history of previous TB drug exposure suggesting that the majority of MDR TB resulted from transmission of drug-resistant M. tuberculosis. The use of non-standardized tuberculosis treatment was common, and the use of antiretroviral therapy infrequent. Compared to those...

[Study of the relationship among expression of Survivin and MRP and the drug resistance in human nasopharyngeal carcinoma].

Science.gov (United States)

Yang, Ning; Zhu, Lepan; Tan, Tan; Hou, Chunyan

2015-02-01

This study aimed to explore the relationship among expression of Survivin and MRP and drug resistance in NPC. Expression of Survivin were detected by immunohistochemistry method in 45 cases of NPC and 24 cases of normal mucous membrane of nasopharynx (NMMN). The relationship between expression of Survivin and pathological factors in NPC were analysized. Expression of Survivin and MRP were detected in 31 patients of NPC with paclitaxel resistance and 20 patients of NPC without paclitaxel resistance. The relation- ship among the expression of Survivin or MRP and paclitaxel resistance in NPC were analysized. The paclitaxel resistance cell line, 5-8F-PTX(+); was established by a step-increased method. The expression of Survivin and MRP were detected by western blot in 5-8F-PTX(+) and 5-8F. The positive were 71. 1% (32/45) in NPC and 8.33% (2/24) in NMMN. And there were significantly differences between them (P MRP were 87.1% (27/31) in NPC patients with paclitaxel resistance and 40.0% (8/20) in NPC patients without paclitaxel resistance, respectively. There were significantly differences between them (P MRP in NPC patients with Paclitaxel resistance. The expression of Survivin and MRP were higher in 5-8F-PTX(+) than in 5-8F. The IC50 of paclitaxel, cDDP, 5-FU and Vincristine were significantly higher in 5-8F-PTX(+) than in 5-8F. There were relationship among the expression of Survivin and difference, metastasis and TNM stages of NPC. Survivin may serves as a molecular marker for development and progress in NPC. There were relationship among the high expression of Survivin and MRP and increasing of drug resistance in NPC.

Decreasing prevalence of multi-drugs resistant Mycobacterium tuberculosis in Nashik City, India

OpenAIRE

More, Arun Punaji; Nagdawane, Ramkrishna Panchamrao; Gangurde, Aniket K

2013-01-01

Objective: In India, increasing prevalence of multi-drug resistant tuberculosis (MDR) has aggravated the control oftuberculosis problem. In many urban and semi-urban regions of India, no surveillance data of multidrug resistance inMycobacterium tuberculosisis available.Methods: A surveillance study on multidrug resistance was carried out in semi-urban and rural regions in and aroundNashik City of Maharashtra, India. The surveillance study was conducted in this region found that the prevalence...

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria.

Science.gov (United States)

Roundhill, E A; Burchill, S A

2012-03-13

Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; PMRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.

Beta-Amyloid Downregulates MDR1-P-Glycoprotein (Abcb1 Expression at the Blood-Brain Barrier in Mice

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Anja Brenn

2011-01-01

Full Text Available Neurovascular dysfunction is an important component of Alzheimer's disease, leading to reduced clearance across the blood-brain barrier and accumulation of neurotoxic β-amyloid (Aβ peptides in the brain. It has been shown that the ABC transport protein P-glycoprotein (P-gp, ABCB1 is involved in the export of Aβ from the brain into the blood. To determine whether Aβ influences the expression of key Aβ transporters, we studied the effects of 1-day subcutaneous Aβ1-40 and Aβ1-42 administration via Alzet mini-osmotic pumps on P-gp, BCRP, LRP1, and RAGE expression in the brain of 90-day-old male FVB mice. Our results demonstrate significantly reduced P-gp, LRP1, and RAGE mRNA expression in mice treated with Aβ1-42 compared to controls, while BCRP expression was not affected. The expression of the four proteins was unchanged in mice treated with Aβ1-40 or reverse-sequence peptides. These findings indicate that, in addition to the age-related decrease of P-gp expression, Aβ1-42 itself downregulates the expression of P-gp and other Aβ-transporters, which could exacerbate the intracerebral accumulation of Aβ and thereby accelerate neurodegeneration in Alzheimer's disease and cerebral β-amyloid angiopathy.

Triorganotin Derivatives Induce Cell Death Effects on L1210 Leukemia Cells at Submicromolar Concentrations Independently of P-glycoprotein Expression

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Viera Bohacova

2018-05-01

Full Text Available The acceleration of drug efflux activity realized by plasma membrane transporters in neoplastic cells, particularly by P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family, represents a frequently observed molecular cause of multidrug resistance (MDR. This multiple resistance represents a real obstacle in the effective chemotherapy of neoplastic diseases. Therefore, identifying cytotoxic substances that are also effective in P-gp overexpressing cells may be useful for the rational design of substances for the treatment of malignancies with developed MDR. Here, we showed that triorganotin derivatives—tributyltin-chloride (TBT-Cl, tributyltin-bromide (TBT-Br, tributyltin-iodide (TBT-I and tributyltin-isothiocyanate (TBT-NCS or triphenyltin-chloride (TPT-Cl and triphenyltin-isothiocyanate (TPT-NCS—could induce the death of L1210 mice leukemia cells at a submicromolar concentration independently of P-gp overexpression. The median lethal concentration obtained for triorganotin derivatives did not exceed 0.5 µM in the induction of cell death of either P-gp negative or P-gp positive L1210 cells. Apoptosis related to regulatory pathway of Bcl-2 family proteins seems to be the predominant mode of cell death in either P-gp negative or P-gp positive L1210 cells. TBT-Cl and TBT-Br were more efficient with L1210 cells overexpressing P-gp than with their counterpart P-gp negative cells. In contrast, TBT-I and TPT-NCS induced a more pronounced cell death effect on P-gp negative cells than on P-gp positive cells. Triorganotin derivatives did not affect P-gp efflux in native cells measured by calcein retention within the cells. Taken together, we assumed that triorganotin derivatives represent substances suitable for suppressing the viability of P-gp positive malignant cells.

Complex interplay between the P-glycoprotein multidrug efflux pump and the membrane: its role in modulating protein function

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Frances Jane Sharom

2014-03-01

Full Text Available Multidrug resistance in cancer is linked to expression of the P-glycoprotein multidrug transporter (Pgp, ABCB1, which exports many structurally diverse compounds from cells. Substrates first partition into the bilayer and then interact with a large flexible binding pocket within the transporter’s transmembrane regions. Pgp has been described as a hydrophobic vacuum cleaner or an outwardly-directed drug/lipid flippase. Recent X-ray crystal structures have shed some light on the nature of the drug-binding pocket and suggested routes by which substrates can enter it from the membrane. Detergents have profound effects on Pgp function, and several appear to be substrates. Biochemical and biophysical studies in vitro, some using purified reconstituted protein, have explored the effects of the membrane environment. They have demonstrated that Pgp is involved in a complex relationship with its lipid environment, which modulates the behaviour of its substrates, as well as various functions of the protein, including ATP hydrolysis, drug binding and drug transport. Membrane lipid composition and fluidity, phospholipid headgroup and acyl chain length all influence Pgp function. Recent studies focusing on thermodynamics and kinetics have revealed some important principles governing Pgp-lipid and substrate-lipid interactions, and how these affect drug binding and transport. In some cells, Pgp is associated with cholesterol-rich microdomains which may modulate its functions. The relationship between Pgp and cholesterol remains an open question; however it clearly affects several aspects of its function in addition to substrate-membrane partitioning. The action of Pgp modulators appears to depend on their membrane permeability, and membrane fluidizers and surfactants reverse drug resistance, likely via an indirect mechanism. A detailed understanding of how the membrane affects Pgp substrates and Pgp’s catalytic cycle may lead to new strategies to combat

Glycoprotein Mucin Molecular Brush on Cancer Cells and its Correlation with Resistance Against Drug Delivery

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Wang, Xin; Shah, Aalok; Campbell, Robert; Wan, Kai-Tak

2012-02-01

Uptake of cytotoxic drugs by typical tumor cells is limited by the dense dendritic network of oligosaccharide mucin chains that forms a mechanical barrier. Atomic force microscopy is used to directly measure the force needed to pierce the mucin layer to reach the cell surface. Measurements are analyzed by deGennes' steric reputation theory. Multi-drug resistant ovarian tumor cells shows significantly larger penetration load compared to the wide type. A pool of pancreatic, lung, colorectal, and breast cells are also characterized. The chemotherapeutic agent, benzyl-α-GalNac, for inhibiting glycosylation is shown to be effective in reducing the mechanical barrier.

Multi drug resistant tuberculosis presenting as anterior mediastinal mass

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Parmarth Chandane

2016-01-01

Full Text Available Enlargement of the mediastinal lymphatic glands is a common presentation of intrathoracic tuberculosis (TB in children. However, usually, the mediastinal TB nodes enlarge to 2.8 ± 1.0 cm. In this report, we describe a case of anterior mediastinal lymphnode TB seen as huge mass (7 cm on computed tomography (CT thorax without respiratory or food pipe compromise despite anterior mediastinum being an enclosed space. CT guided biopsy of the mass cultured Mycobacterium TB complex which was resistant to isoniazide, rifampicin, streptomycin ofloxacin, moxifloxacin, and pyrazinamide. Hence, we report primary multi drug resistant TB presenting as anterior mediastinal mass as a rare case report.

Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

KAUST Repository

Campos, Mônica C.

2017-10-25

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 5–8 million people in Latin America. Although the nitroheterocyclic compound benznidazole has been the front-line drug for several decades, treatment failures are common. Benznidazole is a pro-drug and is bio-activated within the parasite by the mitochondrial nitroreductase TcNTR-1, leading to the generation of reactive metabolites that have trypanocidal activity. To better assess drug action and resistance, we sequenced the genomes of T. cruzi Y strain (35.5 Mb) and three benznidazole-resistant clones derived from a single drug-selected population. This revealed the genome-wide accumulation of mutations in the resistant parasites, in addition to variations in DNA copy-number. We observed mutations in DNA repair genes, linked with increased susceptibility to DNA alkylating and inter-strand cross-linking agents. Stop-codon-generating mutations in TcNTR-1 were associated with cross-resistance to other nitroheterocyclic drugs. Unexpectedly, the clones were also highly resistant to the ergosterol biosynthesis inhibitor posaconazole, a drug proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate that this can result in multi-drug resistance, and indicate that vigilance will be required if benznidazole is used in combination therapy.

In vitro and in vivo analysis of antimicrobial agents alone and in combination against multi-drug resistant Acinetobacter baumannii

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Songzhe eHE

2015-05-01

Full Text Available Objective To investigate the in vitro and in vivo antibacterial activities of tigecycline and other 13 common antimicrobial agents, alone or in combination, against multi-drug resistant Acinetobacter baumannii.MethodsAn in vitro susceptibility test of 101 Acinetobacter baumannii was used to detect minimal inhibitory concentrations (MICs. A mouse lung infection model of multi-drug resistant Acinetobacter baumannii,established by the ultrasonic atomization method, was used to define in vivo antimicrobial activities.Results Multi-drug resistant Acinetobacter baumannii showed high sensitivity to tigecycline (98% inhibition, polymyxin B (78.2% inhibition, and minocycline (74.2% inhibition. However, the use of these antimicrobial agents in combination with other antimicrobial agents produced synergistic or additive effects. In vivo data showed that white blood cell (WBC counts in drug combination groups C (minocycline + amikacin and D (minocycline + rifampicin were significantly higher than in groups A (tigecycline and B (polymyxin B (P < 0.05, after administration of the drugs 24h post-infection. Lung tissue inflammation gradually increased in the model group during the first 24h after ultrasonic atomization infection; vasodilation, congestion with hemorrhage were observed 48h post infection. After three days of anti-infective therapy in groups A, B, C and D, lung tissue inflammation in each group gradually recovered with clear structures. The mortality rates in drug combination groups (groups C and D were much lower than in groups A and B.ConclusionThe combination of minocycline with either rifampicin or amikacin is more effective against multidrug-resistant Acinetobacter baumannii than single-agent tigecycline or polymyxin B. In addition, the mouse lung infection by ultrasonic atomization is a suitable model for drug screening and analysis of infection mechanism.

Evidence for changes in the transcription levels of two putative P-glycoprotein genes in sea lice (Lepeophtheirus salmonis) in response to emamectin benzoate exposure.

Science.gov (United States)

Tribble, Nicholas D; Burka, John F; Kibenge, Frederick S B

2007-05-01

Overexpression of P-glycoproteins (Pgps) is assumed to be a principal mechanism of resistance of nematodes and arthropods to macrocyclic lactones. Quantitative RT-PCR (Q-RT-PCR) was used to demonstrate changes in transcription levels of two putative P-glycoprotein genes, designated here as SL0525 and SL-Pgp1, in sea lice (Lepeophtheirus salmonis) following exposure to emamectin benzoate (EMB). Pre-adult L. salmonis were challenged in an EMB bioassay for 24h and gene expression was studied from lice surviving EMB concentrations of 0, 10, and 30ppb. Gene expression was measured using Q-RT-PCR with elongation factor 1 (eEF1alpha) as an internal reference gene. The results show that both target genes, SL0525 and SL-Pgp1, had significantly increased levels of expression with exposure to 10ppb EMB (p=0.11 and p=0.17, respectively) whereas the group exposed to 30ppb was on the verge of being significant (p=0.053) only in the expression of SL-Pgp1. Gene expression for SL0525 and SL-Pgp1 were increased over five-fold at 10ppb EMB. Therefore, the upregulation of these target genes may offer protection by increasing Pgp expression when lice are exposed to EMB. Our optimized Q-RT-PCR can be used to determine if over-expression of these genes could be the basis for development of resistance in sea lice and thus allow suitable alternative chemotherapeutic options to be assessed.

Technetium-99m sestamibi uptake in human breast carcinoma cell lines displaying glutathione-associated drug-resistance

International Nuclear Information System (INIS)

Kabasakal, L.; Oezker, K.; Hayward, M.; Akansel, G.; Griffith, O.; Isitman, A.T.; Hellman, R.; Collier, D.

1996-01-01

An in vitro study was designed to evaluate the uptake of sestamibi (MIBI) in P-glycoprotein (Pgp) and glutathione-associated (GSH) multidrug-resistant (MDR) cell lines. MIBI uptake was studied in various human breast carcinoma cell lines, i.e. in wild-type (MCF7/wt) cells, in adriamycin-resistant (MCF7/adr) cells which express Pgp and in melphalan-resistant (MCF7/mph) cells with increased levels of GSH. The effects of buthiomine sulphoximine (BSO) and verapamil on MIBI uptake were also studied in the MCF7/mph and MCF7/adr cells respectively. The cells were incubated for 1 h with a dose of 0.1 MBq thallium-201 and technetium-99m MIBI. Both BIBI and 201 Tl uptakes were higher for MCF7/mph cells than for the other cells studied. The mean MIBI uptake in MCF7/adr cells was significantly lower than that in MCF7/wt cells (1.9%±0.5% vs 3.1%.0.6%; P 0.1). This study suggests that the uptake of MIBI is not diminished by glutathione-associated drug resistance and that MIBI uptake in a tumour sample does not necessarly indicate that a cancer is sensitive to drugs. (orig.)

ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal

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Choi Cheol-Hee

2005-10-01

Full Text Available Abstract One of the major problems related with anticancer chemotherapy is resistance against anticancer drugs. The ATP-binding cassette (ABC transporters are a family of transporter proteins that are responsible for drug resistance and a low bioavailability of drugs by pumping a variety of drugs out cells at the expense of ATP hydrolysis. One strategy for reversal of the resistance of tumor cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. In this review, the physiological functions and structures of ABC transporters, and the development of chemosensitizers are described focusing on well-known proteins including P-glycoprotein, multidrug resistance associated protein, and breast cancer resistance protein.

Role of P-glycoprotein on CD69+CD4+ cells in the pathogenesis of proliferative lupus nephritis and non-responsiveness to immunosuppressive therapy.

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Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Tanaka, Yoshiya

2017-01-01

P-glycoprotein (P-gp) expression on activated lymphocytes in systemic lupus erythematosus (SLE) plays a role in active efflux of intracellular drugs, resulting in drug resistance. The role of P-gp-expressing lymphocytes in the pathogenesis of SLE remains unclear. The aim of this study was to determine the importance of P-gp + CD4 + cells in organ manifestations in refractory SLE. The proportion of P-gp + CD4 + cells was determined by flow cytometry in peripheral blood of patients with SLE (n=116) and healthy adults (n=10). Renal biopsy specimens were examined by immunohistochemistry for P-gp expression. CD69 is a marker of CD4 cell activation. The proportion of both P-gp-expressing CD4 + cells and CD69-expressing CD4 + cells in peripheral blood was higher in SLE than control. The proportion of P-gp + CD69 + CD4 + cells correlated with Systemic Lupus Erythematosus Disease Activity Index and was higher in poor responders to corticosteroids. Furthermore, the proportion of P-gp + CD69 + CD4 + cells was significantly higher in proliferative lupus nephritis (LN) with poor response to corticosteroids. The efficacy of immunosuppressive therapy depended on the regulation of the proportion of P-gp + CD69 + CD4 + cells. Marked accumulation of P-gp + CD4 + cells in renal interstitial tissue and high proportion of peripheral P-gp + CD69 + CD4 + cells were noted in patients with proliferative LN. The results showed high proportion of P-gp + CD69 + CD4 + cells in peripheral blood and their accumulation in renal tissue in patients with proliferative LN refractory to CS therapy, suggesting that P-gp expression on activated CD4 + T cells is a potentially useful marker for refractoriness to treatment and a novel target for treatment.

Regional increase in P-glycoprotein function in the blood-brain barrier of patients with chronic schizophrenia : A PET study with [C-11]verapamil as a probe for P-glycoprotein function

NARCIS (Netherlands)

de Klerk, Onno L.; Willemsen, Antoon T. M.; Bosker, Fokko J.; Bartels, Anna L.; Hendrikse, N. Harry; den Boer, Johan A.; Dierckx, Rudy A.

2010-01-01

P-glycoprotein (P-gp), a major efflux pump in the blood-brain barrier (BBB) has a profound effect on entry of drugs, peptides and other substances into the central nervous system (CNS). The brain's permeability can be negatively influenced by modulation of the transport function of P-gp.

Mitochondrial expression and activity of P-glycoprotein under oxidative stress in outer blood-retinal barrier

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Yue-Hong Zhang

2017-07-01

Full Text Available AIM: To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein (P-gp in mitochondria of D407 cells. METHODS: D407 cells were exposed to different ranges of concentrations of H2O2. The mitochondrial location of P-gp in the cells subjected to oxidative stress was detected by confocal analysis. Expression of P-gp in isolated mitochondria was assessed by Western blot. The pump activity of P-gp was evaluated by performing the efflux study on isolated mitochondria with Rhodamine 123 (Rho-123 alone and in the presence of P-gp inhibitor (Tariquidar using flow cytometry analysis. The cells were pretreated with 10 mmol/L N-acetylcysteine (NAC for 30min before exposing to H2O2, and analyzed the mitochondrial extracts by Western blot and flow cytometry. RESULTS: P-gp was co-localized in the mitochondria by confocal laser scanning microscopy, and it was also detected in the mitochondria of D407 cells using Western blot. Exposure to increasing concentrations of H2O2 led to gradually increased expression and location of P-gp in the mitochondria of cells. Rho-123 efflux assay showed higher uptake of Rho-123 on isolated mitochondria in the presence of Tariquidar both in normal and oxidative stress state. H2O2 up-regulated P-gp in D407 cells, which could be reversed by NAC treatment. CONCLUSION: H2O2 could up-regulate the functional expression of P-gp in mitochondria of D407 cells, while antioxidants might suppress oxidative-stress-induced over-expression of functional P-gp. It is indicative that limiting the mitochondrial P-gp transport in retinal pigment epithelium cells would be to improve the effect of mitochondria-targeted antioxidant therapy in age-related macular degeneration-like retinopathy.

Human intestinal P-glycoprotein activity estimated by the model substrate digoxin

DEFF Research Database (Denmark)

Larsen, U L; Hyldahl Olesen, L; Nyvold, Charlotte Guldborg

2007-01-01

P-glycoprotein (Pgp) plays a part in the intestinal uptake of xenobiotics and has been associated with susceptibility to ulcerative colitis. The aim of this study was to examine Pgp activity in relation to age, gender, medical treatment (rifampicin or ketoconazole) and the multidrug resistance (MDR...

Reversal of the multidrug resistance by drug combination using multifunctional liposomes

Science.gov (United States)

Patel, Niravkumar R.

One of the major obstacles to the success of cancer chemotherapy is the multi-drug resistance (MDR) that results due mainly to the over-expression of drug efflux transporter pumps such as P-glycoprotein (P-gp). Highly efficacious third generation P-gp inhibitors, like tariquidar, have shown promising results against MDR. However, P-gp is also expressed in normal tissues like the blood-brain barrier, gastrointestinal tract, liver and kidney. It is therefore important to limit the exposure of P-gp inhibitors to normal tissues and increase their co-localization with anticancer agents in tumor tissues to maximize the efficacy of a P-gp inhibitor. To minimize non-specific binding and increase its delivery to tumor tissues, liposomes, self-assembling phospholipid vesicles, were chosen as a drug delivery vehicle. The liposome has been identified as a system capable of carrying molecules with diverse physicochemical properties. It can also alter the pharmacokinetic profile of loaded molecules which is a concern with both tariquidar and paclitaxel. Liposomes can easily be surface-modified rendering them cell-specific as well as organelle-specific. The main objective of present study was to develop an efficient liposomal delivery system which would deliver therapeutic molecules of interest to tumor tissues and avoid interaction with normal tissues. In this study, the co-delivery of tariquidar and paclitaxel into tumor cells to reverse the MDR using long-circulating cationic liposomes was investigated. SKOV-3TR, the resistant variant of SKOV-3 and MCF-7/ADR, the resistant variant of MCF-7 were used as model cell lines. Uniform liposomal formulations were generated with high incorporation efficiency and no apparent decrease in tariquidar potency towards P-gp. Tariquidar- and paclitaxel- co-loaded long-circulating liposomes showed significant re-sensitization of SKOV-3TR and MCF-7/ADR for paclitaxel in vitro. Further modification of these liposomes with antitumor 2C5 resulted

Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates

DEFF Research Database (Denmark)

Abraham, E H; Shrivastav, B; Salikhova, A Y

2001-01-01

P-glycoprotein is involved with the removal of drugs, most of them cations, from the plasma membrane and cytoplasm. Pgp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central question of this study is whether drug and ATP transport associated...

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein.

Science.gov (United States)

Muñoz-Martínez, Francisco; Reyes, Carolina P; Pérez-Lomas, Antonio L; Jiménez, Ignacio A; Gamarro, Francisco; Castanys, Santiago

2006-01-01

Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.

Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics

Science.gov (United States)

Courtney, Colleen; Chatterjee, Anushree

2014-03-01

``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.

Mechanisms of P-Glycoprotein Modulation by Semen Strychni Combined with Radix Paeoniae Alba

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Li-Li Liu

2017-01-01

Full Text Available Semen Strychni has been extensively used as a Chinese herb, but its therapeutic window is narrowed by the strong toxicity of the compound, which limits its effectiveness. Radix Paeoniae Alba has been reported to reduce the toxic effects and increase the therapeutic effects of Semen Strychni, but the underlying mechanism remains unknown. This research aimed to explore the mechanism through which P-glycoprotein (P-gp is modulated by Semen Strychni combined with Radix Paeoniae Alba in vitro. An MTT assay was used to study cytotoxicity in an MDCK-MDR1 cell model. Rh123 efflux and accumulation were measured to assess P-gp function. The expression levels of MDR1 mRNA and P-gp protein in MDCK-MDR1 cells were investigated. A P-gp ATPase activity assay kit was applied to detect the effect on P-gp ATPase activity. Semen Strychni combined with Radix Paeoniae Alba could induce P-gp-mediated drug transport by inhibiting brucine and strychnine transport in MDCK-MDR1 cells, enhancing the P-gp efflux function, upregulating the P-gp expression and MDR1 mRNA levels, and stimulating P-gp ATPase activity.

ABCB1 (MDR1) polymorphisms and ovarian cancer progression and survival

DEFF Research Database (Denmark)

Johnatty, Sharon E; Beesley, Jonathan; Gao, Bo

2013-01-01

ABCB1 encodes the multi-drug efflux pump P-glycoprotein (P-gp) and has been implicated in multi-drug resistance. We comprehensively evaluated this gene and flanking regions for an association with clinical outcome in epithelial ovarian cancer (EOC)....

Extensively and Pre-Extensively Drug Resistant Tuberculosis in Clinical Isolates of Multi-Drug Resistant Tuberculosis Using Classical Second Line Drugs (Levofloxacin and Amikacin)

International Nuclear Information System (INIS)

Mirza, I. A.; Khan, F. A.; Khan, K. A.; Satti, L.; Ghafoor, T.; Fayyaz, M.

2015-01-01

Objective:To find out the frequency of Extensively Drug Resistant (XDR) and pre-XDR tuberculosis in clinical isolates of Multi-Drug Resistant (MDR) Tuberculosis (TB) by determining the susceptibilities against Levofloxacin and Amikacin (classical second line antituberculosis drugs). Study Design: A descriptive cross-sectional study. Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2011 to August 2013. Methodology: Amikacin (AK) and Levofloxacin (LEVO) were obtained in chemically pure form from Sigma (Taufkirchen, Germany). The breakpoint concentration used for AK was 1.0 micro g/ml and for LEVO 2.0 micro g/ml. Mycobacterial Growth Indicator Tube (MGIT) 960 system was used to carry out drug susceptibility testing as per recommended protocol. Results: A total of 3 MDR-TB isolates (3 percentage) turned out to be XDR-TB based upon simultaneous resistance to injectable second line antituberculosis drug AK and one of the fluoro-quinolones (LEVO). A total of 24 MDR-TB isolates (24 percentage) were found to be pre-XDR based upon resistance to LEVO alone. Treatment status record of patients with XDR and pre-XDRTB isolates revealed that majority of patients had received fluoroquinolones (FQs) during the course of treatment. Conclusion: XDR-TB has started to emerge in MDR-TB isolates in our set up. The worrying sign is the high frequency of pre-XDR tuberculosis. Urgent steps need to be taken to stem the tide of pre-XDR-TB in our population. It is thus recommended to develop facilities to carry out drug susceptibility testing to monitor the status of pre-XDR and XDR-TB in our population. (author)

[Expression and significance of P-gp/mdr1 mRNA, MRP and LRP in non-Hodgkin's lymphoma].

Science.gov (United States)

Li, Le; Su, Li-ping; Ma, Li; Zhao, Jin; Zhu, Lei; Zhou, Yong-an

2009-03-01

To explore the expression and clinical significance of P-glycoprotein (P-gp)/mdr1mRNA, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in newly diagnosed non-Hodgkin's lymphoma. mdr1 mRNA of in 41 patients with non-Hodgkin's lymphoma was assayed by semi-quantitative RT-PCR. The expressions of P-gp, MRP and LRP proteins in lymph node viable blasts were identified by flow cytometry. The results were compared with those obtained from control cases, and the correlation of the changes with clinical outcomes was analyzed. (1) Among the 41 cases, the positive expression of P-gp protein was detected in 8 cases, MRP in 7 cases, LRP in 15 cases, and mdr 1 mRNA in 11 cases. (2) The P-gp and LRP levels in NHL were significantly higher than those in control group, but MRP wasn't. The P-gp over-expression was significantly associated with mdr1mRNA (r = 0.396, P = 0.01). No correlation was showed among the expressions of P-gp, MRP and LRP. (3) Patients with P-gp expression had a poorer outcome of chemotherapy than those with P-gp-negative (P = 0.005). P-gp expression was significantly associated with higher clinical stage (P = 0.046) and elevated serum lactate dehydrogenase level (P = 0.032), but not associated with malignant degree (P = 0.298). MRP had no impact on the outcome of chemotherapy (P = 0.212), and wasn't significantly associated with higher clinical stage (P = 0.369), elevated LDH (P = 0.762) and higher malignant degree (P = 0.451). Patients with LRP expression had a poorer outcome of chemotherapy than those LRP-negative (P = 0.012). LRP expression was significantly associated with higher clinical stage (P = 0.0019), elevated LDH (P = 0.02) and higher malignant degree (P = 0.01). The data of this study indicate that P-gp and LRP expressions but not MRP expression are important in the mechanism of drug resistance associated with a poor clinical outcome in previously untreated NHL.

Advantage and limitations of nitrofurantoin in multi-drug resistant Indian scenario

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Laishram Shakti

2015-01-01

Full Text Available Infections caused by antibiotic resistant pathogens are of significant concern and are associated with higher mortality and morbidity. Nitrofurantoin is a broad-spectrum bactericidal antibiotic and is effectively used to treat urinary tract infections (UTIs caused by E. coli, Klebsiella sp., Enterobacter sp., Enterococcus sp. and Staphylococcus aureus. It interfere with the synthesis of cell wall, bacterial proteins and DNA of both Gram positive and Gram negative pathogens. Nitrofurantoin has been used successfully for treatment and prophylaxis of acute lower urinary tract infections. With the emergence of antibiotic resistance, nitrofurantoin has become the choice of agent for treating UTIs caused by multi-drug resistant pathogens.

3-Aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione for circumvention of anticancer drug resistance.

Science.gov (United States)

Shchekotikhin, Andrey E; Shtil, Alexander A; Luzikov, Yuri N; Bobrysheva, Tatyana V; Buyanov, Vladimir N; Preobrazhenskaya, Maria N

2005-03-15

A series of 3-aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione was synthesized by Mannich reaction or by the transamination of 3-dimethylaminomethyl 4,11-dihydroxy- or 4,11-dimethoxynaphtho[2,3-f]indole-5,10-dione. The potency of novel derivatives was tested on a National Cancer Institute panel of 60 human tumor cell lines as well as in cells with genetically defined determinants of cytotoxic drug resistance, P-glycoprotein (Pgp) expression, and p53 inactivation. Mannich derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione with an additional amino function in their side chain, demonstrated equal cytotoxicity against the parental K562 leukemia cells and their Pgp-positive subline, whereas the latter showed approximately 7-fold resistance to adriamycin, a Pgp transported drug. 3-(1-Piperazinyl)methyl and 3-(quinuclidin-3-yl)aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione killed HCT116 colon carcinoma cells (carrying wild type p53) and their p53-null variant within the similar range of concentrations. We conclude that Mannich modification of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione, especially when cyclic diamine (e.g., piperazine, quinuclidine) is used, confers an important feature to the resulting compounds, namely, the potency for tumor cells otherwise resistant to a variety of anticancer drugs.

Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity

DEFF Research Database (Denmark)

Horvat, B; Multhaupt, H A; Damjanov, I

1993-01-01

We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had...... in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate...... the complexity of glycoconjugate synthesis in mouse vagina and reveal the distinct cycle-specific patterns of individual glycoprotein expression. These cyclic glycoproteins could serve as vaginal biochemical markers for the specific phases of the estrous cycle....

Usefulness of technetium-99m tetrofosmin liver imaging to detect hepatocellular carcinoma and related to expression of P-glycoprotein or multidrug resistance associated protein-a preliminary report

International Nuclear Information System (INIS)

Ding, H.J.; Huang, W.T.; Tsai, C.S.; Chang, C.S.; Kao, A.

2003-01-01

Technetium-99m Tetrofsomin (Tc-TF) has been shown to be useful in identifying several types of tumors, such as breast, lung, and thyroid cancers. There was no report in the literature for Tc-TF uptake in hepatocellular carcinoma (HCC). The aim of this study was to evaluate the usefulness of Tc-TF liver imaging to detect HCC and investigate the relationship between Tc-TF liver imaging findings and P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP) expression. Before any therapy, 22 patients with HCC were enrolled in this study. Tc-TF liver images were performed l0 minutes after intravenous injection of 20mCi Tc-TF. All patients had liver biopsy or surgery within l week after Tc-TF liver imaging. Immunohistochemical study of the biopsy or resected HCC specimens was performed using anti-human Pgp and MRP antibodies. Twenty of the 22 (90.9%) patients showed negative Tc-TF liver imaging results without significant Tc-TF uptake in HCC, whereas only the remaining 2 (9.1%) patients showed positive Tc-TF liver imaging results with significant Tc-TF uptake in HCC. Positive Pgp expression was observed in 13 of 20 patients with negative Tc-TF liver imaging results, whereas positive MRP expression was observed in 6 of the remaining 7 patients with negative both Tc-TF liver imaging results and Pgp expression. However, negative Pgp expression but positive MRP expression was observed in all of the remaining 2 patients with positive Tc-TF liver imaging results. The correlation between Tc-TF liver imaging findings and Pgp expression was significant and better than between Tc-TF liver imaging findings and MRP expression. Pgp or MRP expression in HCC may induce no significant Tc-TF uptake in HCC resulting in negative Tc-TF liver imaging findings. Therefore, Tc-TF liver imaging is potential to be a non-invasive method to predict Pgp or MRP expression in HCC. However, further studies with a larger series of patients and longer follow-up time are necessary to confirm

The identification of new genes related to cisplatin resistance in ovarian adenocarcinoma cell line A2780

International Nuclear Information System (INIS)

Solar, P.; Fedorocko, P.; Sytkowski, A.; Hodorova, I.

2006-01-01

Ovarian cancer cells are usually sensitive to platinum-based chemotherapy, such as cisplatin (CDDP), initially but typically become resistant to the drug over time. The phenomenon of clinical drug resistance represents a serious problem for successful disease treatment, and the molecular mechanism(s) are not fully understood. In search of novel mechanisms that may lead to the development of CDDP chemoresistance we have applied subtractive hybridization based on the PCR-select cDNA subtraction. In current study we have used subtractive hybridization to identify differentially-expressed genes in CDDP resistant CP70 and C200 cells versus CDDP-sensitive A2780 human ovarian adenocarcinoma cells. We have analyzed 256 randomly selected clones. Subtraction efficiency was determined by dot blot and DNA sequencing. Confirmation of differentially expressed cDNAs was done by virtual northern blot analysis, and 17 genes that were differentially expressed in both CDDP resistant cell lines versus CDDP sensitive A2780 cells were identified. The expression of 10 of these genes was undetectable or detected with low expression in sensitive A2780 cells in comparison to resistant ones. These genes included ARHGDIB, RANBP2, ASPH, PRTFDC1, SSX2IP, MBNL1, DNAJC15, MMP10, TCTE1L and one unidentified sequence. Additional 7 genes that were more highly expressed in resistant CP70 and C200 vs. A2780 cells included ANXA2, USP8, HSPCA, TRA1, CNAP1, ATP2B1 and COX2. Interestingly, multi-drug resistance associated p-glycoprotein (p170) was not detected by the western blot in CDDP resistant CP70 and C200 cells. Our identified genes are involved in diverse processes, such as stress response, chromatin condensation, protection from protein degradation, invasiveness of cells, alterations of Ca 2+ homeostasis and others which may contribute to CDDP resistance of ovarian adenocarcinoma cells. Further characterization of these genes and gene products should yield important insights into the biology of

Evaluation of P-glycoprotein expression in pain relevant tissues: understanding translation of efflux from preclinical species to human

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Renu Singh Dhanikula

2016-10-01

Full Text Available Various efflux transporters, such as P-glycoprotein (P-gp are now widely accepted to have profound influence on the disposition of substrates. Nevertheless, there is paucity of information about their expression and functionality in the pain relevant tissues (such as brain, spinal cord and dorsal root ganglia (DRG across various species. Therefore, our attempts were directed at evaluating P-gp expression in these tissues to understand its effect on the central nervous system (CNS disposition. As a means of characterizing the normal tissue distribution of P-gp, immunohistochemistry was performed with two antibodies (C219 and H241 directed against different epitopes of MDR1 gene. Notable expression of P-gp was detected in the DRG of Sprague Dawley rat, Beagle Dog, Cynomolgous monkey as well as human. The expression of P-gp was observed in the CNS tissues with evident species differences, the expression of P-gp in human brain and spinal cord was lower than in rats and dogs but relatively comparable to that in monkeys. However, no species related differences were seen in the expression at the DRG level. Double-labelling using an antibody against a marker of endothelial cells confirmed that P-gp was exclusively localized in capillary endothelial cells. This study highlights the cross species similarities and differences in the expression of P-gp and thus serves as a vital step in understanding the translation of exposure of P-gp substrates to human.

Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

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Filipski, Elisabeth; Berland, Elodie [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Ozturk, Narin [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Istanbul University Faculty of Pharmacy, Department of Pharmacology, Beyazit TR-34116, Istanbul (Turkey); Guettier, Catherine [Assistance Publique-Hôpitaux de Paris, Unité de Chronothérapie, Département de Cancérologie, Hôpital Paul Brousse, Villejuif F-94807 (France); Horst, Gijsbertus T.J. van der [Department of Genetics, Erasmus University Medical Center, 3000 CA Rotterdam (Netherlands); Lévi, Francis [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Assistance Publique-Hôpitaux de Paris, Unité de Chronothérapie, Département de Cancérologie, Hôpital Paul Brousse, Villejuif F-94807 (France); and others

2014-02-01

The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F{sub 1} mice. A three-fold 24 h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p < 0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50 mg/kg/day i.v. × 4 days) as a single agent or combined with P-gp inhibitor PSC833 (6.25 mg/kg/day i.p. × 4 days) at ZT3 or ZT15, respectively corresponding to the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40 mg/kg/day × 4 days) and +/− PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p < 0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p < 0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ∼ 60%. In tumor-bearing mice, body weight loss was ∼ halved in the mice on irinotecan or irinotecan–PSC833 combination at ZT15 as compared to ZT3 (p < 0.001). PSC833–irinotecan at ZT15 increased tumor inhibition by ∼ 40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan. - Highlights: • Irinotecan chronotolerance and chronoefficacy change as drug was applied with PSC833. • P-glycoprotein is an important player of the toxicity and efficacy of irinotecan. • Timing should be considered if chemotherapy is performed with a MDR1 inhibitor.

Tumor p-glycoprotein correlates with efficacy of PF-3758309 in in vitro and in vivo models of colorectal cancer.

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Erica Lynn Bradshaw-Pierce

2013-03-01

Full Text Available P-glycoprotein (P-gp, a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309, a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB1 (P-gp expression was evaluated by microarray for these cell lines. P-gp expression was determined by western blot and activity determined by rhodamine efflux assay. Knock down of P-gp and pharmacologic inhibition of P-gp to restore PF-309 activity was performed in vitro. PF-309 activity was evaluated in vivo in cell line xenograft models and in primary patient derived tumor xenografts (PDTX. Mice were treated with 25 mg/kg PF-309 orally, twice daily. On the last day of treatment, tumor and plasma were collected for PF-309 analysis. Here we show that ABCB1 gene expression correlates with resistance to PF-309 treatment in vitro and the expression and activity of P-gp was verified in a panel of resistant cells. Furthermore, inhibition of P-gp increased the sensitivity of resistant cells, resulting in a 4-100 fold decrease in the IC50s. Eleven cell line xenografts and 12 PDTX models were treated with PF-309. From the cell line xenografts, we found a significant correlation between ABCB1 gene expression profiles and tumor response. We evaluated tumor and plasma concentrations for 8 tumor models (3 cell line xenografts and 5 PDTX models and a significant correlation was found between tumor concentration and response. Additionally, we show that tumor concentration is approximately 4-fold lower in tumors that express P-gp, verified by western blot. Our in vitro and in vivo data strongly suggests that PF-309 efficacy is influenced by the expression of tumor P-gp.

Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

International Nuclear Information System (INIS)

Fuchs, Dominik; Daniel, Volker; Sadeghi, Mahmoud; Opelz, Gerhard; Naujokat, Cord

2010-01-01

Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.

Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

Science.gov (United States)

Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

2016-05-01

Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

Glycoprotein expression by adenomatous polyps of the colon

Science.gov (United States)

Roney, Celeste A.; Xie, Jianwu; Xu, Biying; Jabour, Paul; Griffiths, Gary; Summers, Ronald M.

2008-03-01

Colon cancer is the second leading cause of cancer related deaths in the United States. Specificity in diagnostic imaging for detecting colorectal adenomas, which have a propensity towards malignancy, is desired. Adenomatous polyp specimens of the colon were obtained from the mouse model of colorectal cancer called adenomatous polyposis coli-multiple intestinal neoplasia (APC Min). Histological evaluation, by the legume protein Ulex europaeus agglutinin I (UEA-1), determined expression of the glycoprotein α-L-fucose. FITC-labelled UEA-1 confirmed overexpression of the glycoprotein by the polyps on fluorescence microscopy in 17/17 cases, of which 13/17 included paraffin-fixed mouse polyp specimens. In addition, FITC-UEA-1 ex vivo multispectral optical imaging of 4/17 colonic specimens displayed over-expression of the glycoprotein by the polyps, as compared to non-neoplastic mucosa. Here, we report the surface expression of α-L-fucosyl terminal residues by neoplastic mucosal cells of APC specimens of the mouse. Glycoprotein expression was validated by the carbohydrate binding protein UEA-1. Future applications of this method are the development of agents used to diagnose cancers by biomedical imaging modalities, including computed tomographic colonography (CTC). UEA-1 targeting to colonic adenomas may provide a new avenue for the diagnosis of colorectal carcinoma by CT imaging.

SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

Science.gov (United States)

Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

2009-08-01

S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

Molecular approaches for detection of the multi-drug resistant tuberculosis (MDR-TB in Bangladesh.

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Tafsina Haque Aurin

Full Text Available The principal obstacles in the treatment of tuberculosis (TB are delayed and inaccurate diagnosis which often leads to the onset of the drug resistant TB cases. To avail the appropriate treatment of the patients and to hinder the transmission of drug-resistant TB, accurate and rapid detection of resistant isolates is critical. Present study was designed to demonstrate the efficacy of molecular techniques inclusive of line probe assay (LPA and GeneXpert MTB/RIF methods for the detection of multi-drug resistant (MDR TB. Sputum samples from 300 different categories of treated and new TB cases were tested for the detection of possible mutation in the resistance specific genes (rpoB, inhA and katG through Genotype MTBDRplus assay or LPA and GeneXpert MTB/RIF tests. Culture based conventional drug susceptibility test (DST was also carried out to measure the efficacy of the molecular methods employed. Among 300 samples, 191 (63.7% and 193 (64.3% cases were found to be resistant against rifampicin in LPA and GeneXpert methods, respectively; while 189 (63% cases of rifampicin resistance were detected by conventional DST methods. On the other hand, 196 (65.3% and 191 (63.7% isolates showed isoniazid resistance as detected by LPA and conventional drug susceptibility test (DST, respectively. Among the drug resistant isolates (collectively 198 in LPA and 193 in conventional DST, 189 (95.6% and 187 (96.9% were considered to be MDR as examined by LPA and conventional DST, respectively. Category-II and -IV patients encountered higher frequency of drug resistance compared to those from category-I and new cases. Considering the higher sensitivity, specificity and accuracy along with the required time to results significantly shorter, our study supports the adoption of LPA and GeneXpert assay as efficient tools in detecting drug resistant TB in Bangladesh.

(11) C- and (18) F-Labeled Radioligands for P-Glycoprotein Imaging by Positron Emission Tomography.

NARCIS (Netherlands)

Cantore, Mariangela; Benadiba, Marcel; Elsinga, Philippus; Kwizera, Chantal; Dierckx, Rudi; Colabufo, Nicola A.; Luurtsema, Geert

2016-01-01

P-Glycoprotein (P-gp) is an efflux transporter widely expressed at the human blood-brain barrier. It is involved in xenobiotics efflux and in onset and progression of neurodegenerative disorders. For these reasons, there is great interest in the assessment of P-gp expression and function by

A PROSPECTIVE, OBSERVATIONAL STUDY OF ADVERSE REACTIONS TO DRUG REGIME FOR MULTI-DRUG RESISTANT PULMONARY TUBERCULOSIS IN CENTRAL INDIA.

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Dr. Rohan C. Hire

2014-09-01

Full Text Available Abstract Objective: 1 To assess the adverse drug reactions of second line anti-tubercular drugs used to treat Multi-drug resistant Tuberculosis (MDR TB in central India on the basis of causality, severity and avoidability scales. 2 To study the relationship of type of MDR TB (primary or secondary and presence of diabetes mellitus (DM with mean smear conversion time. Material and Methods: A prospective, observational study was carried out on diagnosed multidrug resistant tuberculosis patients enrolled for DOTS‑Plus regimen at TB and Chest Disease Department from January to December 2012. They were followed for 9 months thereafter and encountered adverse drug reactions (ADRs were noted along with the time of sputum conversion. The data were analysed by Chi-square or Fisher’s exact test and unpaired student’s‘t’ test. Results: Total 64 ADRs were reported in 55 patients out of total 110 patients (n = 110. As per the Naranjo causality assessment of ADRs, 7 patients had “definite” causal relation, 45 had “probable” causal relation and 3 had “possible” causal relation with drugs of DOTS Plus regime. As per the Hartwig’s severity assessment scale, there were total 7 ADRs in Level 1, 6 in Level 2, 33 in Level 3 and 9 in Level 4. Hallas avoidability assessment scale divided the ADRs as 3 being “Definitely avoidable”, 26 “Possibly avoidable”, 23 “Not avoidable” and 3 “unevaluable”. . Mean sputum smear conversion time is significantly higher in patients with secondary type than that of primary type of MDR TB (p = 0.0001 and in patients with DM than those without DM (p <0.0001. Conclusion: ADRs were common in patients of MDR TB on DOTs-Plus drug regime. It was due to lack of availability of safer and equally potent drugs in DOTs-Plus drug regime compared to DOTS regime in non-resistant TB. The frequency and severity of ADRs can be reduced by strict vigilance about known and unknown ADRs, monitoring their laboratory and

Antimicrobial resistance determinant microarray for analysis of multi-drug resistant isolates

Science.gov (United States)

Taitt, Chris Rowe; Leski, Tomasz; Stenger, David; Vora, Gary J.; House, Brent; Nicklasson, Matilda; Pimentel, Guillermo; Zurawski, Daniel V.; Kirkup, Benjamin C.; Craft, David; Waterman, Paige E.; Lesho, Emil P.; Bangurae, Umaru; Ansumana, Rashid

2012-06-01

The prevalence of multidrug-resistant infections in personnel wounded in Iraq and Afghanistan has made it challenging for physicians to choose effective therapeutics in a timely fashion. To address the challenge of identifying the potential for drug resistance, we have developed the Antimicrobial Resistance Determinant Microarray (ARDM) to provide DNAbased analysis for over 250 resistance genes covering 12 classes of antibiotics. Over 70 drug-resistant bacteria from different geographic regions have been analyzed on ARDM, with significant differences in patterns of resistance identified: genes for resistance to sulfonamides, trimethoprim, chloramphenicol, rifampin, and macrolide-lincosamidesulfonamide drugs were more frequently identified in isolates from sources in Iraq/Afghanistan. Of particular concern was the presence of genes responsible for resistance to many of the last-resort antibiotics used to treat war traumaassociated infections.

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells.

Science.gov (United States)

Budworth, J; Gant, T W; Gescher, A

1997-01-01

The aim of this study was to investigate the link between protein kinase C (PKC) and multidrug resistance (mdr) phenotype. The expression of both was studied in doxorubicin-resistant MCF-7/Adr cells as they reverted to the wild-type phenotype when cultured in the absence of drug. The following parameters were measured in cells 4, 10, 15, 20 and 24 weeks after removal of doxorubicin; (1) sensitivity of the cells towards doxorubicin; (2) levels of P-glycoprotein (P-gp) and MDR1 mRNA; (3) levels and cellular localization of PKC isoenzyme proteins alpha, theta and epsilon; and (4) gene copy number of PKC-alpha and MDR1 genes. Cells lost their resistance gradually with time, so that by week 24 they had almost completely regained the drug sensitivity seen in wild-type MCF-7 cells. P-gp levels measured by Western blot mirrored the change in doxorubicin sensitivity. By week 20, P-gp had decreased to 18% of P-gp protein levels at the outset, and P-gp was not detectable at week 24. Similarly, MDR1 mRNA levels had disappeared by week 24. MCF-7/Adr cells expressed more PKCs-alpha and -theta than wild-type cells and possessed a different cellular localization of PKC-epsilon. The expression and distribution pattern of these PKCs did not change for up to 20 weeks, but reverted back to that seen in wild-type cells by week 24. MDR1 gene amplification remained unchanged until week 20, but then was lost precipitously between weeks 20 and 24. The PKC-alpha gene was not amplified in MCF-7/Adr cells. The results suggest that MCF-7/Adr cells lose MDR1 gene expression and PKC activity in a co-ordinate fashion, consistent with the existence of a mechanistic link between MDR1 and certain PKC isoenzymes.

The Alterations in the Expression and Function of P-Glycoprotein in Vitamin A-Deficient Rats as well as the Effect of Drug Disposition in Vivo

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Yubang Wang

2015-12-01

Full Text Available This study was aimed to investigate whether vitamin A deficiency could alter P-GP expression and function in tissues of rats and whether such effects affected the drug distribution in vivo of vitamin A-deficient rats. We induced vitamin A-deficient rats by giving them a vitamin A-free diet for 12 weeks. Then, Abcb1/P-GP expression was evaluated by qRT-PCR and Western blot. qRT-PCR analysis revealed that Abcb1a mRNA levels were increased in hippocampus and liver. In kidney, it only showed an upward trend. Abcb1b mRNA levels were increased in hippocampus, but decreased in cerebral cortex, liver and kidney. Western blot results were in good accordance with the alterations of Abcb1b mRNA levels. P-GP function was investigated through tissue distribution and body fluid excretion of rhodamine 123 (Rho123, and the results proclaimed that P-GP activities were also in good accordance with P-GP expression in cerebral cortex, liver and kidney. The change of drug distribution was also investigated through the tissue distribution of vincristine, and the results showed a significantly upward trend in all indicated tissues of vitamin A-deficient rats. In conclusion, vitamin A deficiency may alter Abcb1/P-GP expression and function in rat tissues, and the alterations may increase drug activity/toxicity through the increase of tissue accumulation.

Immunostimulation of Salmo salar L., and its effect on Lepeophtheirus salmonis (Krøyer) P-glycoprotein mRNA expression following subsequent emamectin benzoate exposure.

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Igboeli, O O; Purcell, S L; Wotton, H; Poley, J; Burka, J F; Fast, M D

2013-03-01

Control of sea lice, Lepeophtheirus salmonis, on farmed Atlantic salmon, Salmo salar, relies heavily on chemotherapeutants. However, reduced efficacy of many treatments and need for integrated sea lice management plans require innovative strategies. Resistance to emamectin benzoate (EMB), a major sea lice parasiticide, has been linked with P-glycoprotein (P-gp) expression. We hypothesized that host immunostimulation would complement EMB treatment outcome. Lepeophtheirus salmonis-infected Atlantic salmon were fed immunostimulatory or control feeds. Sea lice were collected for 24-h EMB bioassays 1 and 2 weeks prior to commencement of EMB treatment of the fish. Two weeks after cessation of immunostimulant-treated feed, EMB was administered at 150 μg kg(-1) fish biomass for 7 days. The bioassay revealed stage, gender and immunostimulant-related differences in EMB EC(50) . Sea lice attached to salmon with a history of immunostimulation exhibited significantly greater survival than those on control feeds, despite similar levels of EMB in host tissues. Lepeophtheirus salmonis from salmon with a history of immunostimulation also exhibited higher P-gp mRNA expression as well as greater survivability compared to controls. Administration of immunostimulants prior to EMB treatment caused increased expression of P-gp mRNA which could have consequently caused decreased efficacy of the parasiticide. © 2013 Blackwell Publishing Ltd.

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents.

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Michaelis, M; Rothweiler, F; Agha, B; Barth, S; Voges, Y; Löschmann, N; von Deimling, A; Breitling, R; Doerr, H Wilhelm; Rödel, F; Speidel, D; Cinatl, J

2012-04-05

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3(r)RITA(10 μM) to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.

Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance

International Nuclear Information System (INIS)

Hembruff, Stacey L; Laberge, Monique L; Villeneuve, David J; Guo, Baoqing; Veitch, Zachary; Cecchetto, Melanie; Parissenti, Amadeo M

2008-01-01

Anthracyclines and taxanes are commonly used in the treatment of breast cancer. However, tumor resistance to these drugs often develops, possibly due to overexpression of drug transporters. It remains unclear whether drug resistance in vitro occurs at clinically relevant doses of chemotherapy drugs and whether both the onset and magnitude of drug resistance can be temporally and causally correlated with the enhanced expression and activity of specific drug transporters. To address these issues, MCF-7 cells were selected for survival in increasing concentrations of doxorubicin (MCF-7 DOX-2 ), epirubicin (MCF-7 EPI ), paclitaxel (MCF-7 TAX-2 ), or docetaxel (MCF-7 TXT ). During selection cells were assessed for drug sensitivity, drug uptake, and the expression of various drug transporters. In all cases, resistance was only achieved when selection reached a specific threshold dose, which was well within the clinical range. A reduction in drug uptake was temporally correlated with the acquisition of drug resistance for all cell lines, but further increases in drug resistance at doses above threshold were unrelated to changes in cellular drug uptake. Elevated expression of one or more drug transporters was seen at or above the threshold dose, but the identity, number, and temporal pattern of drug transporter induction varied with the drug used as selection agent. The pan drug transporter inhibitor cyclosporin A was able to partially or completely restore drug accumulation in the drug-resistant cell lines, but had only partial to no effect on drug sensitivity. The inability of cyclosporin A to restore drug sensitivity suggests the presence of additional mechanisms of drug resistance. This study indicates that drug resistance is achieved in breast tumour cells only upon exposure to concentrations of drug at or above a specific selection dose. While changes in drug accumulation and the expression of drug transporters does occur at the threshold dose, the magnitude of

Multi-drug resistant Acinetobacter infections in critically injured Canadian forces soldiers

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Brisebois Ronald

2007-08-01

Full Text Available Abstract Background Military members, injured in Afghanistan or Iraq, have returned home with multi-drug resistant Acinetobacter baumannii infections. The source of these infections is unknown. Methods Retrospective study of all Canadian soldiers who were injured in Afghanistan and who required mechanical ventilation from January 1 2006 to September 1 2006. Patients who developed A. baumannii ventilator associated pneumonia (VAP were identified. All A. baumannii isolates were retrieved for study patients and compared with A. baumannii isolates from environmental sources from the Kandahar military hospital using pulsed-field gel electrophoresis (PFGE. Results During the study period, six Canadian Forces (CF soldiers were injured in Afghanistan, required mechanical ventilation and were repatriated to Canadian hospitals. Four of these patients developed A. baumannii VAP. A. baumannii was also isolated from one environmental source in Kandahar – a ventilator air intake filter. Patient isolates were genetically indistinguishable from each other and from the isolates cultured from the ventilator filter. These isolates were resistant to numerous classes of antimicrobials including the carbapenems. Conclusion These results suggest that the source of A. baumannii infection for these four patients was an environmental source in the military field hospital in Kandahar. A causal linkage, however, was not established with the ventilator. This study suggests that infection control efforts and further research should be focused on the military field hospital environment to prevent further multi-drug resistant A. baumannii infections in injured soldiers.

Complete genome sequence of Acinetobacter baumannii XH386 (ST208, a multi-drug resistant bacteria isolated from pediatric hospital in China

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Youhong Fang

2016-03-01

Full Text Available Acinetobacter baumannii is an important bacterium that emerged as a significant nosocomial pathogen worldwide. The rise of A. baumannii was due to its multi-drug resistance (MDR, while it was difficult to treat multi-drug resistant A. baumannii with antibiotics, especially in pediatric patients for the therapeutic options with antibiotics were quite limited in pediatric patients. A. baumannii ST208 was identified as predominant sequence type of carbapenem resistant A. baumannii in the United States and China. As we knew, there was no complete genome sequence reproted for A. baumannii ST208, although several whole genome shotgun sequences had been reported. Here, we sequenced the 4087-kilobase (kb chromosome and 112-kb plasmid of A. baumannii XH386 (ST208, which was isolated from a pediatric hospital in China. The genome of A. baumannii XH386 contained 3968 protein-coding genes and 94 RNA-only encoding genes. Genomic analysis and Minimum inhibitory concentration assay showed that A. baumannii XH386 was multi-drug resistant strain, which showed resistance to most of antibiotics, except for tigecycline. The data may be accessed via the GenBank accession number CP010779 and CP010780. Keywords: Acinetobacter baumannii, Multi-drug resistance, Paediatric

Resistance to minor groove binders.

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Colmegna, Benedetta; Uboldi, Sarah; Erba, Eugenio; D'Incalci, Maurizio

2014-03-01

In this paper multiple resistance mechanisms to minor groove binders (MGBs) are overviewed. MGBs with antitumor properties are natural products or their derivatives and, as expected, they are all substrates of P-glycoprotein (P-gp). However, a moderate expression of P-gp does not appear to reduce the sensitivity to trabectedin, the only MGB so far approved for clinical use. Resistance to this drug is often related to transcriptional mechanisms and to DNA repair pathways, particularly defects in transcription-coupled nucleotide excision repair (TC-NER). Therefore tumors resistant to trabectedin may become hypersensitive to UV rays and other DNA damaging agents acting in the major groove, such as Platinum (Pt) complexes. If this is confirmed in clinic, that will provide the rationale to combine trabectedin sequentially with Pt derivates.

Machine learning-, rule- and pharmacophore-based classification on the inhibition of P-glycoprotein and NorA.

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Ngo, T-D; Tran, T-D; Le, M-T; Thai, K-M

2016-09-01

The efflux pumps P-glycoprotein (P-gp) in humans and NorA in Staphylococcus aureus are of great interest for medicinal chemists because of their important roles in multidrug resistance (MDR). The high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of these transmembrane proteins lead us to combining ligand-based approaches, which in the case of this study were machine learning, perceptual mapping and pharmacophore modelling. For P-gp inhibitory activity, individual models were developed using different machine learning algorithms and subsequently combined into an ensemble model which showed a good discrimination between inhibitors and noninhibitors (acctrain-diverse = 84%; accinternal-test = 92% and accexternal-test = 100%). For ligand promiscuity between P-gp and NorA, perceptual maps and pharmacophore models were generated for the detection of rules and features. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening in an attempt to restore drug sensitivity in cancer cells and bacteria.

Drug-resistance patterns of Mycobacterium tuberculosis strains and associated risk factors among multi drug-resistant tuberculosis suspected patients from Ethiopia.

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Mesfin, Eyob Abera; Beyene, Dereje; Tesfaye, Abreham; Admasu, Addisu; Addise, Desalegn; Amare, Miskir; Dagne, Biniyam; Yaregal, Zelalem; Tesfaye, Ephrem; Tessema, Belay

2018-01-01

Multidrug drug-resistant tuberculosis (MDR-TB) is a major health problem and seriously threatens TB control and prevention efforts globally. Ethiopia is among the 30th highest TB burden countries for MDR-TB with 14% prevalence among previously treated cases. The focus of this study was on determining drug resistance patterns of Mycobacterium tuberculosis among MDR-TB suspected cases and associated risk factors. A cross-sectional study was conducted in Addis Ababa from June 2015 to December 2016. Sputum samples and socio-demographic data were collected from 358 MDR-TB suspected cases. Samples were analyzed using Ziehl-Neelsen technique, GeneXpert MTB/RIF assay, and culture using Lowenstein-Jensen and Mycobacterial growth indicator tube. Data were analyzed using SPSS version 23. A total of 226 the study participants were culture positive for Mycobacterium tuberculosis, among them, 133 (58.8%) participants were males. Moreover, 162 (71.7%) had been previously treated for tuberculosis, while 128 (56.6%) were TB/HIV co-infected. A majority [122 (54%)] of the isolates were resistant to any first-line anti-TB drugs. Among the resistant isolates, 110 (48.7%) were determined to be resistant to isoniazid, 94 (41.6%) to streptomycin, 89 (39.4%) to rifampicin, 72 (31.9%) to ethambutol, and 70 (30.9%) to pyrazinamide. The prevalence of MDR-TB was 89 (39.4%), of which 52/89 (58.4%) isolates were resistance to all five first-line drugs. Risk factors such as TB/HIV co-infection (AOR = 5.59, p = 0.00), cigarette smoking (AOR = 3.52, p = 0.045), alcohol drinking (AOR = 5.14, p = 0.001) hospital admission (AOR = 3.49, p = 0.005) and visiting (AOR = 3.34, p = 0.044) were significantly associated with MDR-TB. The prevalence of MDR-TB in the study population was of a significantly high level among previously treated patients and age group of 25-34. TB/HIV coinfection, smoking of cigarette, alcohol drinking, hospital admission and health facility visiting were identified as risk factors

Mechanistic kinetic modeling generates system-independent P-glycoprotein mediated transport elementary rate constants for inhibition and, in combination with 3D SIM microscopy, elucidates the importance of microvilli morphology on P-glycoprotein mediated efflux activity.

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Ellens, Harma; Meng, Zhou; Le Marchand, Sylvain J; Bentz, Joe

2018-06-01

In vitro transporter kinetics are typically analyzed by steady-state Michaelis-Menten approximations. However, no clear evidence exists that these approximations, applied to multiple transporters in biological membranes, yield system-independent mechanistic parameters needed for reliable in vivo hypothesis generation and testing. Areas covered: The classical mass action model has been developed for P-glycoprotein (P-gp) mediated transport across confluent polarized cell monolayers. Numerical integration of the mass action equations for transport using a stable global optimization program yields fitted elementary rate constants that are system-independent. The efflux active P-gp was defined by the rate at which P-gp delivers drugs to the apical chamber, since as much as 90% of drugs effluxed by P-gp partition back into nearby microvilli prior to reaching the apical chamber. The efflux active P-gp concentration was 10-fold smaller than the total expressed P-gp for Caco-2 cells, due to their microvilli membrane morphology. The mechanistic insights from this analysis are readily extrapolated to P-gp mediated transport in vivo. Expert opinion: In vitro system-independent elementary rate constants for transporters are essential for the generation and validation of robust mechanistic PBPK models. Our modeling approach and programs have broad application potential. They can be used for any drug transporter with minor adaptations.

Venetoclax (ABT-199) Might Act as a Perpetrator in Pharmacokinetic Drug–Drug Interactions

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Weiss, Johanna; Gajek, Thomas; Köhler, Bruno Christian; Haefeli, Walter Emil

2016-01-01

Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug–drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug–drug interactions. PMID:26927160

CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

International Nuclear Information System (INIS)

Wang, Xin; Liu, Ying; Wang, Shouju; Shi, Donghong; Zhou, Xianguang; Wang, Chunyan; Wu, Jiang; Zeng, Zhiyong; Li, Yanjun; Sun, Jing; Wang, Jiandong; Zhang, Longjiang; Teng, Zhaogang; Lu, Guangming

2015-01-01

Graphical abstract: - Highlights: • CD44-engineered mesoporous silica nanoparticles are synthesized. • The mechanism of CD44-engineered mesoporous silica nanoparticles is revealed. • This new delivery system increased the drug accumulation in vitro and in vivo. • This new delivery system offers an effective approach to treat multidrug resistance. - Abstract: Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer

Application of 99Tcm-MIBI scintimammography to diagnose P-gP of breast cancer

International Nuclear Information System (INIS)

Zhao Tao

2004-01-01

The article discuss the advances in 99 Tc m -sestamibi scintimammography to diagnose P-glycoprotein of breast cancer in multidrug resistance. As a kind of noninvasive fuctional test imaging technology, SPECT can be used to diagnose P-glycoprotein expression in breast cancer and can make decision for clinical treatment. (authors)

Enhanced cerebral uptake of receptor ligands by modulation of P-glycoprotein function in the blood-brain barrier

NARCIS (Netherlands)

Doze, P; Van Waarde, A; Elsinga, P H; Hendrikse, N H; Vaalburg, W

Low cerebral uptake of some therapeutic drugs can be enhanced by modulation of P-glycoprotein (P-gp), an ATP-driven drug efflux pump at the blood-brain barrier (BBB). We investigated the possibility of increasing cerebral uptake of the beta-adrenergic ligands S-1'-[(18)F]-fluorocarazolol (FCAR) and

Epidemiology of multi-drug resistant staphylococci in cats, dogs and people in Switzerland

OpenAIRE

Decristophoris, Paola Maria Aurelia

2011-01-01

Background: The human relationship with cats and dogs has been suggested to be of potential concern to public health because of the possible role of pets as reservoir of antibiotic resistant microorganisms. Here I suggest the “One Health” interdisciplinary approach to be helpful towards the understanding of the role of pets in antibiotic resistance spreading, considering also the socio-emotional context of the human-pet relationship. Methods: I investigated the presence of multi-drug resis...

Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

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Fu Juanjuan

2011-07-01

Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

Highly effective Cu/Zn-carbon micro/nanofiber-polymer nanocomposite-based wound dressing biomaterial against the P. aeruginosa multi- and extensively drug-resistant strains.

Science.gov (United States)

Ashfaq, Mohammad; Verma, Nishith; Khan, Suphiya

2017-08-01

Pseudomonas aeruginosa (P. aeruginosa) is the most prevalent bacteria in the infections caused by burn, surgery, and traumatic injuries. Emergence of the P. aeruginosa bacterial resistance against various clinical drugs for wound treatment is the major concern nowadays. The present study describes the synthesis of the polyvinyl alcohol (PVA) and cellulose acetate phthalate (CAP) polymeric composite film (~0.2mm thickness) reinforced with the Cu/Zn bimetal-dispersed activated carbon micro/nanofiber (ACF/CNF), as a wound dressing material. The focus is on determining the efficacy of the prepared biomaterial against the multi and extensively drug-resistant P. aeruginosa strains isolated from the burning, surgical, and traumatic injury-wounds. The primary synthesis steps for the biomaterial include the mixing of a blend of CAP powder and the asymmetrically distributed Cu/Zn bimetals in ACF/CNF, into the polymerization reaction mixture of PVA. Biochemical tests showed that the prepared composite material significantly enhanced the in-vitro blood clotting rate, platelet aggregation, and macrophage cell proliferation, indicating the suitability of the material as a fast wound healer. The antibacterial tests performed against the P. aeruginosa strains showed that the material effectively suppressed the bacterial growth, with the bimetal nanoparticles dispersed in the material serving as an antibacterial agent. The PVA/CAP polymer composite served as an encapsulating agent providing a slow release of the nanoparticles, besides increasing the hemostatic properties of the biomaterial. The ACF/CNF served as a support to the dispersed bimetal nanoparticles, which also provided a mechanical and thermal stability to the material. Experimentally demonstrated to be biocompatible, the prepared metal-carbon-polymer nanocomposite in this study is an effective dressing material for the P. aeruginosa-infected wounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Potential pharmacokinetic effect of rifampicin on enrofloxacin in broilers: Roles of P-glycoprotein and BCRP induction by rifampicin.

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Guo, Mengjie; Dai, Xiaohua; Hu, Dongmin; Zhang, Yu; Sun, Yong; Ren, Weilong; Wang, Liping

2016-09-01

P-glycoprotein ( P-GP: , encoding gene Abcb1) and Breast Cancer Resistance Protein ( BCRP: , encoding gene Abcg2) are transport proteins that play a major role in modulating the bioavailability of oral drugs in humans and rodents. It has been shown that rifampicin is the typical inducer of P-gp in rodents by activating the nuclear receptor. However, its effect on Abcb1, Abcg2, CYP3A, and chicken xenobiotic-sensing orphan nuclear receptor ( CXR: ) mRNA expression in broilers is poorly understood. This study explored the effect of rifampicin on mRNA expression of Abcb1, Abcg2, CYP3A37, CXR as well as its effect on the pharmacokinetics of enrofloxacin in broilers. The mRNA levels of Abcb1, Abcg2, CYP3A37, and CXR were significantly increased in the liver (except Abcg2), kidney, jejunum, and ileum (P 0.05) after treated with rifampicin. Further analysis revealed that the variation tendencies of Abcb1, Abcg2, and CYP3A37 expression levels were significantly correlated with CXR mRNA expression levels in liver, kidney, jejunum, and ileum. Coadministration of rifampicin significantly changed the pharmacokinetic behavior of enrofloxacin orally administered by showing clearly lower AUC0-∞, AUC0-t, and Cmax as well as longer Tmax. The bioavailability of orally administered enrofloxacin was decreased from 72.5% to 24.8% by rifampicin. However, rifampicin did not significantly change the pharmacokinetics of enrofloxacin following intravenous administration. Our study shows that rifampicin up-regulated the small intestinal level of P-gp and BCRP and suggests that P-gp and BCRP are key factors that affected pharmacokinetic behavior of orally administered enrofloxacin by limiting its absorption from the intestine in broilers. © 2016 Poultry Science Association Inc.

Characterization of a Novel 99mTc-Carbonyl Complex as a Functional Probe of MDR1 P-Glycoprotein Transport Activity

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Mary Dyszlewski

2002-01-01

Full Text Available Multidrug resistance (MDR mediated by overexpression of MDR1 P-glycoprotein (Pgp is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I-tricarbonyl complex, [99mTc(CO3(MIBI3] + (Tc-CO-MIBI. Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3–1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM. Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(−/− gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

Topicality of the problem of combined course of multi-drug resistant pulmonary tuberculosis with diabetes mellitus

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O. M. Raznatovska

2017-08-01

Full Text Available According to the World Health Organization, today in the world among the infectious chronic diseases one of the leading places and causes of death is multi-drug resistant tuberculosis of the lungs, and chronic non-communicable diseases – diabetes mellitus. The situation is complicated by the fact that the number of patients with combined course of these two heavy separate illnesses that complicate each other increases. It is established that with increasing severity of diabetes mellitus, tuberculosis process in the lungs becomes more complicate and deteriorates, and vice versa, the specific process complicates the course of diabetes mellitus, contributing to the development of diabetic complications. Against this background, the effectiveness of treatment of patients suffering from multi-drug resistant tuberculosis of the lungs in our country remains very low, mainly due to the toxic adverse reactions to antimycobacterial drugs of the reserve line, and in the case of adding diabetes mellitus, it deteriorates even more. The aim of this study was to review the scientific literature to determine the relevance of the study of combined course of multi-drug resistant tuberculosis of the lungs with diabetes mellitus and perspectives of innovative methods of diagnosis of diabetes mellitus. Early diagnosis of pre-diabetes, and autoimmune diseases will allow the use of timely correction techniques that prevents the development of diabetes mellitus, depending on its type, and in the future the development of serious irreversible processes, allow timely applying appropriate methods of correction of the revealed violations. Results. Very little amount of work is dedicated to the problem of combined course of multi-drug resistant tuberculosis of the lungs with diabetes mellitus, regardless of its type, the theme is relevant for today, in Ukraine there are no data regarding its study. This combined course of very difficult in the treatment diseases requires

P-gp and MRP1 Expression in Parathyroid Tumors Related to Histology, Weight and Tc-99m-Sestamibi Imaging Results

NARCIS (Netherlands)

Jorna, F. H.; Hollema, H.; Hendrikse, H. N.; Bart, J.; Brouwers, A. H.; Plukker, J. T. M.

Objective: P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) are membrane efflux pumps that may have a role in the kinetics of Tc-99m-sestamibi (MIBI) in parathyroid tumors. P-gp and MRP1 expression in parathyroid tumors was studied and related to histology, weight and pre- and

Characterization of p38 MAPK isoforms for drug resistance study using systems biology approach.

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Peng, Huiming; Peng, Tao; Wen, Jianguo; Engler, David A; Matsunami, Risë K; Su, Jing; Zhang, Le; Chang, Chung-Che Jeff; Zhou, Xiaobo

2014-07-01

p38 mitogen-activated protein kinase activation plays an important role in resistance to chemotherapeutic cytotoxic drugs in treating multiple myeloma (MM). However, how the p38 mitogen-activated protein kinase signaling pathway is involved in drug resistance, in particular the roles that the various p38 isoforms play, remains largely unknown. To explore the underlying mechanisms, we developed a novel systems biology approach by integrating liquid chromatography-mass spectrometry and reverse phase protein array data from human MM cell lines with computational pathway models in which the unknown parameters were inferred using a proposed novel algorithm called modularized factor graph. New mechanisms predicted by our models suggest that combined activation of various p38 isoforms may result in drug resistance in MM via regulating the related pathways including extracellular signal-regulated kinase (ERK) pathway and NFкB pathway. ERK pathway regulating cell growth is synergistically regulated by p38δ isoform, whereas nuclear factor kappa B (NFкB) pathway regulating cell apoptosis is synergistically regulated by p38α isoform. This finding that p38δ isoform promotes the phosphorylation of ERK1/2 in MM cells treated with bortezomib was validated by western blotting. Based on the predicted mechanisms, we further screened drug combinations in silico and found that a promising drug combination targeting ERK1/2 and NFκB might reduce the effects of drug resistance in MM cells. This study provides a framework of a systems biology approach to studying drug resistance and drug combination selection. RPPA experimental Data and Matlab source codes of modularized factor graph for parameter estimation are freely available online at http://ctsb.is.wfubmc.edu/publications/modularized-factor-graph.php. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

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Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

2016-02-01

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. Published by Elsevier Inc.

Circumvention of acquired resistance to doxorubicin in K562 human leukemia cells by oxatomide.

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Ishikawa, M; Fujita, R; Furusawa, S; Takayanagi, M; Sasaki, K; Satoh, S

2001-10-01

We studied the effect of oxatomide, an antiallergic drug, on the resistance of K562 cells to doxorubicin. Oxatomide synergistically potentiated the cytotoxicity of doxorubicin in doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 1-10 microM, but had hardly any synergistic effect on the parental cell line (K562) at the same concentration. Oxatomide inhibit P-glycoprotein pump-efflux activity and the binding of [3H]-azidopine to the cell-surface protein P-glycoprotein, in a dose-related manner. These results indicate that oxatomide reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

Involvement of HIF-1α activation in the doxorubicin resistance of human osteosarcoma cells.

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Roncuzzi, Laura; Pancotti, Fabia; Baldini, Nicola

2014-07-01

Osteosarcoma is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, survival outcomes remain unsatisfactory, particularly in patients with metastatic and/or recurrent disease. Unfortunately, treatment failure is commonly due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. The aim of the present study was to investigate the role of hypoxia-inducible factor 1α (HIF‑1α) and its signalling pathways as mediators of drug-resistance in human osteosarcoma. Toward this aim, we established two osteosarcoma cell lines selected for resistance to doxorubicin, a drug of choice in the treatment of this tumour. Our results showed that the multidrug resistance (MDR) phenotype was also mediated by HIF-1α, the most important regulator of cell adaptation to hypoxia. Our data showed that this transcription factor promoted the outward transport of intracellular doxorubicin by activating the P-glycoprotein (P-gp) expression in osteosarcoma cells maintained in normoxic conditions. In addition, it hindered doxorubicin-induced apoptosis by regulating the expression of c-Myc and p21. Finally, we observed that the doxorubicin-resistant cells maintained for 2 months of continuous culture in a drug-free medium, lost their drug-resistance and this effect was associated with the absence of HIF-1α expression. The emerging role of HIF-1α in osteosarcoma biology indicates its use as a valuable therapeutic target.

Improved Tumor-Specific Drug Accumulation by Polymer Therapeutics with pH-Sensitive Drug Release Overcomes Chemotherapy Resistance.

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Heinrich, Anne-Kathrin; Lucas, Henrike; Schindler, Lucie; Chytil, Petr; Etrych, Tomáš; Mäder, Karsten; Mueller, Thomas

2016-05-01

The success of chemotherapy is limited by poor selectivity of active drugs combined with occurrence of tumor resistance. New star-like structured N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based drug delivery systems containing doxorubicin attached via a pH-sensitive hydrazone bond were designed and investigated for their ability to overcome chemotherapy resistance. These conjugates combine two strategies to achieve a high drug concentration selectively at the tumor site: (I) high accumulation by passive tumor targeting based on enhanced permeability and retention effect and (II) pH-sensitive site-specific drug release due to an acidic tumor microenvironment. Mice bearing doxorubicin-resistant xenograft tumors were treated with doxorubicin, PBS, poly HPMA (pHPMA) precursor or pHPMA-doxorubicin conjugate at different equivalent doses of 5 mg/kg bodyweight doxorubicin up to a 7-fold total dose using different treatment schedules. Intratumoral drug accumulation was analyzed by fluorescence imaging utilizing intrinsic fluorescence of doxorubicin. Free doxorubicin induced significant toxicity but hardly any tumor-inhibiting effects. Administering at least a 3-fold dose of pHPMA-doxorubicin conjugate was necessary to induce a transient response, whereas doses of about 5- to 6-fold induced strong regressions. Tumors completely disappeared in some cases. The onset of response was differential delayed depending on the tumor model, which could be ascribed to distinct characteristics of the microenvironment. Further fluorescence imaging-based analyses regarding underlying mechanisms of the delayed response revealed a related switch to a more supporting intratumoral microenvironment for effective drug release. In conclusion, the current study demonstrates that the concept of tumor site-restricted high-dose chemotherapy is able to overcome therapy resistance. Mol Cancer Ther; 15(5); 998-1007. ©2016 AACR. ©2016 American Association for Cancer Research.

Collateral sensitivity to cisplatin in KB-8-5-11 drug-resistant cancer cells.

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Doherty, Ben; Lawlor, Denise; Gillet, Jean-Pierre; Gottesman, Michael; O'Leary, John J; Stordal, Britta

2014-01-01

KB-8-5-11 cells are a drug-resistant cervical cell model that overexpresses ABCB1 (P-glycoprotein). KB-8-5-11 has become sensitive to non-ABCB1 substrate cisplatin. Understanding the mechanism of collateral sensitivity to cisplatin may lead to biomarker discovery for platinum sensitivity in patients with cancer. A Taqman low-density array was used to characterize the expression of 380 genes previously associated with chemoresistance. Identified pathways were further analyzed using cytotoxicity assays, metabolomics and western blots. KB-8-5-11 cells were sensitive to CuSO4 and the glutathione inhibitor buthionine sulphoximine. Expression of ATPase, Cu(2+) transporting alpha (ATP7A) and ATP7B were decreased at the protein and gene levels respectively in KB-8-5-11. KB-8-5-11 had decreased gene expression of glutathione S-transferase pi 1 (GSTP1), GSTA4 and GSTK1. Cisplatin treatment significantly lowered total cellular glutathione in parental KB-3-1 cells. Glutathione also tended to be lower in KB-8-5-11 cells compared to KB-3-1 cells. KB-8-5-11 cells have alterations in their copper transporters and glutathione metabolism, contributing to their cisplatin-sensitive phenotype.

Venetoclax (ABT-199 Might Act as a Perpetrator in Pharmacokinetic Drug–Drug Interactions

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Johanna Weiss

2016-02-01

Full Text Available Venetoclax (ABT-199 represents a specific B-cell lymphoma 2 (Bcl-2 inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR cells. We therefore scrutinized its drug–drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1, breast cancer resistance protein (BCRP, and organic anion transporting polypeptides (OATPs was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug–drug interactions.

MicroRNA expression profiles of drug-resistance breast cancer cells and their exosomes.

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Zhong, Shanliang; Chen, Xiu; Wang, Dandan; Zhang, Xiaohui; Shen, Hongyu; Yang, Sujin; Lv, Mengmeng; Tang, Jinhai; Zhao, Jianhua

2016-04-12

Exosomes have been shown to transmit drug resistance through delivering miRNAs. We aimed to explore their roles in breast cancer. Three resistant sublines were established by exposing parental MDA-MB-231 cell line to docetaxel, epirubicin and vinorelbine, respectively. Preneoadjuvant chemotherapy biopsies and paired surgically-resected specimens embedded in paraffin from 23 breast cancer patients were collected. MiRNA expression profiles of the cell lines and their exosomes were evaluated using microarray. The result showed that most miRNAs in exosomes had a lower expression level than that in cells, however, some miRNAs expressed higher in exosomes than in cells, suggesting a number of miRNAs is concentrated in exosomes. Among the dysregulated miRNAs, 22 miRNAs were consistently up-regulated in exosomes and their cells of origin. We further found that 12 of the 22 miRNAs were significantly up-regulated after preneoadjuvant chemotherapy. Further study of the role of these 12 miRNAs in acquisition of drug resistance is needed to clarify their contribution to chemoresistance.

Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines