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Sample records for inhibits p-glycoprotein mediated

  1. Structure-activity relationships of dibenzoylhydrazines for the inhibition of P-glycoprotein-mediated quinidine transport.

    PubMed

    Miyata, Ken-Ichi; Nakagawa, Yoshiaki; Kimura, Yasuhisa; Ueda, Kazumitsu; Akamatsu, Miki

    2016-07-15

    We previously demonstrated that dibenzoylhydrazines (DBHs) are not only P-glycoprotein (P-gp) substrates, but also inhibitors. In the present study, we evaluated the inhibition of P-gp-mediated quinidine transport by two series of DBHs and performed a classical QSAR analysis and docking simulation in order to investigate the mechanisms underlying P-gp substrate/inhibitor recognition. The results of the QSAR analysis identified the hydrophobic factor as the most important for inhibitory activities, while electronic and steric effects also influenced the activities. The different substituent effects observed in each series suggested the different binding modes of each series of DBHs, which was supported by the results of the docking simulation. PMID:27262425

  2. Inhibition of P-glycoprotein-mediated transport by extracts of and monoterpenoids contained in Zanthoxyli Fructus

    SciTech Connect

    Yoshida, Naoko; Takagi, Akiyoshi; Kitazawa, Hidenori; Kawakami, Junichi . E-mail: kawakami-tym@umin.ac.jp; Adachi, Isao

    2005-12-01

    Citrus (rutaceous) herbs are often used in traditional medicine and Japanese cuisine and can be taken concomitantly with conventional medicine. In this study, the effect of various citrus-herb extracts on P-glycoprotein (P-gp)-mediated transport was examined in vitro to investigate a possible interaction with P-gp substrates. Component monoterpenoids of the essential oil in Zanthoxyli Fructus was screened to find novel P-gp inhibitors. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used. Cellular accumulation of [{sup 3}H]digoxin was measured in the presence or absence of P-gp inhibitors or test samples. Aurantii Fructus, Evodiae Fructus, Aurantii Fructus Immaturus, Aurantii Nobilis Pericarpium, Phellodendri Cortex, and Zanthoxyli Fructus were extracted with hot water (decocted) and then fractionated with ethyl acetate. The cell to medium ratio of [{sup 3}H]digoxin accumulation increased significantly in the presence of the decoction of Evodiae Fructus, Aurantii Nobilis Pericarpium, and Zanthoxyli Fructus, and the ethyl acetate fraction of all citrus herbs used. The ethyl acetate fraction of Zanthoxyli Fructus exhibited the strongest inhibition of P-gp among tested samples with an IC{sub 5} value of 166 {mu}g/mL. Then its component monoterpenoids, geraniol, geranyl acetate (R)-(+)-limonene, (R)-(+)-linalool, citronellal (R)-(+)-citronellal, DL-citronellol (S)-(-)-{beta}-citronellol, and cineole, were screened. (R)-(+)-citronellal and (S)-(-)-{beta}-citronellol inhibited P-gp with IC{sub 5} values of 167 {mu}M and 504 {mu}M, respectively. These findings suggest that Zanthoxyli Fructus may interact with P-gp substrates and that some monoterpenoids with the relatively lower molecular weight of about 150 such as (R)-(+)-citronellal can be potent inhibitors of P-gp.

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

    PubMed

    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 < 0.05) change in permeability in the presence of GFJ. Chronic administration of GFJ resulted in a significant decrease in absorptive transport of indinavir, which was even greater than that produced by rifampicin pretreatment. No change in permeability of propranolol, a passive permeability marker, was observed. Further, the decrease in absorptive transport of INDI was reversed by the P-gp inhibitor verapamil. In conclusion, GFJ 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. PMID:16398269

  4. Anthocyanidins but not anthocyanins inhibit P-glycoprotein-mediated calcein extrusion - possible implication for orally administered drugs.

    PubMed

    Vrzal, Radim

    2016-06-01

    P-glycoprotein (P-gp) inhibition represents a promising therapeutic strategy for oncologic patients. The inhibition by naturally occurring anthocyans would bring certain benefits. Unfortunately, due to the low bioavailability and consequently low blood level, they cannot be used for cancer therapy. However, due to the food supplementation, significant concentration can raise up in the intestine, where P-gp is abundantly expressed. As many drugs are orally taken, simultaneous administration might affect the concentration of these drugs in the blood. Here, we found that anthocyanidins (aglycons) but not anthocyanins (glycosides) can significantly inhibit P-gp up to 60% of positive control, verapamil. This inhibitory activity was observed for 500 μm concentrations of malvidin and pelargonidin. We conclude that these compounds may be the source of food-drug interactions either for orally taken drugs or for intravenously administered drugs eliminated via biliary excretion which are the substrates of P-gp. PMID:26821071

  5. neo-Clerodane diterpenoids from Scutellaria barbata mediated inhibition of P-glycoprotein in MCF-7/ADR cells.

    PubMed

    Xue, Gui-Min; Xia, Yuan-Zheng; Wang, Zhi-Min; Li, Ling-Nan; Luo, Jian-Guang; Kong, Ling-Yi

    2016-10-01

    Ten new (1-10) and seventeen known (11-27) neo-clerodane diterpenoids substituted with nicotinoyloxyl were isolated from the plant Scutellaria barbata and their structures were established by extensive spectroscopic analysis. Chemoreversal effects of these neo-clerodane diterpenoids on multidrug resistance were evaluated in breast cancer multidrug-resistant MCF-7/ADR cells that overexpress P-glycoprotein. Four compounds (11, 14, 16, and 18) exhibited better chemoreversal abilities than the classical P-gp inhibitor verapamil and the most potent compound 11 reduced IC50 value of adriamycin in MCF-7/ADR cells from 58.8 μM to 1.3 μM. Mechanistic investigations showed that compound 11 reversed multidrug resistance through suppressing the activity of P-gp and restraining the expression of P-glycoprotein. In the present study, the structure-activity relationships of neo-clerodane diterpenoids were also discussed. PMID:27240278

  6. Inhibition of P-glycoprotein-mediated transport by S-adenosylmethionine and cynarin in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells.

    PubMed

    Angelini, A; Di Pietro, R; Centurione, L; Castellani, M L; Conti, P; Porreca, E; Cuccurullo, F

    2012-01-01

    Multidrug resistance (MDR) to anticancer chemotherapy is often mediated by the overexpression of the plasma membrane drug transporter P-glycoprotein (Pgp) encoded by multidrug resistance gene (MDR1). Various chemosensitizing agents are able to inhibit Pgp activity but their clinical application is limited by their toxicity. Furthermore, hepatotoxicity related to chemotherapy causes delays of treatment in cancer patients and often requires supplementation of anti-tumour therapy with hepatoprotective agents. In this in vitro study, we investigated the effectiveness of an endogenous hepatoprotective agent, S-adenosylmethionine (SAMe), and a natural hepatoprotective compound, Cynarin (Cyn), to inhibit Pgp activity in order to evaluate their potential use as chemosensitizing agents. Human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) expressing high levels of Pgp were treated with two hepatoprotectors at various concentrations (1, 5 and 10 microM) that are clinically achievable, in the presence or absence of three different concentrations of doxo (2, 4 and 8 microM). In order to evaluate the effects of both hepatoprotectors, we measured the intracellular accumulation and cytotoxicity of doxo, the cellular GSH level, ROS production and catalase (CAT) activity. We found that treatment with 2, 4 and 8 microM doxo in the presence of SAMe or Cyn significantly increased the doxo accumulation and cytotoxicity on MES-SA/Dx5 cells, when compared to control cells receiving doxo alone. Moreover, treatment with SAMe or Cyn significantly increased GSH content, greater than 80 percent and 60 percent, respectively) and CAT activity greater than 60 and 150 percent, respectively) in resistant cancer cells, while ROS production was below the values of corresponding untreated control cells. Our in vitro findings provide a rationale for the potential clinical use of these hepatoprotectors both as chemosensitizing agents, to reverse Pgp-mediated MDR, and as antioxidants to

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

    PubMed Central

    Mohseni, Mahsa; Samadi, Nasser; Ghanbari, Parisa; Yousefi, Bahman; Tabasinezhad, Maryam; Sharifi, Simin; Nazemiyeh, Hossein

    2016-01-01

    Objective(s): Chemoresistance remains the main causes of treatment failure and mortality in cancer patients. There is an urgent need to investigate novel approaches to improve current therapeutic modalities and increase cancer patients’ survival. Induction of drug efflux due to overexpression of P-glycoproteins is considered as an important leading cause of multidrug resistance. In this study, we investigated the role of combination treatments of docetaxel and vinblastine in overcoming P-glycoprotein mediated inhibition of apoptosis and induction of cell proliferation in human non-small cell lung carcinoma cells. Materials and Methods: Cell proliferation and apoptosis were assessed using MTT assay and DAPI staining, respectively. P-glycoprotein expression was evaluated in gene and protein levels by Real-time RT-PCR and Western blot analysis, respectively. 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. PMID:27114800

  8. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    SciTech Connect

    Filipski, Elisabeth; Berland, Elodie; Ozturk, Narin; Guettier, Catherine; Horst, Gijsbertus T.J. van der; Lévi, Francis; 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.

  9. Acetaminophen inhibits intestinal p-glycoprotein transport activity.

    PubMed

    Novak, Analia; Carpini, Griselda Delli; Ruiz, María Laura; Luquita, Marcelo G; Rubio, Modesto C; Mottino, Aldo D; Ghanem, Carolina I

    2013-10-01

    Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 μM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP. PMID:23897240

  10. CD44 promotes multi-drug resistance by protecting P-glycoprotein from FBXO21-mediated ubiquitination

    PubMed Central

    Ravindranath, Abhilash K.; Kaur, Swayamjot; Wernyj, Roman P.; Kumaran, Muthu N.; Miletti-Gonzalez, Karl E.; Chan, Rigel; Lim, Elaine; Madura, Kiran; Rodriguez-Rodriguez, Lorna

    2015-01-01

    Here we demonstrate that a ubiquitin E3-ligase, FBXO21, targets the multidrug resistance transporter, ABCB1, also known as P-glycoprotein (P-gp), for proteasomal degradation. We also show that the Ser291-phosphorylated form of the multifunctional protein and stem cell marker, CD44, inhibits FBXO21-directed degradation of P-gp. Thus, CD44 increases P-gp mediated drug resistance and represents a potential therapeutic target in P-gp-positive cells. PMID:26299618

  11. Inhibition of the Multidrug Resistance P-Glycoprotein: Time for a Change of Strategy?

    PubMed Central

    Luk, Frederick; Bebawy, Mary

    2014-01-01

    P-glycoprotein (P-gp) is a key player in the multidrug-resistant phenotype in cancer. The protein confers resistance by mediating the ATP-dependent efflux of an astonishing array of anticancer drugs. Its broad specificity has been the subject of numerous attempts to inhibit the protein and restore the efficacy of anticancer drugs. The general strategy has been to develop compounds that either compete with anticancer drugs for transport or act as direct inhibitors of P-gp. Despite considerable in vitro success, there are no compounds currently available to “block” P-gp–mediated resistance in the clinic. The failure may be attributed to toxicity, adverse drug interaction, and numerous pharmacokinetic issues. This review provides a description of several alternative approaches to overcome the activity of P-gp in drug-resistant cells. These include 1) drugs that specifically target resistant cells, 2) novel nanotechnologies to provide high-dose, targeted delivery of anticancer drugs, 3) compounds that interfere with nongenomic transfer of resistance, and 4) approaches to reduce the expression of P-gp within tumors. Such approaches have been developed through the pursuit of greater understanding of resistance mediators such as P-gp, and they show considerable potential for further application. PMID:24492893

  12. Reversal of P-glycoprotein-mediated multidrug resistance by the novel tetrandrine derivative W6.

    PubMed

    Sun, Hua; Liu, Xiao-Dong; Liu, Qian; Wang, Feng-Peng; Bao, Xiu-Qi; Zhang, Dan

    2015-01-01

    Overexpression of ATP-dependent efflux pump P-glycoprotein (P-gp) is the main cause of multidrug resistance (MDR) and chemotherapy failure in cancer treatment. Inhibition of P-gp-mediated drug efflux is an effective way to overcome cancer drug resistance. The present study investigated the reversal effect of the novel tetrandrine derivative W6 on P-gp-mediated MDR. KBv200, MCF-7/adr and their parental sensitive cell lines KB, MCF-7 were used for reversal study. The intracellular accumulation with P-gp substrates of doxorubicin was determined by flow cytometry. The expression of P-gp and ERK1/2 was investigated by western blot and real-time-PCR (RT-PCR) analysis. ATPase activity of P-gp was performed by P-gp-Glo(TM) assay systems. In comparison with P-gp-negative parental cells, W6 produced a favorable reversal effect in the MDR cells, as determined using the MTT assay. W6 significantly and dose-dependently increased intracellular accumulation of P-gp substrate doxorubicin (DOX) in P-gp overexpressing KBv200 cells, and also inhibited the ATPase activity of P-gp. W6 inhibited P-gp expression in KBv200 cells in a time-dependent manner, but it had no effect on MDR1 expression. In addition, W6 significantly decreased the ERK1/2 activation in KBv200 cells. Our results showed that W6 effectively reversed P-gp-mediated MDR by inhibiting the transport function and expression of P-gp, demonstrating the potential clinical utility of W6. PMID:26235354

  13. Reversion of p-glycoprotein-mediated multidrug resistance in human leukemic cell line by diallyl trisulfide.

    PubMed

    Xia, Qing; Wang, Zhi-Yong; Li, Hui-Qing; Diao, Yu-Tao; Li, Xiao-Li; Cui, Jia; Chen, Xue-Liang; Li, Hao

    2012-01-01

    Multidrug resistance (MDR) is the major obstacle in chemotherapy, which involves multiple signaling pathways. Diallyl trisulfide (DATS) is the main sulfuric compound in garlic. In the present study, we aimed to explore whether DATS could overcome P-glycoprotein-(P-gp-)mediated MDR in K562/A02 cells, and to investigate whether NF-κB suppression is involved in DATS-induced reversal of MDR. MTT assay revealed that cotreatment with DATS increased the response of K562/A02 cells to adriamycin (the resistance reversal fold was 3.79) without toxic side effects. DATS could enhance the intracellular concentration of adriamycin by inhibiting the function and expression of P-gp, as shown by flow cytometry, RT-PCR, and western blot. In addition, DATS resulted in more K562/A02 cell apoptosis, accompanied by increased expression of caspase-3. The expression of NF-κB/p65 (downregulation) was significantly linked to the drug-resistance mechanism of DATS, whereas the expression of IκBα was not affected by DATS. Our findings demonstrated that DATS can serve as a novel, nontoxic modulator of MDR, and can reverse the MDR of K562/A02 cells in vitro by increasing intracellular adriamycin concentration and inducing apoptosis. More importantly, we proved for the first time that the suppression of NF-κB possibly involves the molecular mechanism in the course of reversion by DATS. PMID:22919419

  14. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation

    PubMed Central

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10−6 cm/sec, followed by amodiaquine around 20 x 10−6 cm/sec; both mefloquine and artesunate were around 10 x 10−6 cm/sec. Methylene blue was between 2 and 6 x 10−6 cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

  15. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation.

    PubMed

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10(-6) cm/sec, followed by amodiaquine around 20 x 10(-6) cm/sec; both mefloquine and artesunate were around 10 x 10(-6) cm/sec. Methylene blue was between 2 and 6 x 10(-6) cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

  16. The novel bis-benzylisoquinoline PY35 reverses P-glycoprotein-mediated multidrug resistance.

    PubMed

    Cao, Zhonglian; Wright, Meredith; Cheng, Jiekai; Huang, Xiaoxing; Liu, Li; Wu, Lixing; Yang, Ping

    2014-09-01

    Multidrug resistance (MDR) to chemotherapeutic drugs is the main cause of chemotherapy failure in cancer treatment, and it generally results from expression of ATP-dependent efflux pump P-glycoprotein (P-gp). MDR reversal agents typically act by inhibiting the drug efflux activity of P-gp, thereby increasing intracellular drug levels. PY35 is a novel 5-substituted tetrandrine (Tet) derivative (CN Application No. 201210238709.6). The present study was performed to investigate the ability of PY35 to reverse P-gp-mediated MDR and its mechanism in resistant K562/Adriamycin (ADM), MCF-7/ADM cells and their sensitive cell lines K562 and MCF-7. The ability of PY35 to reverse drug resistance was evaluated by MTT assay. The results showed that PY35 can reverse MDR more effectively than the drug prototype‑Tet. The P-gp function was assessed by the Rhodamine 123 (Rho-123; a P-gp substrate) uptake assay with flow cytometry (FCM) and laser scanning confocal microscopes (LSCM); it showed that the MDR cells pumped Rho-123 out the cells, while their sensitive cells scarcely showed efflux. The presence of PY35 efficiently decreased the efflux of the Rho-123, showing that PY35 can reverse P-gp-mediated MDR by increasing the intracellular concentration of Rho-123. The intracellular accumulation of ADM was analyzed by FCM and showed that the coadministration of PY35 and ADM had clearer accumulation than the treatment of Tet and ADM, and was also more evident than treatment with only ADM. The effect of PY35 on the expression of P-gp was assessed by western blotting. The results indicated that PY35 does not inhibit the expression level of the P-gp. This study indicated that PY35 can effectively reverse P-gp-mediated MDR, not by inhibiting the expression of P-gp, but by the coadministration of PY35 and ADM that could increase the intracellular accumulation of drugs. Thus, PY35 may be a potential inhibitor to overcome drug resistance. PMID:25017650

  17. Inhibition of P-glycoprotein by psychotherapeutic drugs in a canine cell model.

    PubMed

    Schrickx, J A; Fink-Gremmels, J

    2014-10-01

    Drug-drug interactions related to long-term therapies are of increasing concern. Psychotherapeutic drugs, licensed for the use in dogs for the management of separation anxiety and other behavioural disorders, are examples of drugs used in long-term therapies. In an in vitro system with canine P-glycoprotein (P-gp) expressing cell lines, three psychotherapeutic drugs with a different mode of action were tested for their ability to inhibit the canine multidrug transporter P-gp. At 10 μm, the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant clomipramine inhibited P-gp for 41% and 59%, respectively. In contrast, selegeline did not inhibit the function of the canine P-gp. PMID:24602126

  18. P-glycoprotein Mediated Efflux Modulators of Plant Origin: A Short Review.

    PubMed

    Silva, Nuno; Salgueiro, Lígia; Fortuna, Ana; Cavaleiro, Carlos

    2016-05-01

    Drug efflux transporters such as P-glycoprotein (P-gp) help maintain cellular homeostasis but are also major contributors to the development of multidrug resistance (MDR) phenomena. Since P-gp was associated with MDR, several compounds showing potential to inhibit this transporter have been identified. Particular attention has been given to natural products, namely those of plant origin, looking for highly effective and safe P-gp inhibitors with little to no interaction with other cellular or metabolic processes. Here we abridge several examples of plant compounds from distinct classes, polyketides, lignans, anthraquinones, coumarins, alkaloids, mono- and sesqui-terpenes, steroids and limonoids, which have shown the ability to modulate in vitro or in vivo the P-gp activity. PMID:27319155

  19. Oral and inhaled corticosteroids: differences in P-glycoprotein (ABCB1) mediated efflux.

    PubMed

    Crowe, Andrew; Tan, Ai May

    2012-05-01

    There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of only 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (>20×10(-6) cm/s) compared to the inhaled corticosteroids (>5×10(-6) cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. PMID:22464980

  20. Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure

    PubMed Central

    Nicklisch, Sascha C. T.; Rees, Steven D.; McGrath, Aaron P.; Gökirmak, Tufan; Bonito, Lindsay T.; Vermeer, Lydia M.; Cregger, Cristina; Loewen, Greg; Sandin, Stuart; Chang, Geoffrey; Hamdoun, Amro

    2016-01-01

    The world’s oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)–100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals. PMID:27152359

  1. Mixed Micelles of Doxorubicin Overcome Multidrug Resistance by Inhibiting the Expression of P-Glycoprotein.

    PubMed

    Jin, Yan; Zhang, Zhijie; Zhao, Tie; Liu, Xiaodong; Jian, Lingyan

    2015-08-01

    With the goal of overcoming multidrug resistance, DSPE-PEG (polyethylene glycol 2000 grafted with distearoyl phosphatidylethanolamine) and TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) were combined, each with a different inhibiting mechanism for P-glycoprotein (P-gp) expression, to create mixed micelles with the purpose of encapsulating the water-soluble drug, doxorubicin (Dox). As the molar ratio of Dox/DSPE-PEG/TPGS was 1:1:0.2, the encapsulation efficiency and particle size of the micelles were 98.2% and 12.8 nm respectively. Compared to Dox/DSPE-PEG micelles, Dox/DSPE-PEG/TPGS mixed micelles demonstrated enhanced in vitro cytotoxicity, drug uptake, and apoptosis for drug resistant H460/TaxR cancer cells. Western blot results showed that the expression level of P-gp significantly decreased as H460/TaxR cells were incubated with Dox/DSPE-PEG/TPGS mixed micelles. The anti-tumor efficacy in vivo was evaluated using H460/TaxR-bearing mice and showed that Dox/DSPE-PEG/TPGS mixed micelles were more effective at inhibiting tumor growth than Dox/DSPE-PEG micelles and free Dox solution. It was also found that the high efficacy of mixed micelles was associated with the ability to induce dramatic apoptosis of the tumor cells. In summary, through combining different P-gp inhibiting mechanisms, mixed micelles could be a promising nanocarrier for anti-cancer drugs in overcoming multidrug resistance. PMID:26295136

  2. Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure.

    PubMed

    Nicklisch, Sascha C T; Rees, Steven D; McGrath, Aaron P; Gökirmak, Tufan; Bonito, Lindsay T; Vermeer, Lydia M; Cregger, Cristina; Loewen, Greg; Sandin, Stuart; Chang, Geoffrey; Hamdoun, Amro

    2016-04-01

    The world's oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)-100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals. PMID:27152359

  3. Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog

    SciTech Connect

    Akiyama, S.; Cornwell, M.M.; Kuwano, M.; Pastan, I.; Gottesman, M.M.

    1988-02-01

    Multidrug-resistant human KB carcinoma cells express a 170,000-dalton membrane glycoprotein (P-glycoprotein) that can be photoaffinity labeled with the vinblastine analog N-(p-azido-(3-/sup 125/I)salicyl)-N'-(beta-aminoethyl)vindesine. Several agents that suppress the multidrug-resistant phenotype, including N-solanesyl-N,N'-bis(3,4-dimethylbenzyl)ethylenediamine, cepharanthine, quinidine, and reserpine, were found to inhibit photolabeling of P-glycoprotein at doses comparable to those that reverse multidrug resistance. However, the phenothiazines chlorpromazine and trifluoperazine, which also effectively reverse multidrug resistance, were poor inhibitors of the photoaffinity labeling of P-glycoprotein. Chloroquine, propranolol, or atropine, which only partially reversed the drug resistance, also did not inhibit photolabeling. Naphthalene sulfonamide calmodulin inhibitors, W7 and W5, as well as many other drugs that did not circumvent multidrug resistance, did not inhibit photolabeling. These studies suggest that most, but not all, agents that phenotypically suppress multidrug resistance also inhibit drug binding to a site on P-glycoprotein with which a photoaffinity analog of vinblastine interacts.

  4. Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

    SciTech Connect

    Crowe, Andrew Tan, Ai May

    2012-05-01

    There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of only 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

  5. Inhibition of P-glycoprotein in Caco-2 cells: effects of herbal remedies frequently used by cancer patients.

    PubMed

    Engdal, S; Nilsen, O G

    2008-06-01

    1. The herbal products Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic were investigated for in vitro inhibitory potential on P-glycoprotein (P-gp)-mediated transport of digoxin (30 nM) in differentiated and polarized Caco-2 cells. 2. Satisfactory cell functionality was demonstrated through measurements of assay linearity, transepithelial electric resistance (TEER), cytotoxicity, mannitol permeability, and inclusion of the positive inhibition control verapamil. 3. The most potent inhibitors of the net digoxin flux (IC(50)) were mistletoe > Natto K2 > Agaricus > green tea (0.022, 0.62, 3.81, >4.5 mg ml(-1), respectively). Mistletoe also showed the lowest IC(25) value, close to that obtained by verapamil (1.0 and 0.5 microg ml(-1), respectively). The IC(50)/IC(25) ratio was found to be a good parameter for the determination of inhibition profiles. Garlic and noni juice were classified as non-inhibitors. 4. This study shows that mistletoe, Natto K2, Agaricus and green tea inhibit P-gp in vitro. Special attention should be paid to mistletoe due to very low IC(50) and IC(25) values and to Natto K2 due to a low IC(50) value and a low IC(50)/IC(25) ratio. PMID:18570158

  6. Optimization by Molecular Fine Tuning of Dihydro-β-agarofuran Sesquiterpenoids as Reversers of P-Glycoprotein-Mediated Multidrug Resistance.

    PubMed

    Callies, Oliver; Sánchez-Cañete, María P; Gamarro, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Bazzocchi, Isabel L

    2016-03-10

    P-glycoprotein (P-gp) plays a crucial role in the development of multidrug resistance (MDR), a major obstacle for successful chemotherapy in cancer. Herein, we report on the development of a natural-product-based library of 81 dihydro-β-agarofuran sesquiterpenes (2-82) by optimization of the lead compound 1. The compound library was evaluated for its ability to inhibit P-gp-mediated daunomycin efflux in MDR cells. Selected analogues were further analyzed for their P-gp inhibition constant, intrinsic toxicity, and potency to reverse daunomycin and vinblastine resistances. Analogues 6, 24, 28, 59, and 66 were identified as having higher potency than compound 1 and verapamil, a first-generation P-gp modulator. SAR analysis revealed the size of the aliphatic chains and presence of nitrogen atoms are important structural characteristics to modulate reversal activity. The present study highlights the potential of these analogues as modulators of P-gp mediated MDR in cancer cells. PMID:26836364

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

    PubMed

    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. PMID:26686578

  8. Chronic P-glycoprotein inhibition increases the brain concentration of escitalopram: potential implications for treating depression.

    PubMed

    O'Brien, Fionn E; Moloney, Gerard M; Scott, Karen A; O'Connor, Richard M; Clarke, Gerard; Dinan, Timothy G; Griffin, Brendan T; Cryan, John F

    2015-12-01

    Recent preclinical studies have revealed a functionally important role for the drug efflux pump P-glycoprotein (P-gp) at the blood-brain barrier in limiting brain levels and thus antidepressant-like activity of certain antidepressant drugs. Specifically, acute administration of P-gp inhibitors, such as verapamil and cyclosporin A (CsA), has been shown to augment brain concentrations and functional activity of the antidepressant escitalopram in rodents. However, depression is a chronic disorder and current treatments require prolonged administration to elicit their full therapeutic effect. Thus, it is important to investigate whether acute findings in relation to P-gp inhibition translate to chronic paradigms. To this end, the present study investigates whether chronic treatment with the P-gp inhibitor verapamil and the antidepressant escitalopram results in enhanced brain distribution and antidepressant-like effects of escitalopram. Verapamil (10 mg·kg(-1) i.p.) and escitalopram (0.1 mg·kg(-1) i.p.) were administered once daily for 22 days. On the final day of treatment, brain regions and plasma were collected for analysis of cortical and plasma escitalopram concentrations, and to determine the hippocampal expression of genes previously reported to be altered by chronic antidepressant treatment. Verapamil treatment resulted in a greater than twofold increase in brain levels of escitalopram, without altering plasma levels. Neither gene expression analysis nor behavioral testing revealed an augmentation of responses to escitalopram treatment due to verapamil administration. Taken together, these data demonstrate for the first time that P-gp inhibition can yield elevated brain concentrations of an antidepressant after chronic treatment. The functional relevance of these increased brain levels requires further elaboration. PMID:27022464

  9. Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979.

    PubMed

    Dantzig, A H; Shepard, R L; Cao, J; Law, K L; Ehlhardt, W J; Baughman, T M; Bumol, T F; Starling, J J

    1996-09-15

    Overexpression of P-glycoprotein (Pgp) by tumors results in multidrug resistance (MDR) to structurally unrelated oncolytics. MDR cells may be sensitized to these oncolytics when treated with a Pgp modulator. The present study evaluates LY335979 as a modulator both in vitro and in vivo. LY335979 (0.1 microM) fully restored sensitivity to vinblastine, doxorubicin (Dox), etoposide, and Taxol in CEM/VLB100 cells. LY335979 modulated Dox cytotoxicity even when LY335979 (0.5 microM) was removed 24 h prior to the cytotoxicity assay. LY335979 blocked [3H]azidopine photoaffinity labeling of the M(r) approximately 170,000 Pgp in CEM/VLB100 plasma membranes and competitively inhibited equilibrium binding of [3H]vinblastine to Pgp (Ki of approximately 0.06 microM). Treatment of mice bearing P388/ADR murine leukemia cells with LY335979 in combination with Dox or etoposide gave a significant increase in life span with no apparent alteration of pharmacokinetics. LY335979 also enhanced the antitumor activity of Taxol in a MDR human non-small cell lung carcinoma nude mouse xenograft model. Thus, LY335979 is an extremely potent, efficacious modulator that apparently lacks pharmacokinetic interactions with coadministered anticancer drugs and is, therefore, an exciting new agent for clinical evaluation for reversal of Pgp-associated MDR. PMID:8797588

  10. Screening dietary flavonoids for the reversal of P-glycoprotein-mediated multidrug resistance in cancer.

    PubMed

    Mohana, S; Ganesan, M; Agilan, B; Karthikeyan, R; Srithar, G; Beaulah Mary, R; Ananthakrishnan, D; Velmurugan, D; Rajendra Prasad, N; Ambudkar, Suresh V

    2016-07-19

    P-Glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance in cancer cells. Although various screening procedures have been practiced so far to develop first three generations of P-gp inhibitors, their toxicity and drug interaction profiles are still a matter of concern. To address the above important problem of developing safe and effective P-gp inhibitors, we have made systematic computational and experimental studies on the interaction of natural phytochemicals with human P-gp. Molecular docking and QSAR studies were carried out for 40 dietary phytochemicals in the drug-binding site of the transmembrane domains (TMDs) of P-gp. Dietary flavonoids exhibit better interactions with homology modeled human P-gp. Based on the computational analysis, selected flavonoids were tested for their inhibitory potential against P-gp transport function in drug resistant cell lines using calcein-AM and rhodamine 123 efflux assays. It has been found that quercetin and rutin were the highly desirable flavonoids for the inhibition of P-gp transport function and they significantly reduced resistance in cytotoxicity assays to paclitaxel in P-gp overexpressing MDR cell lines. Hence, quercetin and rutin may be considered as potential chemosensitizing agents to overcome multidrug resistance in cancer. PMID:27216424

  11. Glucose modulation induces reactive oxygen species and increases P-glycoprotein-mediated multidrug resistance to chemotherapeutics

    PubMed Central

    Seebacher, N A; Richardson, D R; Jansson, P J

    2015-01-01

    Background and Purpose Cancer cells develop resistance to stress induced by chemotherapy. In tumours, a considerable glucose gradient exists, resulting in stress. Notably, hypoxia-inducible factor-1 (HIF-1) is a redox-sensitive transcription factor that regulates P-glycoprotein (Pgp), a crucial drug-efflux transporter involved in multidrug resistance (MDR). Here, we investigated how glucose levels regulate Pgp-mediated drug transport and resistance. Experimental Approach Human tumour cells (KB31, KBV1, A549 and DMS-53) were incubated under glucose starvation to hyperglycaemic conditions. Flow cytometry assessed reactive oxygen species (ROS) generation and Pgp activity. HIF-1α, NF-κB and Pgp expression were assessed by reverse transcriptase-PCR and Western blotting. Fluorescence microscopy examined p65 distribution and a luciferase-reporter assay assessed HIF-1 promoter-binding activity. The effect of glucose-induced stress on Pgp-mediated drug resistance was examined after incubating cells with the chemotherapeutic and Pgp substrate, doxorubicin (DOX), and performing MTT assays validated by viable cell counts. Key Results Changes in glucose levels markedly enhanced cellular ROS and conferred Pgp-mediated drug resistance. Low and high glucose levels increased (i) ROS generation via NADPH oxidase 4 and mitochondrial membrane destabilization; (ii) HIF-1 activity; (iii) nuclear translocation of the NF-κB p65 subunit; and (iv) HIF-1α mRNA and protein levels. Increased HIF-1α could also be due to decreased prolyl hydroxylase protein under these conditions. The HIF-1α target, Pgp, was up-regulated at low and high glucose levels, which led to lower cellular accumulation of Pgp substrate, rhodamine123, and greater resistance to DOX. Conclusions and Implications As tumour cells become glucose-deprived or exposed to high glucose levels, this increases stress, leading to a more aggressive MDR phenotype via up-regulation of Pgp. PMID:25586174

  12. Design, synthesis and evaluation of novel triazole core based P-glycoprotein-mediated multidrug resistance reversal agents.

    PubMed

    Jiao, Lei; Qiu, Qianqian; Liu, Baomin; Zhao, Tianxiao; Huang, Wenlong; Qian, Hai

    2014-12-15

    A novel series of triazol-N-ethyl-tetrahydroisoquinoline based compounds were designed and synthesized via click chemistry. Most of the synthesized compounds showed P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal activities. Among them, compound 7 with little cytotoxicity towards GES-1 cells (IC50 >80μM) and K562/A02 cells (IC50 >80μM) exhibited more potency than verapamil (VRP) on increasing anticancer drug accumulation in K562/A02 cells. Moreover, compound 7 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 7 in reversing MDR revealed that it could remarkably increase the intracellular accumulation of both rhodamine-123 (Rh123) and adriamycin (ADM) in K562/A02 cells as well as inhibit their efflux from the cells. These results suggested that compound 7 showed more potency than the classical P-gp inhibitor VRP under the same conditions, which may be a promising P-gp-mediated MDR modulator for further development. PMID:25464884

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

    PubMed

    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. PMID:27073052

  14. P-glycoprotein-mediated transport of oxytetracycline in the Caco-2 cell model.

    PubMed

    Schrickx, J; Fink-Gremmels, J

    2007-02-01

    ATP-dependent drug transporters such as P-glycoprotein (P-gp), multi-drug resistance associated protein (MRP2) and breast cancer resistant protein (BCRP) are expressed at the brush border membrane of enterocytes. These efflux transporters excrete their substrates, among other various classes of antibiotics, into the lumen thus reducing net absorption as indicated by a low bioavailability after oral administration. Oxytetracycline (OTC) has been used for decennia in veterinary medicine for its extensive spectrum of antimicrobial activity. A major limitation has been, and still remains, its low bioavailability following oral administration. The present study aimed to investigate to what extent this low bioavailability is attributable to the fact that OTC is a substrate for one or more efflux transporters. As an experimental model to study the transmembrane transport of OTC, differentiated Caco-2 cells grown as monolayers on permeable supports were used. With this model it was shown that the secretion of OTC is slightly higher than its absorption. PSC833, a potent inhibitor of P-gp, decreased the secretion of OTC without affecting its absorption, while the MRP-inhibitor MK571 did not exert any effect. These data indicate that OTC is a substrate for P-gp. The affinity of OTC to these transporters seems to be rather low, as suggested by the low efflux ratio of 1:1.3. In competition experiments, OTC decreased the effluxes of other P-gp substrates such as Rhodamine123 and ivermectin. These findings are of clinical relevance, as they clearly indicate potential drug-drug interactions at the level of P-gp-mediated drug transport. PMID:17217397

  15. Oral Cyclosporin A Inhibits CD4 T cell P-glycoprotein Activity in HIV-Infected Adults Initiating Treatment with Nucleoside Reverse Transcriptase Inhibitors

    PubMed Central

    Hulgan, Todd; Donahue, John P.; Smeaton, Laura; Pu, Minya; Wang, Hongying; Lederman, Michael M.; Smith, Kimberly; Valdez, Hernan; Pilcher, Christopher; Haas, David W.

    2010-01-01

    Purpose P-glycoprotein limits tissue penetration of many antiretroviral drugs. We characterized effects of the P-glycoprotein substrate cyclosporin A on T cell P-glycoprotein activity in HIV-infected AIDS Clinical Trials Group study A5138 participants. Methods We studied P-glycoprotein activity on CD4 and CD8 T cells in 16 participants randomized to receive oral cyclosporin A (n=9) or not (n=7) during initiation antiretroviral therapy (ART) that did not include protease or non-nucleoside reverse transcriptase inhibitors. Results CD4 T cell P-glycoprotein activity decreased by a median of 8 percentage points with cyclosporin A/ART (difference between cyclosporin A/ART versus ART only P=0.001). Plasma trough cyclosporin A concentrations correlated with change in P-glycoprotein activity in several T cell subsets. Conclusions Oral cyclosporin A can inhibit peripheral blood CD4 T cell P-glycoprotein activity. Targeted P-glycoprotein inhibition might enhance delivery of ART to T cells. PMID:19779705

  16. Trantinterol, a novel β2-adrenoceptor agonist, noncompetitively inhibits P-glycoprotein function in vitro and in vivo.

    PubMed

    Wang, Tingting; Sun, Yantong; Ma, Wenxiao; Yang, Zhichao; Yang, Junfeng; Liu, Jingrui; Fan, Hongbo; Yang, Yan; Gu, Jingkai; Fawcett, John Paul; Guo, Yingjie

    2015-01-01

    P-glycoprotein (P-gp)-mediated drug-drug interactions are important factors causing adverse effects of drugs in clinical use. The aim of this study was to determine whether trantinterol (also known as SPFF), a novel β2-adrenoceptor agonist, was a P-gp inhibitor or substrate. The results showed that trantinterol was not a substrate of P-gp but increased rhodamine 123 (Rho 123) uptake by MDCK-MDR1 cells and decreased the efflux transport of both Rho 123 and cyclosporine A (CsA) in bidirectional transport studies across MDCK-MDR1 cell monolayers. This suggested that trantinterol was a P-gp inhibitor but not a P-gp substrate. The mechanism of inhibition was investigated in the P-gp-Glo assay system, where it was found that trantinterol inhibited P-gp ATPase activity in a dose-dependent manner. A subsequent study using the antibody binding assay with the conformation-sensitive P-gp-specific antibody UIC2 confirmed that trantinterol decreased UIC2 binding at 10 μM in contrast to the competitive inhibitor, verapamil. This suggested that trantinterol was a noncompetitive inhibitor of P-gp. Finally, a pharmacokinetic study in rat showed that trantinterol significantly increased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of digoxin and paclitaxel (PAC), and the Cmax of cyclosporine A (CsA). In summary, trantinterol is a potent noncompetitive P-gp inhibitor which may increase the bioavailability of other P-gp substrate drugs coadministered with it. PMID:25389765

  17. Inhibition of P-glycoprotein activity and chemosensitization of multidrug-resistant ovarian carcinoma 2780AD cells by hexanoylglucosylceramide.

    PubMed

    Veldman, R J; Sietsma, H; Klappe, K; Hoekstra, D; Kok, J W

    1999-12-20

    In the present study we show that neutral hexanoyl-(glyco)sphingolipids inhibit P-glycoprotein (Pgp) activity in human ovarian 2780AD cells. By contrast, hexanoylceramide and the gangliosides GM(3) and GM(2) had no effect on Pgp activity, whereas sphingosine had a stimulating effect. In the case of hexanoylglucosylceramide, inhibition of Pgp activity by was reflected by a regained doxorubicin sensitivity of cells, which were grown in medium supplemented with the lipid. Our results lead to the conclusion that a direct transmodulation of Pgp activity by glycolipids occurs, depending on lipid headgroup structure, which can result in reduced resistance to the chemotherapeutic agent doxorubicin. PMID:10600530

  18. Psoralen reverses the P-glycoprotein-mediated multidrug resistance in human breast cancer MCF-7/ADR cells.

    PubMed

    Jiang, Jingru; Wang, Xiaohong; Cheng, Kai; Zhao, Wanzhong; Hua, Yitong; Xu, Chengfeng; Yang, Zhenlin

    2016-06-01

    The resistance of cancer to chemotherapeutic agents is a major obstacle during chemotherapy. Clinical multidrug resistance (MDR) is commonly mediated by membrane drug efflux pumps, including ATP‑binding cassette subfamily B member 1, also termed P-glycoprotein (P-gp). P-gp is a membrane transporter encoded by the MDR1 gene. The current study aimed to investigate the impact of psoralen on the expression and function of P‑gp. The 10% inhibitory concentration (IC10) of psoralen, and its capacity to reduce MDR in adriamycin (ADR)‑resistant MCF‑7/ADR cells were determined using MTT assay. The ability of psoralen to modulate the transport activity of P‑gp in MCF‑7/ADR cells was evaluated by measuring the accumulation and efflux of rhodamine 123 (Rh 123) and adriamycin with flow cytometry. The present study evaluated the mRNA level of MDR1 in MCF‑7 and MCF‑7/ADR cells treated with psoralen using reverse transcription-quantitative polymerase chain reaction. The protein expression level of P‑gp was examined by western blot analysis. The current study demonstrated that the IC10 of psoralen in MCF‑7/ADR cells was 8 µg/ml. At 8 µg/ml, psoralen reduced MDR and the sensitivity of the MCF‑7/ADR cells to ADR compared with untreated cells. Additionally, psoralen significantly increased the intracellular accumulation of ADR and Rh 123. However, the IC10 of psoralen did not affect the protein expression levels of P‑gp or mRNA levels of MDR1 (P>0.05). Psoralen reduces MDR by inhibiting the efflux function of P‑gp, which may be important for increasing the efficiency of chemotherapy and improving the clinical protocols aiming to reverse P-gp-mediated MDR. PMID:27082231

  19. Reversal of P-glycoprotein-mediated multidrug resistance by the murine double minute 2 antagonist nutlin-3.

    PubMed

    Michaelis, Martin; Rothweiler, Florian; Klassert, Denise; von Deimling, Andreas; Weber, Kristoffer; Fehse, Boris; Kammerer, Bernd; Doerr, Hans Wilhelm; Cinatl, Jindrich

    2009-01-15

    Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3 is a MDM2 inhibitor under preclinical investigation as nongenotoxic activator of the p53 pathway for cancer therapy. Here, nutlin-3 was evaluated for its activity alone or in combination with established chemotherapeutic drugs for antitumor action in chemosensitive and chemoresistant neuroblastoma and rhabdomyosarcoma cell lines. Effects of nutlin-3 single treatment were much more pronounced in p53 wild-type cell lines (IC(50)s <3 micromol/L) than in p53-mutated cell lines (IC(50)s >17 micromol/L). In sharp contrast to the expectations, nutlin-3 concentrations that did not affect viability of p53-mutated cell lines strongly increased the efficacy of vincristine in p53-mutated, P-glycoprotein (P-gp)-overexpressing cell lines (decrease in IC(50)s 92- to 3,434-fold). Similar results were obtained for other P-gp substrates. Moreover, nutlin-3 reduced efflux of rhodamine 123 and other fluorescence dyes that are effluxed by P-gp. Investigation of Madin-Darby canine kidney (MDCK) II cells stably transfected with plasmids encoding for P-gp (MDCKII MDR1) or multidrug resistance protein 1 (MRP-1, MDCKII MRP1) revealed that nutlin-3 not only interferes with P-gp but also affects MRP-1-mediated efflux. Kinetic studies and investigation of P-gp-ATPase activity showed that nutlin-3 is likely to act as a P-gp transport substrate. Examination of the nutlin-3 enantiomers nutlin-3a and nutlin-3b revealed that, in contrast to MDM2-inhibitory activity that is limited to nutlin-3a, both enantiomers similarly interfere with P-gp-mediated drug efflux. In conclusion, nutlin-3-induced inhibition of P-gp and MRP-1 was discovered as a novel anticancer mechanism of the substance in this report. PMID:19147553

  20. The use of amlodipine, but not of P-glycoprotein inhibiting calcium channel blockers is associated with clopidogrel poor-response.

    PubMed

    Harmsze, Ankie M; Robijns, Karen; van Werkum, Jochem W; Breet, Nicoline J; Hackeng, Christian M; Ten Berg, Jurrien M; Ruven, Hendrik J T; Klungel, Olaf H; de Boer, Anthonius; Deneer, Vera H M

    2010-05-01

    Clopidogrel is a prodrug that has to be converted in vivo to its active metabolite by cytochrome (CYP)P450 iso-enzymes. As calcium channel blockers (CCBs) are inhibitors of CYP3A4, concomitant use of these drugs might play a role in the wide inter-individual variability in the response to clopidogrel. However, some CCBs also have strong inhibitory effects on the drug transporter P-glycoprotein (Pgp), which mediates clopidogrel's intestinal absorption. It was the aim of this study to evaluate the effect of co-administration of Pgp-inhibiting and non-Pgp-inhibiting CCBs on on-clopidogrel platelet reactivity in patients on dual antiplatelet therapy undergoing elective percutaneous coronary intervention (PCI). In a total of 623 consecutive patients undergoing elective PCI treated with clopidogrel and aspirin, platelet reactivity to 5 and 20 muM adenosine diphospate (ADP) and clopidogrel poor-response (defined as > 70% platelet aggregation to 20 muM ADP) were evaluated by light transmittance aggregometry. A total of 222 patients (35.6%) were on CCB treatment, of which 98 used Pgp-inhibiting CCBs (verapamil, nifedipine, diltiazem, barnidipine) and 124 patients used the non-Pgp-inhibiting CCB amlodipine. Adjusted mean ADP-induced on-clopidogrel platelet reactivity was significantly higher in both users of Pgp-inhibiting CCBs and amlodipine as compared to CCB non-users (all p<0.05). However, only the use of amlodipine was significantly associated with a 2.3-fold increased risk of clopidogrel poor-response. This study demonstrates that concomitant use of Pgp-inhibiting CCBs and amlodipine increases on-clopidogrel platelet reactivity. Only amlodipine was associated with clopidogrel poor-response. The drug-drug interaction between clopidogrel and amlodipine might be more clinically relevant as compared to P-glycoprotein-inhibiting CCBs. PMID:20352154

  1. P-glycoprotein (ABCB1) inhibited network of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum by systems-theoretical analysis.

    PubMed

    Lin, Hong; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Qi, Lianxiu

    2012-10-01

    We constructed the significant low-expression P-glycoprotein (ABCB1) inhibited transport and signal network in chimpanzee compared with high-expression (fold change ≥2) the human left cerebrum in GEO data set, by using integration of gene regulatory activated and inhibited network inference method with gene ontology (GO) analysis. Our result showed that ABCB1 transport and signal upstream network RAB2A inhibited ABCB1, and downstream ABCB1-inhibited SMAD1_2, NCK2, SLC25A46, GDF10, RASGRP1, EGFR, LRPPRC, RASSF2, RASA4, CA2, CBLB, UBR5, SLC25A16, ITGB3BP, DDIT4, PDPN, RAB2A in chimpanzee left cerebrum. We obtained that the different biological processes of ABCB1 inhibited transport and signal network repressed carbon dioxide transport, ER to Golgi vesicle-mediated transport, folic acid transport, mitochondrion transport along microtubule, water transport, BMP signaling pathway, Ras protein signal transduction, transforming growth factor beta receptor signaling pathway in chimpanzee compared with the inhibited network of the human left cerebrum, as a result of inducing inhibition of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum. Our hypothesis was verified by the same and different biological processes of ABCB1 inhibited transport and signal network of chimpanzee compared with the corresponding activated network of chimpanzee and the human left cerebrum, respectively. PMID:22674380

  2. Tetrandrine and fangchinoline, bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells.

    PubMed

    Sun, Yan Fang; Wink, Michael

    2014-01-01

    The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy. PMID:24856768

  3. Influence of P-glycoprotein inhibition on the distribution of the tricyclic antidepressant nortriptyline over the blood-brain barrier.

    PubMed

    Ejsing, Thomas B; Linnet, Kristian

    2005-03-01

    The distribution of the antidepressant drug nortriptyline (NT) and its main metabolite E-10-hydroxy-nortriptyline (E-10-OH-NT) across the blood-brain barrier was considered in relation to inhibition of the multidrug transporter P-glycoprotein (P-gp). Rats received NT in doses of 25 mg/kg orally, 10 mg/kg i.p. or 25 mg/kg i.p. Half the rats were treated with the P-glycoprotein inhibitor cyclosporine A (CsA) (200 mg/kg) 2 h prior to NT administration, and the other half served as a control group. NT and the metabolite were extracted from brain and serum by liquid-liquid extraction and analysed by HPLC with UV-detection. The brain to serum ratio of NT was increased in the CsA treated groups (22.3-26.8) compared with the control groups (16.5-22.7), the difference being statistically significant in two of the three experiments (p<0.05). Increased brain-serum ratios were also found for E-10-OH-NT, but the differences were not statistically significant. These results suggest that inhibition of P-gp by CsA increases the accumulation of NT in the brain. Administration of the antipsychotic drug risperidone (0.5 mg/kg s.c.), which is a P-gp substrate, instead of CsA did not exert any measurable influence on the blood-brain ratio of NT concentrations. In conclusion, the results show that drug-drug interaction at P-gp may influence the intracerebral NT concentration, but apparently, a major inhibition of P-gp is necessary to attain a measurable effect. PMID:15624117

  4. Predicting P-glycoprotein-mediated drug transport based on support vector machine and three-dimensional crystal structure of P-glycoprotein.

    PubMed

    Bikadi, Zsolt; Hazai, Istvan; Malik, David; Jemnitz, Katalin; Veres, Zsuzsa; Hari, Peter; Ni, Zhanglin; Loo, Tip W; Clarke, David M; Hazai, Eszter; Mao, Qingcheng

    2011-01-01

    Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening. PMID:21991360

  5. Predicting P-Glycoprotein-Mediated Drug Transport Based On Support Vector Machine and Three-Dimensional Crystal Structure of P-glycoprotein

    PubMed Central

    Bikadi, Zsolt; Hazai, Istvan; Malik, David; Jemnitz, Katalin; Veres, Zsuzsa; Hari, Peter; Ni, Zhanglin; Loo, Tip W.; Clarke, David M.; Hazai, Eszter; Mao, Qingcheng

    2011-01-01

    Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening. PMID:21991360

  6. P-glycoprotein Mediates Postoperative Peritoneal Adhesion Formation by Enhancing Phosphorylation of the Chloride Channel-3

    PubMed Central

    Deng, Lulu; Li, Qin; Lin, Guixian; Huang, Dan; Zeng, Xuxin; Wang, Xinwei; Li, Ping; Jin, Xiaobao; Zhang, Haifeng; Li, Chunmei; Chen, Lixin; Wang, Liwei; Huang, Shulin; Shao, Hongwei; Xu, Bin; Mao, Jianwen

    2016-01-01

    P-glycoprotein (P-gp) is encoded by the multidrug resistance (MDR1) gene and is well studied as a multi-drug resistance transporter. Peritoneal adhesion formation following abdominal surgery remains an important clinical problem. Here, we found that P-gp was highly expressed in human adhesion fibroblasts and promoted peritoneal adhesion formation in a rodent model. Knockdown of P-gp expression by intraperitoneal injection of MDR1-targeted siRNA significantly reduced both the peritoneal adhesion development rate and adhesion grades. Additionally, we found that operative injury up-regulated P-gp expression in peritoneal fibroblasts through the TGF-β1/Smad signaling pathway and histone H3 acetylation. The overexpression of P-gp accelerated migration and proliferation of fibroblasts via volume-activated Cl- current and cell volume regulation by enhancing phosphorylation of the chloride channel-3. Therefore, P-gp plays a critical role in postoperative peritoneal adhesion formation and may be a valuable therapeutic target for preventing the formation of peritoneal adhesions. PMID:26877779

  7. The functional influences of common ABCB1 genetic variants on the inhibition of P-glycoprotein by Antrodia cinnamomea extracts.

    PubMed

    Sheu, Ming-Jyh; Teng, Yu-Ning; Chen, Ying-Yi; Hung, Chin-Chuan

    2014-01-01

    Antrodia cinnamomea is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of A. cinnamomea (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of ABCB1 genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC50 concentration of 1.51 ± 0.08 µg/mL through non-competitive inhibition. The IC50 concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56 ± 0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications. PMID:24586917

  8. P-glycoprotein Mediates Ceritinib Resistance in Anaplastic Lymphoma Kinase-rearranged Non-small Cell Lung Cancer

    PubMed Central

    Katayama, Ryohei; Sakashita, Takuya; Yanagitani, Noriko; Ninomiya, Hironori; Horiike, Atsushi; Friboulet, Luc; Gainor, Justin F.; Motoi, Noriko; Dobashi, Akito; Sakata, Seiji; Tambo, Yuichi; Kitazono, Satoru; Sato, Shigeo; Koike, Sumie; John Iafrate, A.; Mino-Kenudson, Mari; Ishikawa, Yuichi; Shaw, Alice T.; Engelman, Jeffrey A.; Takeuchi, Kengo; Nishio, Makoto; Fujita, Naoya

    2015-01-01

    The anaplastic lymphoma kinase (ALK) fusion oncogene is observed in 3%–5% of non-small cell lung cancer (NSCLC). Crizotinib and ceritinib, a next-generation ALK tyrosine kinase inhibitor (TKI) active against crizotinib-refractory patients, are clinically available for the treatment of ALK-rearranged NSCLC patients, and multiple next-generation ALK-TKIs are currently under clinical evaluation. These ALK-TKIs exhibit robust clinical activity in ALK-rearranged NSCLC patients; however, the emergence of ALK-TKI resistance restricts the therapeutic effect. To date, various secondary mutations or bypass pathway activation-mediated resistance have been identified, but large parts of the resistance mechanism are yet to be identified. Here, we report the discovery of p-glycoprotein (P-gp/ABCB1) overexpression as a ceritinib resistance mechanism in ALK-rearranged NSCLC patients. P-gp exported ceritinib and its overexpression conferred ceritinib and crizotinib resistance, but not to PF-06463922 or alectinib, which are next-generation ALK inhibitors. Knockdown of ABCB1 or P-gp inhibitors sensitizes the patient-derived cancer cells to ceritinib, in vitro and in vivo. P-gp overexpression was identified in three out of 11 cases with in ALK-rearranged crizotinib or ceritinib resistant NSCLC patients. Our study suggests that alectinib, PF-06463922, or P-gp inhibitor with ceritinib could overcome the ceritinib or crizotinib resistance mediated by P-gp overexpression. PMID:26870817

  9. The multidrug resistant modulator HZ08 reverses multidrug resistance via P-glycoprotein inhibition and apoptosis sensitization in human epidermoid carcinoma cell line KBV200.

    PubMed

    Zhu, Y-L; Cen, J; Zhang, Y-Y; Feng, Y-D; Yang, Y; Li, Y-M; Huang, W-L

    2012-05-01

    Previous studies have demonstrated that the multidrug resistance modulator HZ08 has a strong multidrug resistance reversal effect in vitro and in vivo by inhibiting P-glycoprotein and multidrug resistance-associated protein 1 in K562/A02 and MCF-7/ADM cells, respectively. However, there are many other mechanisms responsible for resistance. In this study, MTT assay was used to examine the cytotoxicity and multidrug resistance reversal of HZ08 in KBV200 cells. It was also used to detect Rh123 and adriamycin accumulation in the presence of HZ08 to assess the effect on P-glycoprotein. Caspase-3 activity was analyzed under the incubation of HZ08 per se and in combination with vincristine. Results showed that HZ08 could increase the activity of caspase-3 with P-glycoprotein inhibition. Further studies revealed that HZ08 increased vincristine-induced apoptosis, characterized as an intrinsic apoptosis pathway with enhanced G2/M phase arrest, since HZ08 had an effect on the intrinsic apoptotic regulator Bcl-2 and Bax. Therefore, the outstanding reversal effect of HZ08 occurs not only through suppressing the P-glycoprotein function but also through activating the intrinsic apoptosis pathway. PMID:22344570

  10. An isoquinoline alkaloid from the Chinese herbal plant Corydalis yanhusuo W.T. Wang inhibits P-glycoprotein and multidrug resistance-associate protein 1.

    PubMed

    Lei, Yu; Tan, Juan; Wink, Michael; Ma, Yonggang; Li, Na; Su, Guannan

    2013-02-15

    Overexpression of P-glycoprotein (P-gp) and multidrug resistance-associate protein 1 (MRP1) is a major mechanism leading to multidrug resistance (MDR) of cancer cells. These transporters expel anti-cancer drugs and greatly impair therapeutic efficacy of chemotherapy. A Chinese herbal plant Yanhusuo (Corydalis yanhusuo W.T. Wang, YHS) is frequently used in functional food and traditional Chinese medicine to improve the efficacy of chemotherapy. The objective of this work was to study effects of glaucine, an alkaloid component of YHS, on P-gp and MRP1 in resistant cancer cells. The resistant cancer cell line, MCF-7/ADR and corresponding parental sensitive cells were employed to determine reversal properties of glaucine. Glaucine inhibits P-gp and MRP1-mediated efflux and activates ATPase activities of the transporters, indicating that it is a substrate and inhibits P-gp and MRP1 competitively. Furthermore, glaucine suppresses expression of ABC transporter genes. It reverses the resistance of MCF-7/ADR to adriamycin and mitoxantrone effectively. PMID:23194502

  11. Furanocoumarin derivatives in Kampo extract medicines inhibit cytochrome P450 3A4 and P-glycoprotein.

    PubMed

    Iwanaga, Kazunori; Hayashi, Manami; Hamahata, Yukimi; Miyazaki, Makoto; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Kakemi, Masawo

    2010-08-01

    Furanocoumarins in grapefruit are known to show inhibitory effects against P-glycoprotein (P-gp) and CYP3A4 in intestinal epithelial cells; however, furanocoumarin derivatives are widely contained in the plants of Rutaceae and Umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of 12 furanocoumarins extracted from plants in the Umbelliferae family against P-gp and CYP3A4 activity. Furthermore, we studied their inhibitory effect on P-gp when furanocoumarins are used as Kampo extract medicine rather than as an isolated single compound. From screening of the CYP3A4 inhibitory effect, notopterol and rivulobirin A, the only dimer types of furanocoumarin, were found to be potent inhibitors of CYP3A4. On the other hand, byakangelicol and rivulobirin A showed strong P-gp inhibition from the screening of P-gp inhibitor evaluated by quinidine permeation through the Caco-2 monolayer; however, the chemical structural relationship of furanocoumarins between P-gp and CYP3A4 inhibitory effects could not be obtained. We also investigated the effect of these furanocoumarins on the transport of digoxin through the Caco-2 monolayer. The inhibitory effect of rivulobirin A was more potent than that of byakangelicol. Application of either Senkyu-cha-cho-san or Sokei-kakketsu-to, which are composed of herbal remedies in the Umbelliferae group, significantly decreased the efflux ratio of digoxin. In conclusion, it was found that some furanocoumarins extracted from the plants in the Umbelliferae family strongly inhibited P-gp and CYP3A4. Kampo extract medicines containing herbal remedies belonging to the Umbelliferae family may cause a drug-drug interaction with P-gp or a CYP3A4 substrate drug. PMID:20463004

  12. Inhibition of P-glycoprotein function by XR9576 in a solid tumour model can restore anticancer drug efficacy.

    PubMed

    Walker, J; Martin, C; Callaghan, R

    2004-03-01

    Resistance to cancer chemotherapy involves both altered drug activity at the designated target and modified intra-tumour pharmacokinetic properties (e.g. uptake, metabolism). The membrane transporter P-glycoprotein (P-gp) plays a major role in pharmacokinetic resistance by preventing sufficient intracellular accumulation of several anticancer agents. Whilst inhibiting P-gp has great potential to restore chemotherapeutic effectiveness in blood-borne cancers, the situation in solid tumours is less clear. Therefore, the degree of resistance tumours pose to the cytotoxicity of vinblastine and doxorubicin was characterised using the multicellular tumour spheroid model. Tumour spheroids were generated from either drug-sensitive MCF7(WT) breast cancer cells or a resistant P-gp-expressing variant (NCI/ADR(Res)). Drug-induced cytotoxicity in tumour spheroids was measured using an outgrowth assay and compared with that observed in monolayer cultures. As anticipated, the 3-D organisation of MCF7(WT) in tumour spheroids was associated with a reduction in the potency of doxorubicin and vinblastine-i.e. the inherent multicellular resistance phenomenon. In contrast, tumour spheroids from NCI/ADR(Res) cells did not display multicellular resistance. However their constitutive expression of P-gp reduced the potency of both anticancer drugs. Moreover, the highly potent P-gp inhibitor, the anthranilic acid derivative, XR9576, was able to restore the cytotoxic efficacy of both drugs in tumour spheroids comprising NCI/ADR(Res) cells. The results suggest that inhibition of P-gp in solid tumours is achievable and that generation of potent inhibitors will provide a significant benefit towards restoration of chemotherapy in solid tissues. PMID:14962729

  13. The Dual Cyclooxygenase/5-Lipoxygenase Inhibitor Licofelone Attenuates P-Glycoprotein-Mediated Drug Resistance in the Injured Spinal Cord

    PubMed Central

    Dulin, Jennifer N.; Moore, Meredith L.

    2013-01-01

    Abstract There are currently no proven effective treatments that can improve recovery of function in spinal cord injury (SCI) patients. Many therapeutic compounds have shown promise in pre-clinical studies, but clinical trials have been largely unsuccessful. P-glycoprotein (Pgp, Abcb1b) is a drug efflux transporter of the blood–spinal cord barrier that limits spinal cord penetration of blood-borne xenobiotics. Pathological Pgp upregulation in diseases such as cancer causes heightened resistance to a broad variety of therapeutic drugs. Importantly, several drugs that have been evaluated for the treatment of SCI, such as riluzole, are known substrates of Pgp. We therefore examined whether Pgp-mediated pharmacoresistance diminishes delivery of riluzole to the injured spinal cord. Following moderate contusion injury at T10 in male Sprague–Dawley rats, we observed a progressive, spatial spread of increased Pgp expression from 3 days to 10 months post-SCI. Spinal cord uptake of i.p.-delivered riluzole was significantly reduced following SCI in wild type but not Abcb1a-knockout rats, highlighting a critical role for Pgp in mediating drug resistance following SCI. Because inflammation can drive Pgp upregulation, we evaluated the ability of the new generation dual anti-inflammatory drug licofelone to promote spinal cord delivery of riluzole following SCI. We found that licofelone both reduced Pgp expression and enhanced riluzole bioavailability within the lesion site at 72 h post-SCI. This work highlights Pgp-mediated drug resistance as an important obstacle to therapeutic drug delivery for SCI, and suggests licofelone as a novel combinatorial treatment strategy to enhance therapeutic drug delivery to the injured spinal cord. PMID:22947335

  14. P-glycoprotein Inhibition by the Agricultural Pesticide Propiconazole and Its Hydroxylated Metabolites: Implications for Pesticide-Drug Interactions.

    EPA Science Inventory

    The human efflux transporter P-glycoprotein (P-gp; MDR1) functions an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure ass...

  15. P-glycoprotein Inhibition by the Agricultural Pesticide Propiconazole and Its Hydroxylated Metabolites: Implications for Pesticide-Drug Interactions

    EPA Science Inventory

    The human efflux transporter P-glycoprotein (P-gp, MDR1) functions an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure ass...

  16. Region-dependent disappearance of vinblastine in rat small intestine and characterization of its P-glycoprotein-mediated efflux system.

    PubMed

    Nakayama, A; Saitoh, H; Oda, M; Takada, M; Aungst, B J

    2000-10-01

    This study was aimed to characterize the absorption behavior of vinblastine (VLB), a well-known substrate of P-glycoprotein (P-gp), from rat small intestine, especially focusing on the regional-dependence of its efflux mediated by P-gp. VLB disappeared from duodenal and ileal loops of male Wistar rats fairly rapidly (30-60% in 30 min). In contrast, its disappearance from the jejunal loop was almost negligible and in some rats >100% of the jejunal dose was recovered. The radioactivity derived from [3H]VLB, which was absorbed from duodenum and ileum, was detected in the jejunal region. The jejunal appearance of radioactivity was increased when unlabeled VLB was present in the region in advance. The basolateral-to-apical transport of [3H]VLB across Caco-2 cell monolayers was greater when unlabeled VLB was added to the apical medium than when VLB-free buffer was applied to the apical side. When verapamil or cyclosporin A, potent modulators of P-gp, was added to the apical medium together with unlabeled VLB, enhanced basolateral-to-apical transport of [3H]VLB was disappeared. It is suggested that VLB absorption is strongly restricted by P-gp, especially in the jejunal region of the rat small intestine, and that the secretory transport via intestinal P-gp may be subject to trans-stimulation. Moreover, intravenously administered methylprednisolone and intramuscularly administered progesterone significantly enhanced the absorption of VLB, suggesting that parenterally administered P-gp modulators could influence the intestinal absorption of P-gp substrates. PMID:11033075

  17. Metalloprobes: Fluorescence imaging of multidrug resistance (MDR1) P-Glycoprotein (Pgp)-mediated functional transport activity in cellulo.

    PubMed

    Sundaram, G S M; Sharma, Monica; Kaganov, Daniel; Cho, Junsang; Harpstrite, Scott E; Sharma, Vijay

    2016-06-01

    Radiolabeled metalloprobes offer sensitive tools for evaluating quantitative accumulation of chemical entities within pooled cell populations. Although beneficial in translational nuclear imaging, this method precludes interrogation of effects resulting from variations at a single cell level, within the same segment of cell population. Compared with radiotracer bioassays, fluorescence imaging offers a cost-efficient technique to assess accumulation of metalloprobes at a single cell level, and determine their intracellular localization under live cell conditions. To evaluate, whether or not radiotracer assay and fluorescence imaging provide complementary information on utility of metalloprobes to assess functional expression of P-glycoprotein (Pgp) on plasma membrane of tumor cells, imaging studies of fluorescent cationic Ga(III)-ENBDMPI (bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-amino-propyl)ethylenediamine) and its neutral counterpart Zn(II)-ENBDMPI are performed. While the uptake profiles of the cationic metalloprobe are inversely proportional to expression of Pgp in tumor cells, the accumulation profiles of the neutral Zn(II)-ENBDMPI in non-MDR and MDR cells are not significantly impacted. The cationic Ga(III)-ENBDMPI maps with Mito-Tracker Red, thereby confirming localization within mitochondria of non-MDR (Pgp-) cells. Depolarization of both plasmalemmal and mitochondrial potentials decreased retention of the cationic Ga(III)-ENBDMPI within the mitochondria. Additionally, LY335979, an antagonist-induced accumulation of the cationic Ga(III) metalloprobe in MDR (Pgp+) cells indicated specificity of the agent. Compared with traits of Ga(III)-ENBDMPI as a Pgp recognized substrate, Zn(II)-ENBDMPI demonstrated uptake in both MDR and non-MDR cells thus indicating the significance of overall molecular charge in mediating Pgp recognition profiles. Combined data indicate that live cell imaging can offer a cost-effective methodology for monitoring

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

    SciTech Connect

    Arora, Annu; Seth, Kavita; Kalra, Neetu; Shukla, Yogeshwer . E-mail: yogeshwer_shukla@hotmail.com

    2005-02-01

    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{sup -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.

  19. Effect of lyophilized grapefruit juice on P-glycoprotein-mediated drug transport in-vitro and in-vivo.

    PubMed

    Ahmed, Iman S; Hassan, Mariame A; Kondo, Takashi

    2015-03-01

    The administration of grapefruit juice (GFJ) has been postulated to inhibit the activity of P-glycoprotein (P-gp) transport system and thus can enhance the uptake of substrate drugs. However, for various reasons, the results obtained have been always swaying between confirmation and refutation. This study aims at re-evaluating the effect of lyophilized freshly-prepared grapefruit juice (LGFJ) prepared from the whole peeled fruit on P-gp activity using the model drug doxorubicin (DOX) in-vitro and timolol maleate (TM) in-vivo. Human uterine sarcoma MES-SA/DX5v cells, grown under nanomolar concentration of DOX and highly expressing P-gp, were used as model cells for in-vitro studies whereas white New Zealand male rabbits were used for in-vivo studies. Results showed that the accumulation of DOX in MES-SA/DX5v cells was increased by 18.3 ± 2.0% in presence of LGFJ compared to control experiments. Results from in-vivo absorption studies showed that the relative oral bioavailability of TM ingested with LGFJ was significantly higher by 70% and 43% compared to the oral bioavailability of TM ingested with saline and a commercial GFJ, respectively. This study as such confirms the inhibitory effects of LGFJ on P-gp efflux proteins and highlights the superiority of using lyophilized freshly prepared juices over the commercially available juices in research studies. Also, the results call for further studies to assess the possibility of co-administrating LGFJ with anti-cancer agents to modulate multidrug resistance in their cellular environment or incorporating LGFJ in solid dosage forms to improve oral bioavailability of drugs. PMID:24303901

  20. Pregnane X receptor mediates the induction of P-glycoprotein by spironolactone in HepG2 cells.

    PubMed

    Rigalli, Juan Pablo; Ruiz, María Laura; Perdomo, Virginia Gabriela; Villanueva, Silvina Stella Maris; Mottino, Aldo Domingo; Catania, Viviana Alicia

    2011-07-11

    We evaluated the effect of spironolactone (SL), a well-known inducer of biotransformation and elimination pathways, on the expression and activity of P-glycoprotein (P-gp/ABCB1/MDR1), a major xenobiotic transporter, in HepG2 cells, as well as the potential mediation of pregnane X nuclear receptor (PXR). Cells were exposed to SL (1, 5, 10, 20 or 50 μM) for 48 h. Expression of P-gp and its mRNA levels were estimated by Western blotting and real time PCR, respectively. P-gp activity was inversely correlated with the ability of the cells to accumulate the model substrate rhodamine 123 (Rh123, 5 μM), in the presence or absence of verapamil (50 μM), a P-gp inhibitor. At the highest dose of SL tested, P-gp and MDR1 mRNA levels were significantly increased (73 and 108%) with respect to control cells. Rh123 accumulation was concomitantly reduced and verapamil was able to abolish this effect, confirming P-gp participation. Additionally, we tested the cytotoxicity of doxorubicin, a model substrate of P-gp, under inducing conditions. HepG2 cells treated with SL exhibited higher viability, i.e. less doxorubicin toxicity, than control cells, consistent with P-gp up-regulation. When HepG2 cells were treated with SL in the presence of ketoconazole (KTZ), a non-specific nuclear receptor inhibitor, the up-regulation of P-gp was suppressed. To further identify the nuclear receptor involved, cells were transfected with a siRNA directed against human PXR, leading to a 74% decrease in PXR protein levels, which totally abolished SL induction of P-gp. We conclude that SL up-regulates P-gp expression, likely at transcriptional level, and its efflux activity in HepG2 cells. This effect is mediated by PXR. Thus, ligands of PXR such as SL may alter the disposition and toxicity of other xenobiotics, including drugs of therapeutic use, that are P-gp substrates. PMID:21459122

  1. Approaching complete inhibition of P-glycoprotein at the human blood-brain barrier: an (R)-[11C]verapamil PET study.

    PubMed

    Bauer, Martin; Karch, Rudolf; Zeitlinger, Markus; Philippe, Cécile; Römermann, Kerstin; Stanek, Johann; Maier-Salamon, Alexandra; Wadsak, Wolfgang; Jäger, Walter; Hacker, Marcus; Müller, Markus; Langer, Oliver

    2015-05-01

    As P-glycoprotein (Pgp) inhibition at the blood-brain barrier (BBB) after administration of a single dose of tariquidar is transient, we performed positron emission tomography (PET) scans with the Pgp substrate (R)-[(11)C]verapamil in five healthy volunteers during continuous intravenous tariquidar infusion. Total distribution volume (VT) of (R)-[(11)C]verapamil in whole-brain gray matter increased by 273 ± 78% relative to baseline scans without tariquidar, which was higher than previously reported VT increases. During tariquidar infusion whole-brain VT was comparable to VT in the pituitary gland, a region not protected by the BBB, which suggested that we were approaching complete Pgp inhibition at the human BBB. PMID:25669913

  2. Approaching complete inhibition of P-glycoprotein at the human blood–brain barrier: an (R)-[11C]verapamil PET study

    PubMed Central

    Bauer, Martin; Karch, Rudolf; Zeitlinger, Markus; Philippe, Cécile; Römermann, Kerstin; Stanek, Johann; Maier-Salamon, Alexandra; Wadsak, Wolfgang; Jäger, Walter; Hacker, Marcus; Müller, Markus; Langer, Oliver

    2015-01-01

    As P-glycoprotein (Pgp) inhibition at the blood–brain barrier (BBB) after administration of a single dose of tariquidar is transient, we performed positron emission tomography (PET) scans with the Pgp substrate (R)-[11C]verapamil in five healthy volunteers during continuous intravenous tariquidar infusion. Total distribution volume (VT) of (R)-[11C]verapamil in whole-brain gray matter increased by 273±78% relative to baseline scans without tariquidar, which was higher than previously reported VT increases. During tariquidar infusion whole-brain VT was comparable to VT in the pituitary gland, a region not protected by the BBB, which suggested that we were approaching complete Pgp inhibition at the human BBB. PMID:25669913

  3. Quantitative Assessment of the Impact of Fluorine Substitution on P-Glycoprotein (P-gp) Mediated Efflux, Permeability, Lipophilicity, and Metabolic Stability.

    PubMed

    Pettersson, Martin; Hou, Xinjun; Kuhn, Max; Wager, Travis T; Kauffman, Gregory W; Verhoest, Patrick R

    2016-06-01

    Strategic replacement of one or more hydrogen atoms with fluorine atom(s) is a common tactic to improve potency at a given target and/or to modulate parameters such as metabolic stability and pKa. Molecular weight (MW) is a key parameter in design, and incorporation of fluorine is associated with a disproportionate increase in MW considering the van der Waals radius of fluorine versus hydrogen. Herein we examine a large compound data set to understand the effect of introducing fluorine on the risk of encountering P-glycoprotein mediated efflux (as measured by MDR efflux ratio), passive permeability, lipophilicity, and metabolic stability. Statistical modeling of the MDR ER data demonstrated that an increase in MW as a result of introducing fluorine atoms does not lead to higher risk of P-gp mediated efflux. Fluorine-corrected molecular weight (MWFC), where the molecular weight of fluorine has been subtracted, was found to be a more relevant descriptor. PMID:27228214

  4. Nanoparticle Mediated P-Glycoprotein Silencing for Improved Drug Delivery across the Blood-Brain Barrier: A siRNA-Chitosan Approach

    PubMed Central

    Malmo, Jostein; Sandvig, Axel; Vårum, Kjell M.; Strand, Sabina P.

    2013-01-01

    The blood-brain barrier (BBB), composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp), expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin. PMID:23372682

  5. Comparison of 99mTc-sestamibi and doxorubicin to monitor inhibition of P-glycoprotein function

    PubMed Central

    Muzzammil, T; Moore, M J; Hedley, D; Ballinger, J R

    2001-01-01

    P-glycoprotein (Pgp) overexpression is a well-recognized factor in resistance to chemotherapy. Doxorubicin flow cytometry is used to monitor Pgp function in haematological specimens and biopsies from other cancers, and radionuclide imaging with sestamibi has recently shown promise for non-invasive monitoring. In the present study the two methods were directly compared in single-cell suspensions of three variants of the human breast carcinoma cell line MCF7: sensitive MCF7/WT, doxorubicin-selected MCF7/AdrR, and MDR1 -gene-transfected MCF7/BC19 cells with doxorubicin resistance factors of 1, 192, and 14, respectively. Accumulation of sestamibi and mean fluorescence of doxorubicin (5.5 μM) were assessed over 60 min in the presence and absence of Pgp modulators GG918 (0.01 to 0.2 μM) and PSC833 (0.05 to 2.0 μM). Accumulation curves for sestamibi and doxorubicin differed among the cell variants under control conditions, with sestamibi showing a significantly greater difference between WT and resistant cells than doxorubicin. Both GG918 and PSC833 reversed uptake deficits to WT levels for sestamibi in MCF7/BC19 cells and doxorubicin in MCF7/BC19 and MCF7/AdrR cells, but failed to show the same effect for sestamibi in MCF7/AdrR cells (∼30% of MCF7/WT level). Thus, both methods clearly distinguished sensitive from resistant MCF7 variants, with the radionuclide method showing greater sensitivity. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11161402

  6. Factors Governing P-Glycoprotein-Mediated Drug–Drug Interactions at the Blood–Brain Barrier Measured with Positron Emission Tomography

    PubMed Central

    2015-01-01

    The adenosine triphosphate-binding cassette transporter P-glycoprotein (ABCB1/Abcb1a) restricts at the blood–brain barrier (BBB) brain distribution of many drugs. ABCB1 may be involved in drug–drug interactions (DDIs) at the BBB, which may lead to changes in brain distribution and central nervous system side effects of drugs. Positron emission tomography (PET) with the ABCB1 substrates (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed direct comparison of ABCB1-mediated DDIs at the rodent and human BBB. In this work we evaluated different factors which could influence the magnitude of the interaction between tariquidar and (R)-[11C]verapamil or [11C]-N-desmethyl-loperamide at the BBB and thereby contribute to previously observed species differences between rodents and humans. We performed in vitro transport experiments with [3H]verapamil and [3H]-N-desmethyl-loperamide in ABCB1 and Abcb1a overexpressing cell lines. Moreover we conducted in vivo PET experiments and biodistribution studies with (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide in wild-type mice without and with tariquidar pretreatment and in homozygous Abcb1a/1b(−/−) and heterozygous Abcb1a/1b(+/−) mice. We found no differences for in vitro transport of [3H]verapamil and [3H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. [3H]-N-Desmethyl-loperamide was transported with a 5 to 9 times higher transport ratio than [3H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for [11C]-N-desmethyl-loperamide than for (R)-[11C]verapamil. Both radiotracers showed tariquidar dose dependent increases in brain distribution with tariquidar half-maximum inhibitory concentrations (IC50) of 1052 nM (95% confidence interval CI: 930–1189) for (R)-[11C]verapamil and 1329 nM (95% CI: 980–1801) for [11C]-N-desmethyl-loperamide. In homozygous Abcb1a/1b(−/−) mice brain

  7. Inhibition of P-glycoprotein enhances transport of imipramine across the blood–brain barrier: microdialysis studies in conscious freely moving rats

    PubMed Central

    O'Brien, FE; Clarke, G; Fitzgerald, P; Dinan, TG; Griffin, BT; Cryan, JF

    2012-01-01

    BACKGROUND AND PURPOSE Recent studies indicate that efflux of antidepressants by the multidrug resistance transporter P-glycoprotein (P-gp) at the blood–brain barrier (BBB) may contribute to treatment-resistant depression (TRD) by limiting intracerebral antidepressant concentrations. In addition, clinical experience shows that adjunctive treatment with the P-gp inhibitor verapamil may improve the clinical outcome in TRD. Therefore, the present study aimed to investigate the effect of P-gp inhibition on the transport of the tricyclic antidepressant imipramine and its active metabolite desipramine across the BBB. EXPERIMENTAL APPROACH Intracerebral microdialysis in rats was used to monitor brain levels of imipramine and desipramine following i.v. imipramine administration, with or without pretreatment with one of the P-gp inhibitors verapamil or cyclosporin A (CsA). Plasma drug levels were also determined at regular intervals. KEY RESULTS Pretreatment with either verapamil or CsA resulted in significant increases in imipramine concentrations in the microdialysis samples, without altering imipramine plasma pharmacokinetics. Furthermore, pretreatment with verapamil, but not CsA, led to a significant elevation in plasma and brain levels of desipramine. CONCLUSIONS AND IMPLICATIONS The present study demonstrated that P-gp inhibition enhanced the intracerebral concentration of imipramine, thus supporting the hypothesis that P-gp activity restricts brain levels of certain antidepressants, including imipramine. These findings may help to explain reports of a beneficial response to adjunctive therapy with verapamil in TRD. PMID:22250926

  8. Inhibition of P-glycoprotein expression and function by anti-diabetic drugs gliclazide, metformin, and pioglitazone in vitro and in situ

    PubMed Central

    Abbasi, Mehran Mesgari; Valizadeh, Hadi; Hamishehkar, Hamed; Zakeri-Milani, Parvin

    2016-01-01

    P-glycoprotein (P-gp) is a trans-membrane drug efflux pump. Several drugs are P-gp substrates. Some drugs may affect the activity of P-gp by inhibiting its function, resulting in significant drug-drug interactions (DDIs). It is critical to understand which drugs are inhibitors of P-gp so that adverse DDIs can be minimized or avoided. This study investigated the effects of gliclazide, metformin, and pioglitazone on the function and expression of P-gp. Rhodamine 123 (Rh 123) efflux assays in Caco-2 cells and western blot testing were used to study in vitro the effect of the drugs on P-gp function and expression. The in situ rat single-pass intestinal permeability model was developed to study the effect of the drugs on P-gp function. Digoxin and verapamil were used as a known substrate and inhibitor of P-gp, respectively. Digoxin levels in intestinal perfusion samples were analyzed by high-performance liquid chromatography. Intestinal effective permeability (Peff) of digoxin in the presence of 0.1, 10, and 500 μM gliclazide, 100 and 7000 μM metformin, and 50 and 300 μM pioglitazone was significantly increased relative to the digoxin treated cells (P < 0.01). P-gp expression was decreased by gliclazide, metformin and pioglitazone. Intracellular accumulation of Rh 123 by the drugs increased, but the differences were not significant relative to the control cells (P > 0.05). It was found that gliclazide, metformin, and pioglitazone inhibited P-gp efflux activity in situ and down-regulated P-gp expression in vitro. Further investigations are necessary to confirm the obtained results and to define the mechanism underlying P-gp inhibition by the drugs. PMID:27499787

  9. Inhibition of P-glycoprotein expression and function by anti-diabetic drugs gliclazide, metformin, and pioglitazone in vitro and in situ.

    PubMed

    Abbasi, Mehran Mesgari; Valizadeh, Hadi; Hamishehkar, Hamed; Zakeri-Milani, Parvin

    2016-01-01

    P-glycoprotein (P-gp) is a trans-membrane drug efflux pump. Several drugs are P-gp substrates. Some drugs may affect the activity of P-gp by inhibiting its function, resulting in significant drug-drug interactions (DDIs). It is critical to understand which drugs are inhibitors of P-gp so that adverse DDIs can be minimized or avoided. This study investigated the effects of gliclazide, metformin, and pioglitazone on the function and expression of P-gp. Rhodamine 123 (Rh 123) efflux assays in Caco-2 cells and western blot testing were used to study in vitro the effect of the drugs on P-gp function and expression. The in situ rat single-pass intestinal permeability model was developed to study the effect of the drugs on P-gp function. Digoxin and verapamil were used as a known substrate and inhibitor of P-gp, respectively. Digoxin levels in intestinal perfusion samples were analyzed by high-performance liquid chromatography. Intestinal effective permeability (Peff) of digoxin in the presence of 0.1, 10, and 500 μM gliclazide, 100 and 7000 μM metformin, and 50 and 300 μM pioglitazone was significantly increased relative to the digoxin treated cells (P < 0.01). P-gp expression was decreased by gliclazide, metformin and pioglitazone. Intracellular accumulation of Rh 123 by the drugs increased, but the differences were not significant relative to the control cells (P > 0.05). It was found that gliclazide, metformin, and pioglitazone inhibited P-gp efflux activity in situ and down-regulated P-gp expression in vitro. Further investigations are necessary to confirm the obtained results and to define the mechanism underlying P-gp inhibition by the drugs. PMID:27499787

  10. Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein.

    PubMed

    Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro; Ogihara, Takuo

    2016-08-01

    P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. PMID:27286705

  11. Novel polymer micelle mediated co-delivery of doxorubicin and P-glycoprotein siRNA for reversal of multidrug resistance and synergistic tumor therapy.

    PubMed

    Zhang, Chun-Ge; Zhu, Wen-Jing; Liu, Yang; Yuan, Zhi-Qiang; Yang, Shu-di; Chen, Wei-Liang; Li, Ji-Zhao; Zhou, Xiao-Feng; Liu, Chun; Zhang, Xue-Nong

    2016-01-01

    Co-delivery of chemotherapeutics and siRNA with different mechanisms in a single system is a promising strategy for effective cancer therapy with synergistic effects. In this study, a triblock copolymer micelle was prepared based on the polymer of N-succinyl chitosan-poly-L-lysine-palmitic acid (NSC-PLL-PA) to co-deliver doxorubicin (Dox) and siRNA-P-glycoprotein (P-gp) (Dox-siRNA-micelle). Dox-siRNA-micelle was unstable in pH 5.3 medium than in pH 7.4 medium, which corresponded with the in vitro rapid release of Dox and siRNA in acidic environments. The antitumor efficacy of Dox-siRNA-micelle in vitro significantly increased, especially in HepG2/ADM cells, which was due to the downregulation of P-gp. Moreover, almost all the Dox-siRNA-micelles accumulated in the tumor region beyond 24 h post-injection, and the co-delivery system significantly inhibited tumor growth with synergistic effects in vivo. This study demonstrated the effectiveness of Dox-siRNA-micelles in tumor-targeting and MDR reversal, and provided a promising strategy to develop a co-delivery system with synergistic effects for combined cancer therapy. PMID:27030638

  12. Borneol Depresses P-Glycoprotein Function by a NF-κB Signaling Mediated Mechanism in a Blood Brain Barrier in Vitro Model

    PubMed Central

    Fan, Xiang; Chai, Lijuan; Zhang, Han; Wang, Yuefei; Zhang, Boli; Gao, Xiumei

    2015-01-01

    P-glycoprotein (P-gp) on brain microvascular endothelial cells (BMECs) that form the blood brain barrier (BBB), influences transportation of substances between blood and brain. The objective of this study was to characterize the effects of borneol on P-gp efflux function on BBB and explore the potential mechanisms. We established an in vitro BBB model comprised of rat BMECs and astrocytes to measure the effects of borneol on the known P-gp substrates transport across BBB, and examined the function and expression of P-gp in BMECs and the signaling pathways regulating P-gp expression. Borneol increased intracellular accumulation of Rhodamine 123, enhanced verapamil and digoxin across the BBB in vitro model, and depressed mdr1a mRNA and P-gp expression. Borneol could activate nuclear factor-κB (NF-κB) and inhibition of NF-κB with MG132 (carbobenzoxy-Leu-Leu-leucinal) and SN50 (an inhibitory peptide) obscuring the P-gp decreases induced by borneol. These data suggested that borneol depresses P-gp function in BMECs by a NF-κB signaling medicated mechanism in a BBB in vitro model. PMID:26593909

  13. Novel polymer micelle mediated co-delivery of doxorubicin and P-glycoprotein siRNA for reversal of multidrug resistance and synergistic tumor therapy

    PubMed Central

    Zhang, Chun-ge; Zhu, Wen-jing; Liu, Yang; Yuan, Zhi-qiang; Yang, Shu-di; Chen, Wei-liang; Li, Ji-zhao; Zhou, Xiao-feng; Liu, Chun; Zhang, Xue-nong

    2016-01-01

    Co-delivery of chemotherapeutics and siRNA with different mechanisms in a single system is a promising strategy for effective cancer therapy with synergistic effects. In this study, a triblock copolymer micelle was prepared based on the polymer of N-succinyl chitosan–poly-L-lysine–palmitic acid (NSC–PLL–PA) to co-deliver doxorubicin (Dox) and siRNA–P-glycoprotein (P-gp) (Dox–siRNA-micelle). Dox–siRNA-micelle was unstable in pH 5.3 medium than in pH 7.4 medium, which corresponded with the in vitro rapid release of Dox and siRNA in acidic environments. The antitumor efficacy of Dox–siRNA-micelle in vitro significantly increased, especially in HepG2/ADM cells, which was due to the downregulation of P-gp. Moreover, almost all the Dox–siRNA-micelles accumulated in the tumor region beyond 24 h post-injection, and the co-delivery system significantly inhibited tumor growth with synergistic effects in vivo. This study demonstrated the effectiveness of Dox–siRNA-micelles in tumor-targeting and MDR reversal, and provided a promising strategy to develop a co-delivery system with synergistic effects for combined cancer therapy. PMID:27030638

  14. Apical-to-basolateral transport of amyloid-β peptides through blood-brain barrier cells is mediated by the receptor for advanced glycation end-products and is restricted by P-glycoprotein.

    PubMed

    Candela, Pietra; Gosselet, Fabien; Saint-Pol, Julien; Sevin, Emmanuel; Boucau, Marie-Christine; Boulanger, Eric; Cecchelli, Roméo; Fenart, Laurence

    2010-01-01

    Several studies have highlighted the close relationship between Alzheimer's disease (AD) and alterations in the bidirectional transport of amyloid-β (Aβ) peptides across the blood-brain barrier (BBB). The brain capillary endothelial cells (BCECs) that compose the BBB express the receptors and transporters that enable this transport process. There is significant in vivo evidence to suggest that P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) restrict Aβ peptides entry into the brain, whereas the receptor for advanced glycation end-products (RAGE) seems to mediate apical-to-basolateral passage across the BBB. However, deciphering the molecular mechanisms underlying these in vivo processes requires further in vitro characterization. Using an in vitro BBB model and specific competition experiments against RAGE, we have observed a significant decrease in apical-to-basolateral (but not basolateral-to-apical) transport of Aβ1-40 and Aβ1-42 peptides through BCECs. This transport is a caveolae-dependent process and fits with the apical location of RAGE observed in confocal microscopy experiments. Inhibition of P-gp and BCRP using different inhibitors increases transport of Aβ peptides suggesting that these efflux pumps are involved in Aβ peptide transport at the BCECs level. Taken as a whole, these results demonstrate the involvement of the caveolae-dependent transcytosis of Aβ peptides through the BBB in a RAGE-mediated transport process, reinforcing the hypothesis whereby this receptor is a potential drug target in AD. PMID:20858979

  15. Role of P-glycoprotein in mediating rivastigmine effect on amyloid-β brain load and related pathology in Alzheimer's disease mouse model.

    PubMed

    Mohamed, Loqman A; Keller, Jeffrey N; Kaddoumi, Amal

    2016-04-01

    Recently, we showed that rivastigmine decreased amyloid-β (Aβ) brain load in aged rats by enhancing its clearance across the blood-brain barrier (BBB) via upregulation of P-glycoprotein (P-gp) and low-density lipoprotein receptor-related protein 1 (LRP1). Here, we extend our previous work to clarify P-gp role in mediating rivastigmine effect on Aβ brain levels and neuroprotection in a mouse model of Alzheimer's disease (AD) that expresses different levels of P-gp. APPSWE mice were bred with mdr1a/b knockout mice to produce littermates that were divided into three groups; APP(+)/mdr1(+/+), APP(+)/mdr1(+/-) and APP(+)/mdr1(-/-). Animals received rivastigmine treatment (0.3mg/kg/day) or vehicle for 8weeks using Alzet osmotic mini-pumps. ELISA analysis of brain homogenates for Aβ showed rivastigmine treatment to significantly decrease Aβ brain load in APP(+)/mdr1(+/+) by 25% and in APP(+)/mdr1(+/-) mice by 21% compared to their vehicle treated littermates, but not in APP(+)/mdr1(-/-) mice. In addition, rivastigmine reduced GFAP immunostaining of astrocytes by 50% and IL-1β brain level by 43% in APP(+)/mdr1(+/+) mice, however its effect was less pronounced in P-gp knockout mice. Moreover, rivastigmine demonstrated a P-gp expression dependent neuroprotective effect that was highest in APP(+)/mdr1(+/+)>APP(+)/mdr1(+/-)>APP(+)/mdr1(-/-) as determined by expression of synaptic markers PSD-95 and SNAP-25 using Western blot analysis. Collectively, our results suggest that P-gp plays important role in mediating rivastigmine non-cholinergic beneficial effects, including Aβ brain load reduction, neuroprotective and anti-inflammatory effects in the AD mouse models. PMID:26780497

  16. ANTIPSYCHOTICS REVERSE P-GLYCOPROTEIN-MEDIATED DOXORUBICIN RESISTANCE IN HUMAN UTERINE SARCOMA MES-SA/Dx5 CELLS: A NOVEL APPROACH TO CANCER CHEMOTHERAPY.

    PubMed

    Angelini, A; Ciofani, G; Conti, P

    2015-01-01

    Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) remains one of the major obstacles to effective cancer chemotherapy. Several chemosensitizers have been used in vivo and in vitro to reverse MDR but have exhibited several unwanted side effects. Antipsychotics are often administered to treat psychiatric disorders such as delirium, anxiety and sleep disorders in cancer patients during chemotherapy. The present in vitro study, examined the effects of two common antipsychotic compounds, haloperidol and risperidone, and a natural compound such as theobromine on reversing MDR Pgp-mediated, to evaluate their potential use as chemosensitizing agents. The human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) that overexpress Pgp (100-fold), were treated with the antipsychotic alone (1, 10 and 20 μM) or in combination with different concentrations of doxo (2, 4 and 8 μM). The accumulation and cytotoxicity of doxo (MTT assay) and cellular GSH content (GSH assay) in comparison with verapamil, a well-known Pgp inhibitor, used as reference molecule were examined. It was found that the three compounds significantly enhanced the intracellular accumulation of doxo in resistant cancer cells, when compared with cells receiving doxo alone (p<0.05). Furthermore, compounds showed strong potency to increase doxo cytotoxicity toward resistant MES-SA/Dx5 cells, when compared with untreated control cells. The antipsychotic compounds also significantly increased GSH content at all concentrations (> 30%) in resistant cells, when compared to untreated control cells (p<0.05). These findings suggest that the antipsychotics or their derivatives might represent a novel class of reversal agents for overcoming MDR in cancer therapy, in particular theobromine showed to be an effective Pgp inhibitor with the lowest toxicity. PMID:26122223

  17. Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation

    SciTech Connect

    Piwnica-Worms, D.; Vallabhaneni, V.R.; Kronauge, J.F.

    1995-09-26

    Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an {approximately}170 integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis(2-methoxyisobutyl isonitrile) technetium(I) (Tc-SESTAMIBI), a {gamma}-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (T{sub 1/2} {approx} 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. 70 refs., 7 figs., 2 tabs.

  18. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    PubMed

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. PMID:26918948

  19. Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells.

    PubMed

    Sampson, Kathleen E; Brinker, Amanda; Pratt, Jennifer; Venkatraman, Neetu; Xiao, Yongling; Blasberg, Jim; Steiner, Toni; Bourner, Maureen; Thompson, David C

    2015-02-01

    Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 μM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-2',7'-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches. PMID:25388687

  20. Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A, a compound isolated from Hedyotis corymbosa.

    PubMed

    Yue, Grace Gar-Lee; Kin-Ming Lee, Julia; Cheng, Ling; Chung-Lap Chan, Ben; Jiang, Lei; Fung, Kwok-Pui; Leung, Ping-Chung; Bik-San Lau, Clara

    2012-06-01

    Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study. The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [(3)H]-thymidine incorporation assay. Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC(50) = 43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC(50) > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells. Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor. PMID:22352391

  1. Population pharmacokinetic modelling of non-linear brain distribution of morphine: influence of active saturable influx and P-glycoprotein mediated efflux

    PubMed Central

    Groenendaal, D; Freijer, J; de Mik, D; Bouw, M R; Danhof, M; de Lange, E C M

    2007-01-01

    Background and purpose: Biophase equilibration must be considered to gain insight into the mechanisms underlying the pharmacokinetic-pharmacodynamic (PK-PD) correlations of opioids. The objective was to characterise in a quantitative manner the non-linear distribution kinetics of morphine in brain. Experimental approach: Male rats received a 10-min infusion of 4 mg kg−1 of morphine, combined with a continuous infusion of the P-glycoprotein (Pgp) inhibitor GF120918 or vehicle, or 40 mg kg−1 morphine alone. Unbound extracellular fluid (ECF) concentrations obtained by intracerebral microdialysis and total blood concentrations were analysed using a population modelling approach. Key results: Blood pharmacokinetics of morphine was best described with a three-compartment model and was not influenced by GF120918. Non-linear distribution kinetics in brain ECF was observed with increasing dose. A one compartment distribution model was developed, with separate expressions for passive diffusion, active saturable influx and active efflux by Pgp. The passive diffusion rate constant was 0.0014 min−1. The active efflux rate constant decreased from 0.0195 min−1 to 0.0113 min−1 in the presence of GF120918. The active influx was insensitive to GF120918 and had a maximum transport (Nmax/Vecf) of 0.66 ng min−1 ml−1 and was saturated at low concentrations of morphine (C50=9.9 ng ml−1). Conclusions and implications: Brain distribution of morphine is determined by three factors: limited passive diffusion; active efflux, reduced by 42% by Pgp inhibition; low capacity active uptake. This implies blood concentration-dependency and sensitivity to drug-drug interactions. These factors should be taken into account in further investigations on PK-PD correlations of morphine. PMID:17471182

  2. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

    SciTech Connect

    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong; Li, Yunman; Huang, Wenlong

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein

  3. Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

    SciTech Connect

    Zaja, Roko; Caminada, Daniel; Loncar, Jovica; Fent, Karl; Smital, Tvrtko

    2008-03-01

    The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 {mu}M doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin

  4. Stereoselective Property of 20(S)-Protopanaxadiol Ocotillol Type Epimers Affects Its Absorption and Also the Inhibition of P-Glycoprotein

    PubMed Central

    Wang, Wenyan; Wu, Xiangmeng; Wang, Li; Meng, Qingguo; Liu, Wanhui

    2014-01-01

    Stereoselectivity has been proved to be tightly related to drug action including pharmacodynamics and pharmacokinetics. (20S,24R)-epoxy-dammarane-3,12,25-triol (24R-epimer) and (20S,24S)-epoxy-dammarane-3,12,25-triol (24S-epimer), a pair of 20(S)-protopanaxadiol (PPD) ocotillol type epimers, were the main metabolites of PPD. Previous studies have shown that 24R-epimer and 24S-epimer had stereoselectivity in pharmacological action and pharmacokinetics. In the present study, the aim was to further study the pharmacokinetic characteristics of both epimers, investigate their absorption mechanism and analyze the selectivity effects of ocotillol type side chain and C24 stereo-configuration on P-glycoprotein (P-gp) in vivo and in vitro. Results showed that the absolute bioavailability of 24R-epimer was about 14-fold higher than that of 24S-epimer, and a linear kinetic characteristic was acquired in doses of 5–20 mg/kg for both epimers after oral administration. Furthermore, the apparent permeability coefficients of 24R-epimer were 5–7 folds higher than that of 24S-epimer having lower efflux ratios in Caco-2 cell models. Moreover, both 24R-epimer and 24S-epimer had similar inhibitory effects on P-gp by increasing cellular retention of rhodamine 123 in Caco-2 cells and decreasing efflux of digoxin across Caco-2 cell monolayers. In situ in vivo experiments showed that the inhibition of 24R-epimer on P-gp was stronger than that of 24S-epimer by single-pass intestinal perfusion of rhodamine 123 in rats. Western blot analyses demonstrated that both epimers had no action on P-gp expression in Caco-2 cells. In conclusion, with respect to the stereoselectivity, C24 S-configuration of the ocotillol type epimers processed a poor transmembrane permeability and could be distinguished by P-gp. Sharing a dammarane skeleton, both 24R-epimer and 24S-epimer were potent inhibitors of P-gp. This study provides a new case of stereoselective pharmacokinetics of chiral compounds which

  5. Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp

    SciTech Connect

    Li, Xue Hu, Jinping Wang, Baolian Sheng, Li Liu, Zhihao Yang, Shuang Li, Yan

    2014-03-01

    Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb–drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F{sub 1} [20(S)-GF{sub 1}] exhibited significant inhibition (> 50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC{sub 50} = 9.42 μM), followed by 18β-GA (IC{sub 50} = 21.78 μM), 20(S)-GF{sub 1} (IC{sub 50} = 76.08 μM) and DAG (IC{sub 50} = 77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with K{sub m} and V{sub max} values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF{sub 1} exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure–inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC{sub 0−t} and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF{sub 1} and Rh{sub 1} at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb–drug interactions in humans. - Highlights: • Emodin, 18

  6. SDZ 280-446, a novel semi-synthetic cyclopeptolide: in vitro and in vivo circumvention of the P-glycoprotein-mediated tumour cell multidrug resistance.

    PubMed Central

    Loor, F.; Boesch, D.; Gavériaux, C.; Jachez, B.; Pourtier-Manzanedo, A.; Emmer, G.

    1992-01-01

    SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species (mouse, human, Chinese hamster) representing four different cell lineages (monocytic leukaemia, nasopharyngeal epithelial carcinoma, colon epithelial carcinoma, ovary fibroblastoid carcinoma), and using four different drug classes (colchicine, vincristine, daunomycin/doxorubicin and etoposide). By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers (including verapamil, quinidine and amiodarone which have already entered clinical trials in MDR reversal). Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4 h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly

  7. Grape Seed Procyanidin Reversal of P-glycoprotein Associated Multi-Drug Resistance via Down-regulation of NF-κB and MAPK/ERK Mediated YB-1 Activity in A2780/T Cells

    PubMed Central

    Wang, Sheng-qi; Duan, Lian; Huo, Qi-lu; Ren, Fei; Li, Guo-feng

    2013-01-01

    The expression and function of P-glycoprotein (P-gp) is associated with the phenotype of multi-drug resistance (MDR), leading chemotherapy failure of patients suffered with cancer. Grape seed procyanidin(GSP) is a natural polyphenol supplement with anti-inflammatory effect. Present study assessed a new use of GSP on the MDR reversal activity and its possible molecular mechanisms in MDR1-overpressing paclitaxel resistant ovarian cancer cells. Our results showed GSP significantly enhanced the cytotoxicity of paclitaxel and adriamycin in paclitaxel resistant A2780/T cells but its parental A2780 cells. Furthermore, GSP strongly inhibited P-gp expression by blocking MDR1 gene transcription, as well as, increased the intracellular accumulation of the P-gp substrate rhodamine-123 in A2780/T cells. Nuclear factor-κB(NF-κB) activity, IκB degradation level and NF-κB/p65 nuclear translocation induced by lipopolysaccharide (LPS) and receptor activator for nuclear factor-κB ligand (RANKL) were markedly inhibited by pre-treatment with GSP. Meanwhile, GSP inhibited MAPK/ERK pathway by decreasing the phosphorylation of ERK1/2, resulting in reduced the Y-box binding protein 1 (YB-1) activation with blocking its nuclear translocation. Moreover, the up-regulation of P-gp expression, the activation of AKT/NF-κB and MAPK/ERK pathway induced by LPS was attenuated by GSP administration. Compared with PDTC and U1026, inhibitor of NF-κB and MAPK/ERK respectively, GSP showed the same tendency of down-regulating NF-κB and MAPK/ERK mediated YB-1 activities. Thus, GSP reverses P-gp associated MDR by inhibiting the function and expression of P-gp through down-regulation of NF-κB activity and MAPK/ERK pathway mediated YB-1 nuclear translocation, offering insight into the mechanism of reversing MDR by natural polyphenol supplement compounds. GSP could be a new potential MDR reversal agent used for combination therapy with chemotherapeutics in clinic. PMID:23967153

  8. Dose-response assessment of tariquidar and elacridar and regional quantification of P-glycoprotein inhibition at the rat blood-brain barrier using (R)-[11C]verapamil PET

    PubMed Central

    Kuntner, Claudia; Bankstahl, Jens P.; Bankstahl, Marion; Stanek, Johann; Wanek, Thomas; Stundner, Gloria; Karch, Rudolf; Brauner, Rebecca; Meier, Martin; Ding, Xiaoqi; Müller, Markus; Löscher, Wolfgang; Langer, Oliver

    2013-01-01

    Purpose Overactivity of the multidrug efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) is believed to play an important role in resistance to central nervous system drug treatment. (R)-[11C]verapamil (VPM) PET can be used to measure the function of P-gp at the BBB, but low brain uptake of VPM hampers the mapping of regional differences in cerebral P-gp function and expression. The aim of this study was to evaluate the dose-response relationship of two potent P-gp inhibitors and to investigate if increased brain uptake of VPM mediated by P-gp inhibition can be used to assess regional differences in P-gp activity. Methods Two groups of Sprague-Dawley rats (n=12) underwent single VPM PET scans at 120 min after administration of different doses of the P-gp inhibitors tariquidar and elacridar. In an additional 6 rats, paired VPM PET scans were performed before and after administration of 3 mg/kg tariquidar. Results Inhibitor administration resulted in an up to 11-fold increase in VPM brain distribution volumes (DV) with ED50 values of 3.0±0.2 and 1.2±0.1 mg/kg for tariquidar and elacridar, respectively. In paired PET scans, 3 mg/kg tariquidar resulted in regionally different enhancement of brain activity distribution, with lowest DV in cerebellum and highest DV in thalamus. Conclusion Our data show that tariquidar and elacridar are able to increase VPM brain distribution in rat brain up to 11-fold over baseline at maximum effective doses, with elacridar being about 3 times more potent than tariquidar. Regional differences in tariquidar-induced modulation of VPM brain uptake point to regional differences in cerebral P-gp function and expression in rat brain. PMID:20016890

  9. Non-alkaloids extract from Stemona sessilifolia enhances the activity of chemotherapeutic agents through P-glycoprotein-mediated multidrug-resistant cancer cells.

    PubMed

    Han, Lu; Ma, Yang-Mei; An, Li; Zhang, Qiao; Wang, Chang-Li; Zhao, Qing-Chun

    2016-01-01

    One of the major impediments to the successful treatment of cancer is the development of resistant cancer cells, which could cause multidrug resistance (MDR), and overexpression of ABCB1/P-glycoprotein (P-gp) is one of the most common causes of MDR in cancer cells. Recently, natural products or plant-derived chemicals have been investigated more and more widely as potential multidrug-resistant (MDR) reversing agents. The current study demonstrated for the first time that non-alkaloids extract from Stemona sessilifolia significantly reversed the resistance of chemotherapeutic agents, adriamycin, paclitaxel and vincristine to MCF-7/ADR cells compared with MCF-7/S cells in a dose-dependent manner. The results obtained from these studies indicated that the non-alkaloids extract from S. sessilifolia plays an important role in reversing MDR of cancer as a P-gp modulator in vitro and may be effective in the treatment of multidrug-resistant cancers. PMID:26190165

  10. Kinetic analysis of human and canine P-glycoprotein-mediated drug transport in MDR1-MDCK cell model: approaches to reduce false-negative substrate classification.

    PubMed

    Li, Jibin; Wang, Ying; Hidalgo, Ismael J

    2013-09-01

    Madin-Darby canine kidney (MDCK) cells transfected with the multidrug resistance 1 (MDR1) gene, MDR1-MDCK, are widely used as an in vitro model to classify compounds as human P-glycoprotein (hPgp) substrates or nonsubstrates. Because MDCK cells express endogenous canine Pgp (cPgp), which is prone to downregulation after transfection with hPgp, this situation could lead to false-negative classification of hPgp substrates. The aim of this study was to investigate factors that influence hPgp substrate classification in MDR1-MDCK model and to seek ways to reduce false classification. Three-compartment models were used to derive flux equations describing the drug transport processes; factors influencing hPgp substrate classification were evaluated by simulations. Pgp functionality was assessed by determining the bidirectional permeability of a series of test compounds. Expressions of hPgp and cPgp were measured by quantitative polymerase chain reaction (qPCR). Kinetic model analysis revealed that the current net flux ratio calculation for hPgp substrate classification is influenced by endogenous cPgp expression as well as hPgp-cPgp expression ratio; the effect was more pronounced in low hPgp-cPgp region and diminished in high ratio region. On the basis of kinetic considerations, this study provides a rational experimental approach and appropriate mathematical corrections to minimize the potential occurrence of false-negative classification of new molecular entities. PMID:23558561

  11. Resistant mechanisms of anthracyclines--pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues.

    PubMed

    Kubota, T; Furukawa, T; Tanino, H; Suto, A; Otan, Y; Watanabe, M; Ikeda, T; Kitajima, M

    2001-01-01

    Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing "permeability" of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdr1 gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of "ATP-Binding Cassette transporter"(ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc.htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express P-gp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear. PMID:11791127

  12. P-glycoprotein- and organic anion-transporting polypeptide-mediated transport of periplocin may lead to drug–herb/drug–drug interactions

    PubMed Central

    Liang, Sheng; Deng, Fengchun; Xing, Haiyan; Wen, He; Shi, Xiaoyan; Martey, Orleans Nii; Koomson, Emmanuel; He, Xin

    2014-01-01

    Periplocin, an active and toxic component of the traditional Chinese herbal medicine Periploca sepium Bge, is a cardiac glycoside compound that has been implicated in various clinical accidents. This study investigated the role of transporters in the intestinal absorption and biliary excretion of periplocin, as well as the possible metabolic mechanism of periplocin in liver S9. In a bidirectional transport assay using Madin–Darby canine kidney (MDCK) and MDCK multidrug-resistance protein (MRP)-1 cell monolayers, both in situ intestinal and liver-perfusion models were used to evaluate the role of efflux and uptake transporters on the absorption and biliary excretion of periplocin. In addition, in vitro metabolism of periplocin was investigated by incubating with human/rat liver S9 homogenate fractions to evaluate its metabolic mechanisms in liver metabolic enzymes. The results showed that P-glycoprotein (P-gp) was involved in the intestinal absorption of periplocin, whereas MRP2 and breast cancer-resistance protein were not. The efflux function of P-gp may be partly responsible for the low permeability and bioavailability of periplocin. Moreover, both inhibitors of P-gp and organic anion-transporting polypeptides (OATPs) increased periplocin biliary excretion. No obvious indications of metabolism were observed in the in vitro incubation system, which suggests that periplocin did not interact with the hepatic drug metabolic enzymes. The results of this study showed that the efflux and uptake transporters P-gp and OATPs were involved in the absorption and biliary excretion of periplocin, which may partially account for its low permeability and bioavailability. As a toxic compound, potential drug–herb/herb–herb interactions based on OATPs and P-gp should be taken into account when using P. sepium Bge in the clinic. PMID:24872678

  13. 6,7-Dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tetrahydroisoquinolines as superior reversal agents for P-glycoprotein-mediated multidrug resistance.

    PubMed

    Liu, Baomin; Qiu, Qianqian; Zhao, Tianxiao; Jiao, Lei; Li, Yunman; Huang, Wenlong; Qian, Hai

    2015-02-01

    P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major obstacle for successful cancer chemotherapy. Based on our previous study, 17 novel compounds with the 6,7-dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tetrahydroisoquinoline scaffold were designed and synthesized. Among them, 2-[(1-{4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl]phenyl}-1H-1,2,3-triazol-4-yl)methoxy]-N-(p-tolyl)benzamide (compound 7 h) was identified as a potent modulator of P-gp-mediated MDR, with high potency (EC50 =127.5 ± 9.1 nM), low cytotoxicity (TI>784.3), and long duration (>24 h) in reversing doxorubicin (DOX) resistance in K562/A02 cells. Compound 7 h also enhanced the effects of other MDR-related cytotoxic agents (paclitaxel, vinblastine, and daunorubicin), increased the accumulation of DOX and blocked P-gp-mediated rhodamine 123 efflux function in K562/A02 MDR cells. Moreover, 7 h did not have any effect on cytochrome (CYP3A4) activity. These results indicate that 7 h is a relatively safe modulator of P-gp-mediated MDR that has good potential for further development. PMID:25470220

  14. Inhibition of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 Regulates the Hepatobiliary Excretion and Plasma Exposure of Thienorphine and Its Glucuronide Conjugate

    PubMed Central

    Kong, Ling-Lei; Shen, Guo-Lin; Wang, Zhi-Yuan; Zhuang, Xiao-Mei; Xiao, Wei-Bin; Yuan, Mei; Gong, Ze-Hui; Li, Hua

    2016-01-01

    Thienorphine (TNP) is a novel partial opioid agonist that has completed phase II clinical evaluation as a promising drug candidate for the treatment of opioid dependence. Previous studies have shown that TNP and its glucuronide conjugate (TNP-G) undergo significant bile excretion. The purpose of this study was to investigate the roles of efflux transporters in regulating biliary excretion and plasma exposure of TNP and TNP-G. An ATPase assay suggested that TNP and TNP-G were substrates of P-gp and MRP2, respectively. The in vitro data from rat hepatocytes showed that bile excretion of TNP and TNP-G was regulated by the P-gp and MRP2 modulators. The accumulation of TNP and TNP-G in HepG2 cells significantly increased by the treatment of mdr1a or MRP2 siRNA for P-gp or MRP2 modulation. In intact rats, the bile excretion, and pharmacokinetic profiles of TNP and TNP-G were remarkably changed with tariquidar and probenecid pretreatment, respectively. Tariquidar increased the Cmax and AUC0-t and decreased MRT and T1/2 of TNP, whereas probenecid decreased the plasma exposure of TNP-G and increased its T1/2. Knockdown P-gp and MRP2 function using siRNA significantly increased the plasma exposure of TNP and TNP-G and reduced their mean retention time in mice. These results indicated the important roles of P-gp and MRP2 in hepatobiliary excretion and plasma exposure of TNP and TNP-G. Inhibition of the efflux transporters may affect the pharmacokinetics of TNP and result in a drug-drug interaction between TNP and the concomitant transporter inhibitor or inducer in clinic. PMID:27555820

  15. Forced expression of heat shock protein 27 (Hsp27) reverses P-glycoprotein (ABCB1)-mediated drug efflux and MDR1 gene expression in Adriamycin-resistant human breast cancer cells.

    PubMed

    Kanagasabai, Ragu; Krishnamurthy, Karthikeyan; Druhan, Lawrence J; Ilangovan, Govindasamy

    2011-09-23

    Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer. PMID:21784846

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

    PubMed Central

    Chen, Hsin-Hung; Huang, Wen-Chia; Chiang, Wen-Hsuan; Liu, Te-I; Shen, Ming-Yin; Hsu, Yuan-Hung; Lin, Sung-Chyr; Chiu, Hsin-Cheng

    2015-01-01

    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 enhanced passive tumor accumulation due to the loose intercellular junctions of endothelial cells lining inside blood vessels at tumor site, and the lack of lymphatic drainage. The growth of MCF-7/MDR xenografted tumor on Balb/c nude mice was inhibited to ~400 mm3 in volume as compared with the free DOX treatment group, 1,140 mm3, and the group treated with 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] solid lipid nanoparticles, 820 mm3. Analysis of the body weight of nude mice and the histology of organs and tumor after the

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

    PubMed

    Chen, Hsin-Hung; Huang, Wen-Chia; Chiang, Wen-Hsuan; Liu, Te-I; Shen, Ming-Yin; Hsu, Yuan-Hung; Lin, Sung-Chyr; Chiu, Hsin-Cheng

    2015-01-01

    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 enhanced passive tumor accumulation due to the loose intercellular junctions of endothelial cells lining inside blood vessels at tumor site, and the lack of lymphatic drainage. The growth of MCF-7/MDR xenografted tumor on Balb/c nude mice was inhibited to ~400 mm(3) in volume as compared with the free DOX treatment group, 1,140 mm(3), and the group treated with 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] solid lipid nanoparticles, 820 mm(3). Analysis of the body weight of nude mice and the histology of organs and tumor after the

  18. Synthesis of new steroidal inhibitors of P-glycoprotein-mediated multidrug resistance and biological evaluation on K562/R7 erythroleukemia cells.

    PubMed

    de Ravel, Marc Rolland; Alameh, Ghina; Melikian, Maxime; Mahiout, Zahia; Emptoz-Bonneton, Agnès; Matera, Eva-Laure; Lomberget, Thierry; Barret, Roland; Rocheblave, Luc; Walchshofer, Nadia; Beltran, Sonia; El Jawad, Lucienne; Mappus, Elisabeth; Grenot, Catherine; Pugeat, Michel; Dumontet, Charles; Le Borgne, Marc; Cuilleron, Claude Yves

    2015-02-26

    A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/β-pregnane-3,20-dione or 5β-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/β-pregnane-3,20-dione, among which the 5β-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 μM versus 1.2 and 10.6 μM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5β-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 μM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport. PMID:25634041

  19. Cysteines Introduced into Extracellular Loops 1 and 4 of Human P-Glycoprotein That Are Close Only in the Open Conformation Spontaneously Form a Disulfide Bond That Inhibits Drug Efflux and ATPase Activity*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2014-01-01

    P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 Å apart) because they are separated widely (30.4 Å apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity. PMID:25053414

  20. Co-administration strategy to enhance brain accumulation of vandetanib by modulating P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp1/Abcg2) mediated efflux with m-TOR inhibitors.

    PubMed

    Minocha, Mukul; Khurana, Varun; Qin, Bin; Pal, Dhananjay; Mitra, Ashim K

    2012-09-15

    The objectives of this study were (i) to characterize the interaction of vandetanib with P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) in vitro and in vivo (ii) to study the modulation of P-gp and BCRP mediated efflux of vandetanib with specific transport inhibitors and m-TOR inhibitors, everolimus and temsirolimus. Cellular accumulation and bi-directional transport studies in MDCKII cell monolayers were conducted to delineate the role of efflux transporters on disposition of vandetanib. Brain distribution studies were conducted in male FVB wild-type mice with vandetanib administered intravenously either alone or in the presence of specific inhibitors and m-TOR inhibitors. In vitro studies suggested that vandetanib is a high affinity substrate of Bcrp1 but is not transported by P-gp. Interestingly, in vivo brain distribution studies in FVB wild type mice indicated that vandetanib penetration into the brain is restricted by both Bcrp1 and P-gp mediated active efflux at the blood brain barrier (BBB). Co-administration of elacridar, a dual P-gp/BCRP inhibitor increased the brain to plasma concentration ratio of vandetanib upto 5 fold. Of the two m-TOR pathway inhibitors examined; everolimus showed potent effect on modulating vandetanib brain penetration whereas no significant affect on vandetanib brain uptake was observed following temsirolimus co-administration. This finding could be clinically relevant as everolimus can provide synergistic pharmacological effect in addition to primary role of vandetanib efflux modulation at BBB for the treatment of brain tumors. PMID:22633931

  1. P-glycoprotein is a major determinant of norbuprenorphine brain exposure and antinociception.

    PubMed

    Brown, Sarah M; Campbell, Scott D; Crafford, Amanda; Regina, Karen J; Holtzman, Michael J; Kharasch, Evan D

    2012-10-01

    Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of μ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(-/-) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(-/-) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(-/-) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine. PMID:22739506

  2. P-Glycoprotein Is a Major Determinant of Norbuprenorphine Brain Exposure and Antinociception

    PubMed Central

    Brown, Sarah M.; Campbell, Scott D.; Crafford, Amanda; Regina, Karen J.; Holtzman, Michael J.

    2012-01-01

    Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of μ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(−/−) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(−/−) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(−/−) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine. PMID

  3. Generating inhibitors of P-glycoprotein: where to, now?

    PubMed

    Crowley, Emily; McDevitt, Christopher A; Callaghan, Richard

    2010-01-01

    The prominent role for the drug efflux pump ABCB1 (P-glycoprotein) in mediating resistance to chemotherapy was first suggested in 1976 and sparked an incredible drive to restore the efficacy of anticancer drugs. Achieving this goal seemed inevitable in 1982 when a series of calcium channel blockers were demonstrated to restore the efficacy of chemotherapy agents. A large number of other compounds have since been demonstrated to restore chemotherapeutic sensitivity in cancer cells or tissues. Where do we stand almost three decades since the first reports of ABCB1 inhibition? Unfortunately, in the aftermath of extensive fundamental and clinical research efforts the situation remains gloomy. Only a small handful of compounds have reached late stage clinical trials and none are in routine clinical usage to circumvent chemoresistance. Why has the translation process been so ineffective? One factor is the multifactorial nature of drug resistance inherent to cancer tissues; ABCB1 is not the sole factor. However, expression of ABCB1 remains a significant negative prognostic indicator and is closely associated with poor response to chemotherapy in many cancer types. The main difficulties with restoration of sensitivity to chemotherapy reside with poor properties of the ABCB1 inhibitors: (1) low selectivity to ABCB1, (2) poor potency to inhibit ABCB1, (3) inherent toxicity and/or (4) adverse pharmacokinetic interactions with anticancer drugs. Despite these difficulties, there is a clear requirement for effective inhibitors and to date the strategies for generating such compounds have involved serendipity or simple chemical syntheses. This chapter outlines more sophisticated approaches making use of bioinformatics, combinatorial chemistry and structure informed drug design. Generating a new arsenal of potent and selective ABCB1 inhibitors offers the promise of restoring the efficacy of a key weapon in cancer treatment--chemotherapy. PMID:19949934

  4. Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

    PubMed Central

    Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Kumar, Pradeep; Tyagi, Charu; du Toit, Lisa C.; Pillay, Viness

    2015-01-01

    The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

  5. Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

    PubMed Central

    Melchior, Donald L.; Sharom, Frances J.; Evers, Raymond; Wright, George E.; Chu, Joseph W.K.; Wright, Stephen E.; Chu, Xiaoyan; Yabut, Jocelyn

    2012-01-01

    Introduction P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Methods Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Results Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2 = 0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. Discussion This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 minutes, and requires minimal quantities of test

  6. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    SciTech Connect

    Vogler, Meike; Dickens, David; Dyer, Martin J.S.; Owen, Andrew; Pirmohamed, Munir; Cohen, Gerald M.

    2011-05-06

    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  7. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

    PubMed

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain. PMID:27000241

  8. Relevance of p-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation.

    PubMed Central

    Fricker, G.; Drewe, J.; Huwyler, J.; Gutmann, H.; Beglinger, C.

    1996-01-01

    1. The interaction of cyclosporin A (CyA) with p-glycoprotein during intestinal uptake was investigated by a combination of in vitro experiments with human Caco-2 cells and an intubation study in healthy volunteers. 2. CyA uptake into the cells was not saturable and exhibited only a low temperature sensitivity, suggesting passive diffusion. When the permeation of CyA across Caco-2 monolayers from the apical to the basolateral side was determined, overall transport had an apparently saturable component up to a concentration of 1 microM. At higher concentrations permeation increased over-proportionally. Calculation of the kinetic parameters of apical to basolateral permeation suggested a diffusional process with a KD of 0.5 microliter min-1 per filter, which was overlayed by an active system in basolateral to apical direction with a KM of 3.8 microM and a Jmax of 6.5 picomol min-1 per filter. 3. CyA permeation was significantly higher when the drug was given from the basolateral side as compared to the permeation from the apical side. Apical to basolateral transport of CyA was increased in the presence of vinblastine, daunomycin and a non-immunosuppressive CyA-derivative. All compounds inhibit p-glycoprotein-mediated transport processes. Basolateral to apical permeation of CyA showed a dose-dependent decrease in the presence of vinblastine. Permeation of daunomycin across Caco-2 cell monolayers was also higher from the basolateral to the apical side than vice versa. Basolateral to apical permeation was decreased in the presence of SDZ PSC 833 and cyclosporin A. 4. Western blot analysis of Caco-2 cells with the monoclonal antibody C219 confirmed the presence of p-glycoprotein in the used cell system. 5. When the absorption of CyA in the gastrointestinal (GI)-tract of healthy volunteers was determined, a remarkable decrease of the plasma AUC could be observed dependent on the location of absorption in the rank order stomach > jejunum/ileum > colon. The decrease in

  9. Annotating Human P-Glycoprotein Bioassay Data

    PubMed Central

    Zdrazil, Barbara; Pinto, Marta; Vasanthanathan, Poongavanam; Williams, Antony J; Balderud, Linda Zander; Engkvist, Ola; Chichester, Christine; Hersey, Anne; Overington, John P; Ecker, Gerhard F

    2012-01-01

    Abstract Huge amounts of small compound bioactivity data have been entering the public domain as a consequence of open innovation initiatives. It is now the time to carefully analyse existing bioassay data and give it a systematic structure. Our study aims to annotate prominent in vitro assays used for the determination of bioactivities of human P-glycoprotein inhibitors and substrates as they are represented in the ChEMBL and TP-search open source databases. Furthermore, the ability of data, determined in different assays, to be combined with each other is explored. As a result of this study, it is suggested that for inhibitors of human P-glycoprotein it is possible to combine data coming from the same assay type, if the cell lines used are also identical and the fluorescent or radiolabeled substrate have overlapping binding sites. In addition, it demonstrates that there is a need for larger chemical diverse datasets that have been measured in a panel of different assays. This would certainly alleviate the search for other inter-correlations between bioactivity data yielded by different assay setups. PMID:23293680

  10. Modulation of P-glycoprotein ATPase activity by some phytoconstituents.

    PubMed

    Najar, I A; Sachin, B S; Sharma, S C; Satti, N K; Suri, K A; Johri, R K

    2010-03-01

    In the present investigation 16 phytoconstituents, which are active moieties found in several medicinal herbs, have been evaluated for their P-glycoprotein (P-gp) stimulation/inhibition profiles using a P-gp-dependent ATPase assay in rat jejunal membrane (in vitro). Acteoside, agnuside, catechin, chlorogenic acid, picroside -II and santonin showed an inhibitory effect. Negundoside, picroside -I and oleanolic acid caused a stimulatory effect. Andrographolide, apocyanin, berberine, glycyrrhizin, magniferin and piperine produced a biphasic response (stimulation at low concentration and inhibition at high concentration). The results suggested that a possible interaction of these phytoconstituents at the level of P-gp, could be an important parameter in determining their role in several key pharmacodynamic events. PMID:19653312

  11. P-glycoprotein in autoimmune rheumatic diseases.

    PubMed

    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. PMID:25712147

  12. Inhibitory effects of neochamaejasmin B on P-glycoprotein in MDCK-hMDR1 cells and molecular docking of NCB binding in P-glycoprotein.

    PubMed

    Pan, Lanying; Hu, Haihong; Wang, Xiangjun; Yu, Lushan; Jiang, Huidi; Chen, Jianzhong; Lou, Yan; Zeng, Su

    2015-01-01

    Stellera chamaejasme L. (Thymelaeaceae) is widely distributed in Mongolia, Tibet and the northern parts of China. Its roots are commonly used as "Langdu", which is embodied in the Pharmacopoeia of the P.R. China (2010) as a toxic Traditional Chinese Medicine. It is claimed to have antivirus, antitumor and antibacterial properties in China and other Asian countries. Studies were carried out to characterize the inhibition of neochamaejasmin B (NCB) on P-glycoprotein (P-gp, ABCB1, MDR1). Rhodamine-123 (R-123) transport and accumulation studies were performed in MDCK-hMDR1 cells. ABCB1 (MDR1) mRNA gene expression and P-gp protein expression were analyzed. Binding selectivity studies based on molecular docking were explored. R-123 transport and accumulation studies in MDCK-hMDR1 cells indicated that NCB inhibited the P-gp-mediated efflux in a concentration-dependent manner. RT-PCR and Western blot demonstrated that the P-gp expression was suppressed by NCB. To investigate the inhibition type of NCB on P-gp, Ki and Ki' values were determined by double-reciprocal plots in R-123 accumulation studies. Since Ki was greater than Ki', the inhibition of NCB on P-gp was likely a mixed type of competitive and non-competitive inhibition. The results were confirmed by molecular docking in our current work. The docking data indicated that NCB had higher affinity to P-gp than to Lig1 ((S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one). PMID:25679052

  13. Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay.

    PubMed

    Jouan, Elodie; Le Vée, Marc; Mayati, Abdullah; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-01-01

    In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug-drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context, the present study was designed to investigate the use of the fluorescent dye rhodamine 123 as a reference P-gp substrate probe for characterizing P-gp inhibitory potential of 16 structurally-unrelated drugs known to interact with P-gp. 14/16 of these P-gp inhibitors were found to increase rhodamine 123 accumulation in P-gp-overexpressing MCF7R cells, thus allowing the determination of their P-gp inhibitory potential, i.e., their half maximal inhibitor concentration (IC50) value towards P-gp-mediated transport of the dye. These IC50 values were in the range of variability of previously reported IC50 for P-gp and can be used for the prediction of clinical P-gp inhibition according to Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). Therefore, the data demonstrated the feasibility of the use of rhodamine 123 for evaluating the P-gp inhibitory potential of drugs. PMID:27077878

  14. Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay

    PubMed Central

    Jouan, Elodie; Le Vée, Marc; Mayati, Abdullah; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-01-01

    In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug–drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context, the present study was designed to investigate the use of the fluorescent dye rhodamine 123 as a reference P-gp substrate probe for characterizing P-gp inhibitory potential of 16 structurally-unrelated drugs known to interact with P-gp. 14/16 of these P-gp inhibitors were found to increase rhodamine 123 accumulation in P-gp-overexpressing MCF7R cells, thus allowing the determination of their P-gp inhibitory potential, i.e., their half maximal inhibitor concentration (IC50) value towards P-gp-mediated transport of the dye. These IC50 values were in the range of variability of previously reported IC50 for P-gp and can be used for the prediction of clinical P-gp inhibition according to Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). Therefore, the data demonstrated the feasibility of the use of rhodamine 123 for evaluating the P-gp inhibitory potential of drugs. PMID:27077878

  15. Homology modelling of human P-glycoprotein.

    PubMed

    Domicevica, Laura; Biggin, Philip C

    2015-10-01

    P-glycoprotein (P-gp) is an ATP-binding cassette transporter that exports a huge range of compounds out of cells and is thus one of the key proteins in conferring multi-drug resistance in cancer. Understanding how it achieves such a broad specificity and the series of conformational changes that allow export to occur form major, on-going, research objectives around the world. Much of our knowledge to date has been derived from mutagenesis and assay data. However, in recent years, there has also been great progress in structural biology and although the structure of human P-gp has not yet been solved, there are now a handful of related structures on which homology models can be built to aid in the interpretation of the vast amount of experimental data that currently exists. Many models for P-gp have been built with this aim, but the situation is complicated by the apparent flexibility of the system and by the fact that although many potential templates exist, there is large variation in the conformational state in which they have been crystallized. In this review, we summarize how homology modelling has been used in the past, how models are typically selected and finally illustrate how MD simulations can be used as a means to give more confidence about models that have been generated via this approach. PMID:26517909

  16. B4GALT family mediates the multidrug resistance of human leukemia cells by regulating the hedgehog pathway and the expression of p-glycoprotein and multidrug resistance-associated protein 1

    PubMed Central

    Zhou, H; Ma, H; Wei, W; Ji, D; Song, X; Sun, J; Zhang, J; Jia, L

    2013-01-01

    β-1, 4-Galactosyltransferase gene (B4GALT) family consists of seven members, which encode corresponding enzymes known as type II membrane-bound glycoproteins. These enzymes catalyze the biosynthesis of different glycoconjugates and saccharide structures, and have been recognized to be involved in various diseases. In this study, we sought to determine the expressional profiles of B4GALT family in four pairs of parental and chemoresistant human leukemia cell lines and in bone marrow mononuclear cells (BMMC) of leukemia patients with multidrug resistance (MDR). The results revealed that B4GALT1 and B4GALT5 were highly expressed in four MDR cells and patients, altered levels of B4GALT1 and B4GALT5 were responsible for changed drug-resistant phenotype of HL60 and HL60/adriamycin-resistant cells. Further data showed that manipulation of these two gene expression led to increased or decreased activity of hedgehog (Hh) signaling and proportionally mutative expression of p-glycoprotein (P-gp) and MDR-associated protein 1 (MRP1) that are both known to be related to MDR. Thus, we propose that B4GALT1 and B4GALT5, two members of B4GALT gene family, are involved in the development of MDR of human leukemia cells, probably by regulating the activity of Hh signaling and the expression of P-gp and MRP1. PMID:23744354

  17. The Connection between the Toxicity of Anthracyclines and Their Ability to Modulate the P-Glycoprotein-Mediated Transport in A549, HepG2, and MCF-7 Cells

    PubMed Central

    Szwed, Marzena; Rychlik, Błażej

    2014-01-01

    Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of solid tumors. We compared the resistance of the most popular solid tumors, breast adenocarcinoma (MCF-7 cell line) and nonsmall cell lung (A549 cell line) hepatocellular liver carcinoma (HepG2 cells), to aclarubicin (ACL) and doxorubicin (DOX). This research aimed at determining the relation between the toxicity of ACL and DOX, their cell accumulation, and then effect on P-glycoprotein functionality. ACL is more cytotoxic for tumor cells compared to DOX. The intracellular concentration of drugs in cancer cells was dependent on the dose of the drugs and the time of incubation. The P-gp inhibitor Verapamil (V) increased DOX accumulation in all tested cell lines. By contrast, the intracellular level of ACL was not affected by this modifying agent. The assessment of the uptake of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) or Rhodamine 123 (R123) allows the evaluation of the different influence of drugs on P-gp activity which is in agreement with the estimation of expression measured by MDR-1 shift assay. These data suggest that ACL is less P-gp dependent than DOX and consequently may be used in a clinical setting to increase treatment efficacy in resistant human tumors. PMID:24574923

  18. P-glycoprotein activity and biological response

    SciTech Connect

    Vaalburg, W. . E-mail: w.vaalburg@pet.umcg.nl; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

    2005-09-01

    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.

  19. Convallatoxin: a new P-glycoprotein substrate.

    PubMed

    Gozalpour, Elnaz; Greupink, Rick; Bilos, Albert; Verweij, Vivienne; van den Heuvel, Jeroen J M W; Masereeuw, Rosalinde; Russel, Frans G M; Koenderink, Jan B

    2014-12-01

    Digitalis-like compounds (DLCs), such as digoxin and digitoxin that are derived from digitalis species, are currently used to treat heart failure and atrial fibrillation, but have a narrow therapeutic index. Drug-drug interactions at the transporter level are frequent causes of DLCs toxicity. P-glycoprotein (P-gp, ABCB1) is the primary transporter of digoxin and its inhibitors influence pharmacokinetics and disposition of digoxin in the human body; however, the involvement of P-gp in the disposition of other DLCs is currently unknown. In present study, the transport of fourteen DLCs by human P-gp was studied using membrane vesicles originating from human embryonic kidney (HEK293) cells overexpressing P-gp. DLCs were quantified by liquid chromatography-mass spectrometry (LC-MS). The Lily of the Valley toxin, convallatoxin, was identified as a P-gp substrate (Km: 1.1±0.2 mM) in the vesicular assay. Transport of convallatoxin by P-gp was confirmed in rat in vivo, in which co-administration with the P-gp inhibitor elacridar, resulted in increased concentrations in brain and kidney cortex. To address the interaction of convallatoxin with P-gp on a molecular level, the effect of nine alanine mutations was compared with the substrate N-methyl quinidine (NMQ). Phe343 appeared to be more important for transport of NMQ than convallatoxin, while Val982 was particularly relevant for convallatoxin transport. We identified convallatoxin as a new P-gp substrate and recognized Val982 as an important amino acid involved in its transport. These results contribute to a better understanding of the interaction of DLCs with P-gp. PMID:25264938

  20. Identification of members of the P-glycoprotein multigene family

    SciTech Connect

    Ng, W.F.; Sarangi, F.; Zastawny, R.L.; Veinot-Drebot, L.; Ling, V. )

    1989-03-01

    Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, the authors found that the hamster P-glycoprotein gene family consists of three genes. They also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. The propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgpl and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. They speculate that the hamster pgpl and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.

  1. Re-evaluation of the role of P-glycoprotein in in vitro drug permeability studies with the bovine brain microvessel endothelial cells.

    PubMed

    Hakkarainen, Jenni J; Rilla, Kirsi; Suhonen, Marjukka; Ruponen, Marika; Forsberg, Markus M

    2014-03-01

    1.  Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2.  BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3.  P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4.  In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates. PMID:23924297

  2. Comparison of steroid substrates and inhibitors of P-glycoprotein by 3D-QSAR analysis

    NASA Astrophysics Data System (ADS)

    Li, Yan; Wang, Yong-Hua; Yang, Ling; Zhang, Shu-Wei; Liu, Chang-Hou; Yang, Sheng-Li

    2005-01-01

    Steroid derivatives show a complex interaction with P-glycoprotein (Pgp). To determine the essential structural requirements of a series of structurally related and functionally diverse steroids for Pgp-mediated transport or inhibition, a three-dimensional quantitative structure activity relationship study was performed by comparative similarity index analysis modeling. Twelve models have been explored to well correlate the physiochemical features with their biological functions with Pgp on basis of substrate and inhibitor datasets, in which the best predictive model for substrate gave cross-validated q2=0.720, non-cross-validated r2=0.998, standard error of estimate SEE=0.012, F=257.955, and the best predictive model for inhibitor gave q2=0.536, r2=0.950, SEE=1.761 and F=45.800. The predictive ability of all models was validated by a set of compounds that were not included in the training set. The physiochemical similarities and differences of steroids as Pgp substrate and inhibitor, respectively, were analyzed to be helpful in developing new steroid-like compounds.

  3. Binding of modulators to mouse and human multidrug resistance P-glycoprotein. A computational study.

    PubMed

    Jara, Gabriel E; Vera, D Mariano A; Pierini, Adriana B

    2013-11-01

    The human multidrug resistance (MDR) P-glycoprotein (P-gp) mediates the extrusion of chemotherapeutic drugs from cancer cells. Modulators are relevant pharmaceutical targets since they are intended to control or to inhibit its pumping activity. In the present work, a common binding site for Rhodamine 123 and modulators with different modulation activity was found by molecular docking over the crystal structure of the mouse P-gp. The modulators involved a family of compounds, including derivatives of propafenone (3-phenylpropiophenone nucleus) and XR9576 (tariquidar). Our results showed that the relative binding energies estimated by molecular docking were in good correlation with the experimental activities. Preliminary classical molecular dynamics results on selected P-gp/modulator complexes were also performed in order to understand the nature of the prevalent molecular interactions and the possible main molecular features that characterize a modulator. Besides, the results obtained with a human P-gp homology model from the mouse structure are also presented and analyzed. Our observations suggest that the hydrophobicity and molecular flexibility are the main features related to the inhibitory activity. The latter factor would increase the modulator ability to fit the aromatic rings inside the transmembrane domain. PMID:24095875

  4. Predicting Binding to P-Glycoprotein by Flexible Receptor Docking

    PubMed Central

    Dolghih, Elena; Bryant, Clifford; Renslo, Adam R.; Jacobson, Matthew P.

    2011-01-01

    P-glycoprotein (P-gp) is an ATP-dependent transport protein that is selectively expressed at entry points of xenobiotics where, acting as an efflux pump, it prevents their entering sensitive organs. The protein also plays a key role in the absorption and blood-brain barrier penetration of many drugs, while its overexpression in cancer cells has been linked to multidrug resistance in tumors. The recent publication of the mouse P-gp crystal structure revealed a large and hydrophobic binding cavity with no clearly defined sub-sites that supports an “induced-fit” ligand binding model. We employed flexible receptor docking to develop a new prediction algorithm for P-gp binding specificity. We tested the ability of this method to differentiate between binders and nonbinders of P-gp using consistently measured experimental data from P-gp efflux and calcein-inhibition assays. We also subjected the model to a blind test on a series of peptidic cysteine protease inhibitors, confirming the ability to predict compounds more likely to be P-gp substrates. Finally, we used the method to predict cellular metabolites that may be P-gp substrates. Overall, our results suggest that many P-gp substrates bind deeper in the cavity than the cyclic peptide in the crystal structure and that specificity in P-gp is better understood in terms of physicochemical properties of the ligands (and the binding site), rather than being defined by specific sub-sites. PMID:21731480

  5. Interaction of Common Azole Antifungals with P Glycoprotein

    PubMed Central

    Wang, Er-jia; Lew, Karen; Casciano, Christopher N.; Clement, Robert P.; Johnson, William W.

    2002-01-01

    Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and exposure to azole antifungal therapeutics and partially explain the clinical drug interactions observed with some antifungals. Using a whole-cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the abilities of the most widely prescribed orally administered azole antifungals to inhibit the function of this transporter. In a cell line presenting an overexpressed amount of the human P-gp transporter, itraconazole and ketoconazole inhibited P-gp function with 50% inhibitory concentrations (IC50s) of ∼2 and ∼6 μM, respectively. Cyclosporin A was inhibitory with an IC50 of 1.4 μM in this system. Uniquely, fluconazole had no effect in this assay, a result consistent with known clinical interactions. The effects of these azole antifungals on ATP consumption by P-gp (representing transport activity) were also assessed, and the Km values were congruent with the IC50s. Therefore, exposure of tissue to the azole antifungals may be modulated by human P-gp, and the clinical interactions of azole antifungals with other drugs may be due, in part, to inhibition of P-gp transport. PMID:11751127

  6. Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil

    SciTech Connect

    Safa, A.R. )

    1988-10-01

    Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido(3,5-{sup 3}H)benzoyl)aminomethyl verapamil and N-(p-azido(3-{sup 125}I)salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistance DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein. Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil. Photoaffinity labeling was also inhibited by 50 {mu}M concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 {mu}M compared to that by verapamil at 8 {mu}M. These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.

  7. Inhibition of Glutathione Peroxidase Mediates the Collateral Sensitivity of Multidrug-resistant Cells to Tiopronin*

    PubMed Central

    Hall, Matthew D.; Marshall, Travis S.; Kwit, Alexandra D. T.; Miller Jenkins, Lisa M.; Dulcey, Andrés E.; Madigan, James P.; Pluchino, Kristen M.; Goldsborough, Andrew S.; Brimacombe, Kyle R.; Griffiths, Gary L.; Gottesman, Michael M.

    2014-01-01

    Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic. PMID:24930045

  8. Evaluation of genipin on human cytochrome P450 isoenzymes and P-glycoprotein in vitro.

    PubMed

    Gao, Li-Na; Zhang, Ye; Cui, Yuan-Lu; Yan, Kuo

    2014-10-01

    Genipin is obtained from the fruit of Gardenia jasminoides Ellis and acts as an herbal medicine or functional food in East Asia. In addition to produce natural colorant, it possesses widely antiinflammatory, antithrombotic, antidepressive and anticarcinogenic activities. However, little research focuses on the potential of genipin for drug-drug interactions. In this study, effects of genipin on mRNA and protein expression of cytochrome P450 (CYP) 2C19, CYP2D6 and CYP3A4 were detected by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) and Western blot, respectively, in human hepatoma HepG2 cells. Enzyme activities of which were detected by luminogenic CYP assay in vitro. Moreover, effect of genipin on P-glycoprotein expression was analyzed by Western blot. Results showed that genipin possessed a significant induction on CYP2D6 and a remarkable inhibition on CYP2C19 and CYP3A4 not only from the expression of mRNA and protein (P<0.05 or P<0.01), but the level of enzyme activity. Moreover, a concentration-dependent induction of genipin on P-glycoprotein expression was observed. In conclusion, caution should be exercised with respect to the induction or inhibition of genipin on CYP isoenzymes and the strong induction on P-glycoprotein. PMID:25073096

  9. The isolation of minor-occurring furanocoumarins in grapefruit and analysis of their inhibition of cyp 3a4 and p-glycoprotein transport of talinolol from caco-2 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of grapefruit juice on the disposition of certain prescription drugs in humans have been mainly attributed to the inhibition of intestinal cytochrome P450 3A4 (CYP 3A4) by linear furanocoumarins. A number of the main furanocoumarins in grapefruit juice have been identified and analyzed ...

  10. Conformational changes of P-glycoprotein by nucleotide binding.

    PubMed Central

    Wang, G; Pincheira, R; Zhang, M; Zhang, J T

    1997-01-01

    P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[beta,gamma-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport. PMID:9396736

  11. Effects of Kaempferia parviflora extracts and their flavone constituents on P-glycoprotein function.

    PubMed

    Patanasethanont, Denpong; Nagai, Junya; Yumoto, Ryoko; Murakami, Teruo; Sutthanut, Khaetthareeya; Sripanidkulchai, Bung-Orn; Yenjai, Chavi; Takano, Mikihisa

    2007-01-01

    The purpose of this study was to examine the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on P-glycoprotein (P-gp)-mediated transport in LLC-GA5-COL150, a transfectant cell line of a porcine kidney epithelial cell line LLC-PK1 with human MDR1 cDNA. Ethanol extract obtained from Kaempferia parviflora rhizome significantly increased the accumulation of rhodamine 123 and daunorubicin, P-gp substrates, in LLC-GA5-COL150 cells, but not in LLC-PK1 cells. The aqueous extract also increased the accumulation in LLC-GA5-COL150 cells with lower potency than the ethanol extract. The effects of flavone derivatives isolated from the rhizome of Kaempferia parviflora on P-gp function were examined. Among six flavones tested, 3,5,7,3',4'-pentamethoxyflavone most potently increased the accumulation of rhodamine 123 and daunorubicin in LLC-GA5-COL150 cells in a concentration-dependent manner. In addition, 5,7-dimethoxyflavone to lesser degree increased rhodamine 123 accumulation in LLC-GA5-COL150 cells. In contrast, the other four flavone derivatives had no significant effect on the accumulation of rhodamine 123 in LLC-GA5-COL150 cells in a concentration range tested. These results indicate that extracts and flavone derivatives from the rhizome of Kaempferia parviflora can inhibit P-gp function, which may be useful for overcoming P-gp-mediated multidrug resistance and improving the oral bioavailability of anticancer agents. PMID:17031860

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

    PubMed Central

    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

  13. Selective inhibition of human cytochrome P450 3A4 by N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-(1, 1-dimethylethylaminocarbonyl)-4-[(furo[2, 3-b]pyridin-5-yl)methyl]piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethy lpentanamide and P-glycoprotein by valspodar in gene transfectant systems.

    PubMed

    Kawahara, I; Kato, Y; Suzuki, H; Achira, M; Ito, K; Crespi, C L; Sugiyama, Y

    2000-10-01

    Our previous report showed that L754.394 and valspodar (PSC833) are potent inhibitors of midazolam hydroxylation in human jejunum microsomes and vectorial transport of vinblastine in Caco-2 cells, respectively. In the present study, to directly examine the interactions of these compounds as well as other substrates with CYP3A4 and P-glycoprotein (P-gp), we performed in vitro inhibition studies using recombinant CYP3A4-expressed microsomes and an MDR1-transfected cell line, LLC-MDR1, respectively. In CYP3A4-expressed microsomes, both L754.394 and ketoconazole, at a concentration less than 0.5 microM, are the most potent inhibitors of the formation of 1'-hydroxymidazolam, a major metabolite of midazolam formed by CYP3A4. The greatest inhibitory effect on the transcellular transport of digoxin in LLC-MDR1 cells was observed in the presence of valspodar (<0.1 microM), followed by verapamil. From a comparison of the IC(50) values, it was shown that L754.394 and valspodar exhibited the highest selectivity for CYP3A4 and P-gp, respectively. To demonstrate such specificity, both midazolam hydroxylation and digoxin transport were observed in CYP3A4 transfected Caco-2 cells, which coexpress both P-gp and CYP3A4, in the presence or absence of L754.394 (0.5 microM) and valspodar (1.0 microM). L754.394 almost completely inhibited midazolam hydroxylation, but not digoxin transport, whereas almost complete inhibition of digoxin transport was observed in the presence of valspodar, but inhibition of the hydroxylation was minimal. Thus, the present study has demonstrated that L754.394 has a specific inhibitory effect on CYP3A4, whereas valspodar is specific for P-gp. PMID:10997946

  14. Aβ40 Reduces P-Glycoprotein at the Blood–Brain Barrier through the Ubiquitin–Proteasome Pathway

    PubMed Central

    Zhong, Yu; Wolf, Andrea; LeVine, Harry; Miller, David S.; Bauer, Björn

    2016-01-01

    Failure to clear amyloid-β (Aβ) from the brain is in part responsible for Aβ brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aβ across the blood–brain barrier is the efflux transporter P-glycoprotein (P-gp) in the luminal plasma membrane of the brain capillary endothelium. P-gp is reduced at the blood–brain barrier in AD, which has been shown to be associated with Aβ brain accumulation. However, the mechanism responsible for P-gp reduction in AD is not well understood. Here we focused on identifying critical mechanistic steps involved in reducing P-gp in AD. We exposed isolated rat brain capillaries to 100 nm Aβ40, Aβ40, aggregated Aβ40, and Aβ42. We observed that only Aβ40 triggered reduction of P-gp protein expression and transport activity levels; this occurred in a dose- and time-dependent manner. To identify the steps involved in Aβ-mediated P-gp reduction, we inhibited protein ubiquitination, protein trafficking, and the ubiquitin–proteasome system, and monitored P-gp protein expression, transport activity, and P-gp-ubiquitin levels. Thus, exposing brain capillaries to Aβ40 triggers ubiquitination, internalization, and proteasomal degradation of P-gp. These findings may provide potential therapeutic targets within the blood–brain barrier to limit P-gp degradation in AD and improve Aβ brain clearance. SIGNIFICANCE STATEMENT The mechanism reducing blood–brain barrier P-glycoprotein (P-gp) in Alzheimer's disease is poorly understood. In the present study, we focused on defining this mechanism. We demonstrate that Aβ40 drives P-gp ubiquitination, internalization, and proteasome-dependent degradation, reducing P-gp protein expression and transport activity in isolated brain capillaries. These findings may provide potential therapeutic avenues within the blood–brain barrier to limit P-gp degradation in Alzheimer's disease and improve Aβ brain clearance. PMID:26865616

  15. Co-formulation of P-glycoprotein Substrate and Inhibitor in Nanocarriers: An Emerging Strategy for Cancer Chemotherapy.

    PubMed

    Saneja, Ankit; Dubey, Ravindra Dhar; Alam, Noor; Khare, Vaibhav; Gupta, Prem N

    2014-01-01

    Scientific community is striving to understand the role of P-glycoprotein (P-gp) in drug discovery programs due to its impact on pharmacokinetic and multi-drug resistance (MDR) of anticancer drugs. A number of efforts to resolve the crystal structure and understanding the mechanism of P-gp mediated efflux have been made. Several generations of Pgp inhibitors have been developed to tackle this multi-specific efflux protein. Unfortunately, these inhibitors lack selectivity, exhibit poor solubility and severe pharmacokinetic interactions restricting their clinical use. The nanocarrier drug delivery systems (NDDS) are receiving increasing attention for P-gp modulating activity of pharmaceutical excipients which are used in their fabrication. In addition, NDDS can enhance the solubility and exhibited ability to bypass P-gp mediated efflux. The co-formulation of P-gp inhibitors and substrate anticancer drugs in single drug delivery system offers the advantage of bypassing P-gp mediated drug efflux as well as inhibiting the P-gp. Moreover, severe pharmacokinetic interactions between P-gp inhibitor and substrate anticancer drugs could be avoided by using this strategy. In this article we describe the co-formulation strategies using nanocarriers for modulation of pharmacokinetics as well as multi-drug resistance of anticancer drugs along with the challenges in this area. PMID:24720364

  16. P-glycoprotein activity in the blood-brain barrier is affected by virus-induced neuroinflammation and antipsychotic treatment.

    PubMed

    Doorduin, Janine; de Vries, Erik F J; Dierckx, Rudi A; Klein, Hans C

    2014-10-01

    A large percentage of schizophrenic patients respond poorly to antipsychotic treatment. This could be explained by inefficient drug transport across the blood-brain barrier due to P-glycoprotein mediated efflux. P-glycoprotein activity and expression in the blood-brain barrier can be affected by inflammation and pharmacotherapy. We therefore investigated the effect of herpes simplex virus type-1 (HSV-1) induced neuroinflammation and antipsychotic treatment on P-glycoprotein activity. Rats were inoculated with HSV-1 or PBS (control) on day 0 and treated with saline, clozapine or risperidone from day 0 up until day 4 post-inoculation. Positron emission tomography with the P-glycoprotein substrate [11C]verapamil was used to assess P-glycoprotein activity at day 6 post-inoculation. Disease symptoms in HSV-1 inoculated rats increased over time and were not significantly affected by treatment. The volume of distribution (VT) of [11C]verapamil was significantly lower (10-22%) in HSV-1 inoculated rats than in control rats. In addition, antipsychotic treatment significantly affected the VT of [11C]verapamil in all brain regions, although this effect was drug dependent. In fact, VT of [11C]verapamil was significantly increased (22-39%) in risperidone treated rats in most brain regions when compared to clozapine treated rats and in midbrain when compared to saline treated rats. No interaction between HSV-1 inoculation and antipsychotic treatment on VT of [11C]verapamil was found. In this study we demonstrated that HSV-1 induced neuroinflammation increased and risperidone treatment decreased P-glycoprotein activity. This finding is of importance for the understanding of treatment resistance in schizophrenia, and warrants further investigation of the underlying mechanism and the importance in clinical practice. PMID:24973705

  17. Blockade of P-Glycoprotein Decreased the Disposition of Phenformin and Increased Plasma Lactate Level

    PubMed Central

    Choi, Min-Koo; Song, Im-Sook

    2016-01-01

    This study aimed to investigate the in vivo relevance of P-glycoprotein (P-gp) in the pharmacokinetics and adverse effect of phenformin. To investigate the involvement of P-gp in the transport of phenformin, a bi-directional transport of phenformin was carried out in LLC-PK1 cells overexpressing P-gp, LLC-PK1-Pgp. Basal to apical transport of phenformin was 3.9-fold greater than apical to basal transport and became saturated with increasing phenformin concentration (2–75 μM) in LLC-PK1-Pgp, suggesting the involvement of P-gp in phenformin transport. Intrinsic clearance mediated by P-gp was 1.9 μL/min while passive diffusion clearance was 0.31 μL/min. Thus, P-gp contributed more to phenformin transport than passive diffusion. To investigate the contribution of P-gp on the pharmacokinetics and adverse effect of phenformin, the effects of verapamil, a P-gp inhibitor, on the pharmacokinetics of phenformin were also examined in rats. The plasma concentrations of phenformin were increased following oral administration of phenformin and intravenous verapamil infusion compared with those administerd phenformin alone. Pharmacokinetic parameters such as Cmax and AUC of phenformin increased and CL/F and Vss/F decreased as a consequence of verapamil treatment. These results suggested that P-gp blockade by verapamil may decrease the phenformin disposition and increase plasma phenformin concentrations. P-gp inhibition by verapamil treatment also increased plasma lactate concentration, which is a crucial adverse event of phenformin. In conclusion, P-gp may play an important role in phenformin transport process and, therefore, contribute to the modulation of pharmacokinetics of phenformin and onset of plasma lactate level. PMID:26797108

  18. Blockade of P-Glycoprotein Decreased the Disposition of Phenformin and Increased Plasma Lactate Level.

    PubMed

    Choi, Min-Koo; Song, Im-Sook

    2016-03-01

    This study aimed to investigate the in vivo relevance of P-glycoprotein (P-gp) in the pharmacokinetics and adverse effect of phenformin. To investigate the involvement of P-gp in the transport of phenformin, a bi-directional transport of phenformin was carried out in LLC-PK1 cells overexpressing P-gp, LLC-PK1-Pgp. Basal to apical transport of phenformin was 3.9-fold greater than apical to basal transport and became saturated with increasing phenformin concentration (2-75 μM) in LLC-PK1-Pgp, suggesting the involvement of P-gp in phenformin transport. Intrinsic clearance mediated by P-gp was 1.9 μL/min while passive diffusion clearance was 0.31 μL/min. Thus, P-gp contributed more to phenformin transport than passive diffusion. To investigate the contribution of P-gp on the pharmacokinetics and adverse effect of phenformin, the effects of verapamil, a P-gp inhibitor, on the pharmacokinetics of phenformin were also examined in rats. The plasma concentrations of phenformin were increased following oral administration of phenformin and intravenous verapamil infusion compared with those administerd phenformin alone. Pharmacokinetic parameters such as Cmax and AUC of phenformin increased and CL/F and Vss/F decreased as a consequence of verapamil treatment. These results suggested that P-gp blockade by verapamil may decrease the phenformin disposition and increase plasma phenformin concentrations. P-gp inhibition by verapamil treatment also increased plasma lactate concentration, which is a crucial adverse event of phenformin. In conclusion, P-gp may play an important role in phenformin transport process and, therefore, contribute to the modulation of pharmacokinetics of phenformin and onset of plasma lactate level. PMID:26797108

  19. Disease control by regulation of P-glycoprotein on lymphocytes in patients with rheumatoid arthritis

    PubMed Central

    Tsujimura, Shizuyo; Tanaka, Yoshiya

    2015-01-01

    The main purpose of treatment of rheumatoid arthritis (RA) with disease modifying antirheumatic drugs (DMARDs) is to control activation of lymphocytes, although some patients do not respond adequately to such treatment. Among various mechanisms of multidrug resistance, P-glycoprotein (P-gp), a member of ATP-binding cassette transporters, causes drug-resistance by efflux of intracellular drugs. Certain stimuli, such as tumor necrosis factor-α, activate lymphocytes and induce P-gp expression on lymphocytes, as evident in active RA. Studies from our laboratories showed spontaneous nuclear accumulation of human Y-box-binding protein-1, a multidrug resistance 1 transcription factor, in unstimulated lymphocytes, and surface overexpression of P-gp on peripheral lymphocytes of RA patients with high disease activity. The significant correlation between P-gp expression level and RA disease activity is associated with active efflux of drugs from the lymphocyte cytoplasm and in drug-resistance. However, the use of biological agents that reduce P-gp expression as well as P-gp antagonists (e.g., cyclosporine) can successfully reduce the efflux of corticosteroids from lymphocytes in vitro, suggesting that both types of drugs can be used to overcome drug-resistance and improve clinical outcome. We conclude that lymphocytes activated by various stimuli in RA patients with highly active disease acquire P-gp-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. Expression of P-gp on lymphocytes is a promising marker of drug resistance and a suitable therapeutic target to prevent drug resistance in patients with active RA. PMID:26618109

  20. Liposomes Coloaded with Elacridar and Tariquidar To Modulate the P-Glycoprotein at the Blood-Brain Barrier.

    PubMed

    Nieto Montesinos, Rita; Béduneau, Arnaud; Lamprecht, Alf; Pellequer, Yann

    2015-11-01

    This study prepared three liposomal formulations coloaded with elacridar and tariquidar to overcome the P-glycoprotein-mediated efflux at the blood-brain barrier. Their pharmacokinetics, brain distribution, and impact on the model P-glycoprotein substrate, loperamide, were compared to those for the coadministration of free elacridar plus free tariquidar. After intravenous administration in rats, elacridar and tariquidar in conventional liposomes were rapidly cleared from the bloodstream. Their low levels in the brain did not improve the loperamide brain distribution. Although elacridar and tariquidar in PEGylated liposomes exhibited 2.6 and 1.9 longer half-lives than free elacridar and free tariquidar, respectively, neither their Kp for the brain nor the loperamide brain distribution was improved. However, the conjugation of OX26 F(ab')2 fragments to PEGylated liposomes increased the Kps for the brain of elacridar and tariquidar by 1.4- and 2.1-fold, respectively, in comparison to both free P-gp modulators. Consequently, the Kp for the brain of loperamide increased by 2.7-fold. Moreover, the plasma pharmacokinetic parameters and liver distribution of loperamide were not modified by the PEGylated OX26 F(ab')2 immunoliposomes. Thus, this formulation represents a promising tool for modulating the P-glycoprotein-mediated efflux at the blood-brain barrier and could improve the brain uptake of any P-glycoprotein substrate that is intended to treat central nervous system diseases. PMID:26390138

  1. MMP2-Sensitive PEG-Lipid Copolymers: A New Type of Tumor-Targeted P-Glycoprotein Inhibitor.

    PubMed

    Dai, Zhi; Yao, Qing; Zhu, Lin

    2016-05-25

    Low tumor targetability and multidrug resistance (MDR) are two major impediments to the success of cancer treatments. Nanomaterials which possess high tumor targetability and the ability to reverse the MDR are rare. This report describes a new type of self-assembling polyethylene glycol-phosphoethanolamine-based copolymers (PEG-pp-PE) which showed both the matrix metalloproteinase 2 (MMP2)-sensitive tumor-targeted drug delivery and ability to inhibit the P-glycoprotein (P-gp)-mediated drug efflux. In this study, we synthesized a series of the homologous analogues of PEG-pp-PE copolymers and investigated the influence of their structures, including PEG lengths and peptide linkers, on the drug efflux, and identified the underlying mechanisms. We found that the whole structure (PEG-peptide-lipid) rather than any parts of the copolymers was key for the P-gp inhibition and a delicate balance between the hydrophilic and lipophilic segments of the PEG-pp-PE copolymers was needed for better modulating the P-gp-mediated drug efflux. The best copolymer, PEG2k-pp-PE, showed even higher P-gp inhibition effect than the d-α-tocopherol polyethylene glycol 1000 succinate (TPGS1k). We also found that the P-gp inhibition capability of PEG-pp-PE copolymers was highly associated with the P-gp down-regulation, the increase in the plasma membrane fluidity, and the inhibition of the P-gp ATPase activity. Besides, the excellent physicochemical properties, high drug loading, MMP2-dependent drug release, and improved drug efficacy in the MDR cancer cells suggested that the PEG-pp-PE copolymers might have great potential for building tumor-targeted drug delivery systems for treating drug-resistant cancers. PMID:27145021

  2. Marine Natural Products with P-Glycoprotein Inhibitor Properties

    PubMed Central

    Lopez, Dioxelis; Martinez-Luis, Sergio

    2014-01-01

    P-glycoprotein (P-gp) is a protein belonging to the ATP-binding cassette (ABC) transporters superfamily that has clinical relevance due to its role in drug metabolism and multi-drug resistance (MDR) in several human pathogens and diseases. P-gp is a major cause of drug resistance in cancer, parasitic diseases, epilepsy and other disorders. This review article aims to summarize the research findings on the marine natural products with P-glycoprotein inhibitor properties. Natural compounds that modulate P-gp offer great possibilities for semi-synthetic modification to create new drugs and are valuable research tools to understand the function of complex ABC transporters. PMID:24451193

  3. New insight into p-glycoprotein as a drug target.

    PubMed

    Breier, Albert; Gibalova, Lenka; Seres, Mario; Barancik, Miroslav; Sulova, Zdenka

    2013-01-01

    Multidrug resistance (MDR) of cancer tissue is a phenomenon in which cancer cells exhibit reduced sensitivity to a large group of unrelated drugs with different mechanisms of pharmacological activity. Mechanisms that reduce cell sensitivity to damage induced by a variety of chemicals were found to be caused by diverse, albeit well-defined, phenotypic alterations. The molecular basis of MDR commonly involves overexpression of the plasma membrane drug efflux pump - P-glycoprotein (P-gp). This glycoprotein is an ABCB1 member of the ABC transporter family. Cells that develop MDR of this type express massive amounts of P-gp that can induce a drug resistance of more than 100 times higher than normal cells to several drugs, which are substrates of P-gp. Expression of P-gp could be inherent to cancer cells with regard to the specialized tissues from which the cells originated. This is often designated as intrinsic Pgp- mediated MDR. However, overexpression of P-gp may be induced by selection and/or adaptation of cells during exposure to anticancer drugs; this particular example is known as acquired P-gp-mediated MDR. Drugs that are potential inducers of P-gp are often substrates of this transporter. However, several substances that have been proven to not be transportable by P-gp (such as cisplatin or alltrans retinoic acid) could induce minor improvements in P-gp overexpression. It is generally accepted that the drug efflux activity of Pgp is a major cause of reduced cell sensitivity to several compounds. However, P-gp may have side effects that are independent of its drug efflux activity. Several authors have described a direct influence of P-gp on the function of proteins involved in regulatory pathways, including apoptotic progression (such as p53, caspase-3 and Pokemon). Moreover, alterations of cell regulatory pathways, including protein expression, glycosylation and phosphorylation, have been demonstrated in cells overexpressing P-gp, which may consequently induce

  4. Astragaloside IV reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines

    PubMed Central

    WANG, PEI-PEI; XU, DU-JUAN; HUANG, CAN; WANG, WEI-PING; XU, WEN-KE

    2014-01-01

    Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside IV (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that ASIV enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy. PMID:24676670

  5. Astragaloside Ⅳ reduces the expression level of P-glycoprotein in multidrug-resistant human hepatic cancer cell lines.

    PubMed

    Wang, Pei-Pei; Xu, Du-Juan; Huang, Can; Wang, Wei-Ping; Xu, Wen-Ke

    2014-06-01

    Astragaloside is a saponin widely used in traditional Chinese medicine and has been reported to be a potent multidrug resistance (MDR) reversal agent. The present study investigated the role of astragaloside Ⅳ (ASIV) in the regulation of P-glycoprotein (P-gp, encoded by the mdr1 gene) and its effect on the reversal of MDR. The activity of ASIV was evaluated using human hepatic cancer cells Bel-7402 and the corresponding 5-fluorouracil (5-FU) resistant cells Bel-7402/FU. ASIV (0.08 mg/ml) potentiated the cytotoxicity of 5-FU which was demonstrated using the MTT assay on Bel-7402/FU cells. ASIV reduced the expression of P-gp as was revealed by immunocytochemistry. Accumulation and efflux studies with the P-gp substrate, rhodamine 123 (Rh123), demonstrated that ASIV inhibited P-gp-mediated drug efflux. Furthermore, it was demonstrated that ASⅣ enhanced the drug accumulation of 5-FU using a high performance liquid chromatography (HPLC) assay for drug resistant cells. Furthermore, ASIV may downregulate the expression of P-gp, which was examined using western blot analysis and polymerase chain reaction. In conclusion, the results of the present study indicated that ASIV reverses the drug resistance of Bel-7402/FU cells by downregulating the expression of mdr1. ASIV may represent a potent modulator of P-gp-mediated MDR in hepatic cancer therapy. PMID:24676670

  6. The P-glycoprotein inhibitor GF120918 modulates Ca2+-dependent processes and lipid metabolism in Toxoplasma gondii.

    PubMed

    Bottova, Iveta; Sauder, Ursula; Olivieri, Vesna; Hehl, Adrian B; Sonda, Sabrina

    2010-01-01

    Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development. PMID:20386707

  7. The P-glycoprotein Inhibitor GF120918 Modulates Ca2+-Dependent Processes and Lipid Metabolism in Toxoplasma Gondii

    PubMed Central

    Bottova, Iveta; Sauder, Ursula; Olivieri, Vesna; Hehl, Adrian B.; Sonda, Sabrina

    2010-01-01

    Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development. PMID:20386707

  8. THE EFFECTS OF HIV INFECTION ON THE EXPRESSION OF THE DRUG EFFLUX PROTEINS P-GLYCOPROTEIN AND BREAST CANCER RESISTANCE PROTEIN IN A HUMAN INTESTINE MODEL

    PubMed Central

    Ellis, Kelstan; Marlin, Jerry; Taylor, Tracey AH; Fitting, Sylvia; Hauser, Kurt F.; Rice, Greg

    2015-01-01

    Objectives In HIV infection, decreased penetration of antiretroviral drugs is postulated to contribute to HIV persistence within lymphoid rich regions of the gastrointestinal (GI) tract. However, mechanistic explanations for this phenomenon remain unclear. Specifically, investigations of HIV effects on drug efflux proteins within intestinal models are minimal. Methods Using an in vitro co-culture model of the GI tract, effects of HIV infection on drug efflux proteins, P-glycoprotein and Breast Cancer Resistance Protein (BCRP) were evaluated. The influence of the HIV-1 protein, Tat, and oxidative stress on P-glycoprotein and BCRP also was evaluated. Key Findings P-glycoprotein expression demonstrated an HIV-induced upregulation in Caco-2 cells over time for cells grown in co-culture with resting lymphocytes. BCRP overall expression increased with HIV exposure in activated primary human lymphocytes co-cultured with Caco-2 cells. Tat treatment resulted in no significant alterations in P-glycoprotein (43% increase), BCRP expression, or oxidative stress. Conclusions HIV exposure within an in vitro intestinal model resulted in increases in, P-glycoprotein and BCRP in a cell specific manner. Additionally, observed changes were not mediated by Tat. Collectively, these results suggest that alterations in BCRP and P-glycoprotein may contribute, in part, to decreased antiretroviral concentrations within the gastrointestinal tract in HIV infection. PMID:25557407

  9. Induction of cytochrome P450 3A4 and P-glycoprotein by the isoxazolyl-penicillin antibiotic flucloxacillin.

    PubMed

    Huwyler, Jörg; Wright, Matthew B; Gutmann, Heike; Drewe, Juergen

    2006-02-01

    Clinical findings indicate that co-administration of the isoxazolyl-penicillin flucloxacillin with cyclosporine may reduce the plasma concentrations of cyclosporine. We have explored in the present study if induction of cytochrome P450 3A4 or P-glycoprotein may offer a mechanistic explanation of the observed effects. Flucloxacillin is neither an inhibitor nor a substrate of drug metabolizing cytochrome P450 isoenzymes (CYP3A4, 1A2, 2C9, 2C19 and 2D6) or P-glycoprotein as shown by an in vitro assay for CYP inhibition, a fluorescent indicator assay for P-glycoprotein inhibition and a functional P-glycoprotein ATPase assay. However, incubation of human LS 180 colorectal adenocarcinoma cells with flucloxacillin led to a dose-dependent induction of MDR1 as well as of CYP3A4 mRNA, which was also confirmed in primary human hepatocytes. At high concentrations, flucloxacillin activated the human Pregnane-X-Receptor, PXR, a ligand-dependent transcription factor that is the target of many drugs that induce CYP3A4, with consequences for the metabolism of other drugs. Liver microsomes from control rats or rats, which received for 3 consecutive days 100 mg/kg of oral flucloxacillin, were used to study the metabolism and metabolite pattern of midazolam, a model substrate of CYP 3A4. There was a trend towards a higher intrinsic microsomal clearance of midazolam using microsomes from flucloxacillin treated rats. In addition, there was a significant increase in the formation of the principal midazolam metabolites 1-hydroxy midazolam, 4-hydroxy midazolam and 1,4-dihydroxy midazolam as compared to controls. These findings indicate that flucloxacillin has the potential to induce expression of both CYP3A4 as well as P-glycoprotein, most likely through activation of the nuclear hormone receptor PXR. This would offer an explanation for the observed clinical drug-drug interactions between the antibiotic and cyclosporine. PMID:16472102

  10. DUSP1 induces paclitaxel resistance through the regulation of p-glycoprotein expression in human ovarian cancer cells.

    PubMed

    Kang, Yu-Seon; Seok, Hyun-Jeong; Jeong, Eun-Jeong; Kim, Yuna; Yun, Seok-Joong; Min, Jeong-Ki; Kim, Sun Jin; Kim, Jang-Seong

    2016-09-01

    The heterogeneity and genetic instability of ovarian cancer cells often lead to the development of drug resistance, closely related with the increased cancer-related mortality. In this study, we investigated the role of dual-specificity phosphatase 1 (DUSP1) in the development of the resistance in human ovarian cancer cells against paclitaxel. Overexpression of DUSP1 in HeyA8 human ovarian cancer cells (HeyA8-DUSP1) up-regulated the expression of the drug efflux pump, p-glycoprotein. Consequently, HeyA8-DUSP1 cells are highly resistant to paclitaxel, with the resistance comparable to that of a multi-drug resistance cell line (HeyA8-MDR). Moreover, over expression of DUSP1 significantly increased the activation of p38 MAPK, leaving the activation of ERK1/2 and JNK1/2 unaffected. Pharmacological suppression of p38 MAPK activity prevents the up-regulation of p-glycoprotein expression and the consequent resistance against paclitaxel in HeyA8-DUSP1 cells. By contrast, HeyA8-MDR cells expressed a significantly higher level of DUSP1, but treatment with small interference RNA against DUSP1 significantly suppressed the expression of p-glycoprotein and the resistance against paclitaxel in HeyA8-MDR cells. Ectopic expression of MKK3, an upstream activator of p38 MAPK, significantly up-regulated the expression of p-glycoprotein and increased the consequent resistance against paclitaxel in HeyA8 cells. Collectively, these data indicated that DUSP1 may induce the resistance against paclitaxel through the p38 MAPK-mediated overexpression of p-glycoprotein in human ovarian cancer cells. PMID:27422607

  11. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine

    PubMed Central

    Sparreboom, Alex; van Asperen, Judith; Mayer, Ulrich; Schinkel, Alfred H.; Smit, Johan W.; Meijer, Dirk K. F.; Borst, Piet; Nooijen, Willem J.; Beijnen, Jos H.; van Tellingen, Olaf

    1997-01-01

    In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(−/−) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(−/−) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in mdr1a(−/−) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(−/−) mice. The cumulative fecal excretion (0–96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(−/−) mice. Biliary excretion was not significantly different in wt and mdr1a(−/−) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(−/−) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen. PMID:9050899

  12. Modulation of the multidrug resistance P-glycoprotein: Detection with technetium-99m-sestamibi in vivo

    SciTech Connect

    Luker, G.D.; Fracasso, P.M.; Dobkin, J.; Piwnica-Worms, D.

    1997-03-01

    Overexpression of the multidrug resistance (MDR1) P-glycoprotein (Pgp) has been documented in nearly all forms of human cancers and increased levels of Pgp in some tumors correlate with poor response to treatment. Technetium-99m-sestamibi has recently been validated as a Pgp transport substrate. Pgp is also normally expressed along the biliary canalicular surface of hepatocytes and the luminal side of proximal tubule cells in the kidney, while not expressed in heart. Focused on these organs with known Pgp status, we present the findings on {sup 99m}Tc-sestamibi showed normal, prompt clearance of the radiotracer from the liver and kidneys relative to the heart. After administration of the Pgp modulator, {sup 99m}Tc-sestamibi was selectively retained in the liver and kidneys. Hepatobiliary and renal clearance of {sup 99m}Tc-sestamibi are Pgp-mediated, and inhibition of Pgp transport in these organs can be successfully imaged using {sup 99m}Tc-sestamibi in patients. Similar results might be expected with this and related radiopharmaceuticals for functional imaging of Pgp transport and modulation in tumors. 34 refs., 2 figs.

  13. Inhibitory Potential of Antifungal Drugs on ATP-Binding Cassette Transporters P-Glycoprotein, MRP1 to MRP5, BCRP, and BSEP.

    PubMed

    Lempers, Vincent J C; van den Heuvel, Jeroen J M W; Russel, Frans G M; Aarnoutse, Rob E; Burger, David M; Brüggemann, Roger J; Koenderink, Jan B

    2016-06-01

    Inhibition of ABC transporters is a common mechanism underlying drug-drug interactions (DDIs). We determined the inhibitory potential of antifungal drugs currently used for invasive fungal infections on ABC transporters P-glycoprotein (P-gp), MRP1 to MRP5, BCRP, and BSEP in vitro Membrane vesicles isolated from transporter-overexpressing HEK 293 cells were used to investigate the inhibitory potential of antifungal drugs (250 μM) on transport of model substrates. Concentration-inhibition curves were determined if transport inhibition was >60%. Fifty percent inhibitory concentrations (IC50s) for P-gp and BCRP were both 2 μM for itraconazole, 5 and 12 μM for hydroxyitraconazole, 3 and 6 μM for posaconazole, and 3 and 11 μM for isavuconazole, respectively. BSEP was strongly inhibited by itraconazole and hydroxyitraconazole (3 and 17 μM, respectively). Fluconazole and voriconazole did not inhibit any transport for >60%. Micafungin uniquely inhibited all transporters, with strong inhibition of MRP4 (4 μM). Anidulafungin and caspofungin showed strong inhibition of BCRP (7 and 6 μM, respectively). Amphotericin B only weakly inhibited BCRP-mediated transport (127 μM). Despite their wide range of DDIs, azole antifungals exhibit selective inhibition on efflux transporters. Although echinocandins display low potential for clinically relevant DDIs, they demonstrate potent in vitro inhibitory activity. This suggests that inhibition of ABC transporters plays a crucial role in the inexplicable (non-cytochrome P450-mediated) DDIs with antifungal drugs. PMID:27001813

  14. Silencing of P-glycoprotein increases mortality in temephos-treated Aedes aegypti larvae.

    PubMed

    Figueira-Mansur, J; Ferreira-Pereira, A; Mansur, J F; Franco, T A; Alvarenga, E S L; Sorgine, M H F; Neves, B C; Melo, A C A; Leal, W S; Masuda, H; Moreira, M F

    2013-12-01

    Re-emergence of vector-borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P-glycoproteins (P-gps) are ATP-dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P-glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P-gps. The basal ATPase activity of ATP-binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P-gp (AaegP-gp) gene expression was detected in temephos-treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP-gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)-mediated silencing of the AaegP-gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide-induced P-gp expression can be involved in the modulation of insecticide efflux. PMID:23980723

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

    PubMed

    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. PMID:27129170

  16. MAPK1 of Leishmania donovani Modulates Antimony Susceptibility by Downregulating P-Glycoprotein Efflux Pumps

    PubMed Central

    Garg, Mansi

    2015-01-01

    Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance in Leishmania donovani. The L. donovani MAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug. PMID:25870075

  17. Structure-activity relationships and in silico models of P-glycoprotein (ABCB1) inhibitors.

    PubMed

    Liu, Hongming; Ma, Zhiguo; Wu, Baojian

    2013-11-01

    1. The efflux pump p-glycoprotein (P-gp/ABCB1) has received enormous attention in drug (xenobiotic) disposition due to its role in modulation of the drug availability and in protection of sensitive organs. 2. P-gp mediated efflux is one of main mechanisms for multidrug resistance in cancer cells. A main approach to reverse the resistance and restore the drug efficacy is to use specific inhibitors of P-gp that suppress the efflux activity. 3. This review summarizes the binding capabilities of known chemical inhibitors based on the analyses of structure-activity relationships, and computational modeling of the inhibitors as well as the binding site of P-gp protein. 4. The molecular models will facilitate the design of lead inhibitors as drug candidates. Also, it helps scientists in early drug discovery phase to synthesize chemical series with better understanding of their P-gp binding liabilities. PMID:23617855

  18. Pharmacokinetic Interactions of Herbs with Cytochrome P450 and P-Glycoprotein

    PubMed Central

    Cho, Hyun-Jong

    2015-01-01

    The concurrent use of drugs and herbal products is becoming increasingly prevalent over the last decade. Several herbal products have been known to modulate cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) which are recognized as representative drug metabolizing enzymes and drug transporter, respectively. Thus, a summary of knowledge on the modulation of CYP and P-gp by commonly used herbs can provide robust fundamentals for optimizing CYP and/or P-gp substrate drug-based therapy. Herein, we review ten popular medicinal and/or dietary herbs as perpetrators of CYP- and P-gp-mediated pharmacokinetic herb-drug interactions. The main focus is placed on previous works on the ability of herbal extracts and their phytochemicals to modulate the expression and function of CYP and P-gp in several in vitro and in vivo animal and human systems. PMID:25632290

  19. Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans.

    PubMed

    Janssen, I Jana I; Krücken, Jürgen; Demeler, Janina; von Samson-Himmelstjerna, Georg

    2015-08-01

    P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Expression of Parascaris pgp-11 in two transgenic lines significantly decreased ivermectin susceptibility in a motility (thrashing) assay conducted in liquid medium. The EC50 values increased by 3.2- and 4.6-fold in the two lines relative to a transgenic control strain. This is the first report on the successful functional analysis of a parasitic nematode Pgp in the model organism C. elegans. PMID:25905032

  20. In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture

    PubMed Central

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

    2015-01-01

    Failure of cancer chemotherapies is often linked to the over expression of ABC efflux transporters like the multidrug resistance P-glycoprotein (P-gp). P-gp expression in cells leads to the elimination of a variety of chemically unrelated, mostly cytotoxic compounds. Administration of chemotherapeutics during therapy frequently selects for cells that over express P-gp and are therefore capable of robustly exporting diverse compounds, including chemotherapeutics, from the cells. P-gp thus confers multidrug resistance to a majority of drugs currently available for the treatment of cancers and diseases like HIV/AIDS. The search for P-gp inhibitors for use as co-therapeutics to combat multidrug resistances has had little success to date. In a previous study (Brewer et al., Mol Pharmacol 86: 716–726, 2014), we described how ultrahigh throughput computational searches led to the identification of four drug-like molecules that specifically interfere with the energy harvesting steps of substrate transport and inhibit P-gp catalyzed ATP hydrolysis in vitro. In the present study, we demonstrate that three of these compounds reversed P-gp-mediated multidrug resistance of cultured prostate cancer cells to restore sensitivity comparable to naïve prostate cancer cells to the chemotherapeutic drug, paclitaxel. Potentiation concentrations of the inhibitors were <3 μmol/L. The inhibitors did not exhibit significant toxicity to noncancerous cells at concentrations where they reversed multidrug resistance in cancerous cells. Our results indicate that these compounds with novel mechanisms of P-gp inhibition are excellent leads for the development of co-therapeutics for the treatment of multidrug resistances. PMID:26516582

  1. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier

    PubMed Central

    Hartz, Anika M. S.; Bauer, Björn; Block, Michelle L.; Hong, Jau-Shyong; Miller, David S.

    2008-01-01

    Here, we report that diesel exhaust particles (DEPs), a major constituent of urban air pollution, affect blood-brain barrier function at the tissue, cellular, and molecular levels. Isolated rat brain capillaries exposed to DEPs showed increased expression and transport activity of the key drug efflux transporter, P-glycoprotein (6 h EC50 was ∼5 μg/ml). Up-regulation of P-glycoprotein was abolished by blocking transcription or protein synthesis. Inhibition of NADPH oxidase or pretreatment of capillaries with radical scavengers ameliorated DEP-induced P-glycoprotein up-regulation, indicating a role for reactive oxygen species in signaling. DEP exposure also increased brain capillary tumor necrosis factor-α (TNF-α) levels. DEP-induced P-glycoprotein up-regulation was abolished when TNF-receptor 1 (TNF-R1) was blocked and was not evident in experiments with capillaries from TNF-R1 knockout mice. Inhibition of JNK, but not NF-κB, blocked DEP-induced P-glycoprotein up-regulation, indicating a role for AP-1 in the signaling pathway. Consistent with this, DEPs increased phosphorylation of c-jun. Together, our results show for the first time that a component of air pollution, DEPs, alters blood-brain barrier function through oxidative stress and proinflammatory cytokine production. These experiments disclose a novel blood-brain barrier signaling pathway, with clear implications for environmental toxicology, CNS pathology, and the pharmacotherapy of CNS disorders.—Hartz, A. M. S., Bauer, B., Block, M. L., Hong, J.-S., Miller, D.-S. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier. PMID:18474546

  2. Inhibitory Effects of Green Tea and (–)-Epigallocatechin Gallate on Transport by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K and P-Glycoprotein

    PubMed Central

    Singer, Katrin; Hoier, Eva; Müller, Fabian; Glaeser, Hartmut; König, Jörg; Fromm, Martin F.

    2015-01-01

    Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC50 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC50 values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates. PMID:26426900

  3. [Modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui].

    PubMed

    Sun, Ya-Bin; Li, Guo-Feng; Tang, Zhong-Kun; Wu, Bing-Yi

    2010-04-01

    To investigate the modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui with ussing chamber and rt-pcr, Rhodamine 123 (R123), a P-gp substrate and fluorescein sodium (CF), a model drug of non-P-gp substrate transported by a passive diffusion were taken as investigational drugs. Because these two drugs can be easily assayed and widely used in various research fields. The permeability of R123 or CF via Wistar rat jejunum membranes was evaluated by in vitro ussing chamber after oral administration of four different decoctions of Glycyrrhiza inflata and Kansui for 1 week. And the concentration of R123 or CF was determined by the fluorospectrophotometry in the receiving solution. Meanwhile the expression of mdr1a in P-glycoprotein was detected by real-time fluorescent quantitative PCR. After oral administration of combined decoction of the single drug, the absorptive directed permeability of R123 increased significantly (P < 0.01). On the other hand, Kansui and combine decoction of the two drugs also decrease the permeability of secretory directed transport (P < 0.05). No action of Glycyrrhiza inflata was found on the secretory transport of R123 [Papp = (2.56 +/- 0.38) x 10(-5), cm x s(-1)] across the jejunum tissues, while Papp of control group was found [Papp = (2.35 +/- 0.27) x 10(-5), cm x s(-1)]. After oral administration of Kansui decoction for 1 week and 2 weeks, the levels of mdr1a expression in Wistar rats were lower than that of the control group, but there were no significant difference in the results. Meanwhile, Glycyrrhiza inflata had no effect on transport of CF across the jejunum tissues, though the other three groups could decrease the permeability of CF, as compared with control group. Kansui may slightly inhibit P-glycoprotein function in the intestinal membrane. For another, some compositions in Kansui inhibit P-glycoprotein function, and some others strengthen the tight junction between cells in the

  4. Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein

    PubMed Central

    2015-01-01

    P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug–drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ18O4-labeled ATP, no positional isotope exchange is detectable at the bridging β-phosphorus–O−γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three 18O/two 18O/one 18O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO42– (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states. PMID:24506763

  5. P-Glycoprotein and Drug Resistance in Systemic Autoimmune Diseases

    PubMed Central

    Picchianti-Diamanti, Andrea; Rosado, Maria Manuela; Scarsella, Marco; Laganà, Bruno; D’Amelio, Raffaele

    2014-01-01

    Autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are chronic inflammatory disorders of unknown etiology characterized by a wide range of abnormalities of the immune system that may compromise the function of several organs, such as kidney, heart, joints, brain and skin. Corticosteroids (CCS), synthetic and biologic immunosuppressive agents have demonstrated the capacity to improve the course of autoimmune diseases. However, a significant number of patients do not respond or develop resistance to these therapies over time. P-glycoprotein (P-gp) is a transmembrane protein that pumps several drugs out of the cell, including CCS and immunosuppressants; thus, its over-expression or hyper-function has been proposed as a possible mechanism of drug resistance in patients with autoimmune disorders. Recently, different authors have demonstrated that P-gp inhibitors, such as cyclosporine A (CsA) and its analogue Tacrolimus, are able to reduce P-gp expression and or function in SLE, RA and PsA patients. These observations suggest that P-gp antagonists could be adopted to revert drug resistance and improve disease outcome. The complex inter-relationship among drug resistance, P-gp expression and autoimmunity still remains elusive. PMID:24658440

  6. [Classification models of structure - P-glycoprotein activity of drugs].

    PubMed

    Grigorev, V Yu; Solodova, S L; Polianczyk, D E; Raevsky, O A

    2016-01-01

    Thirty three classification models of substrate specificity of 177 drugs to P-glycoprotein have been created using of the linear discriminant analysis, random forest and support vector machine methods. QSAR modeling was carried out using 2 strategies. The first strategy consisted in search of all possible combinations from 1÷5 descriptors on the basis of 7 most significant molecular descriptors with clear physico-chemical interpretation. In the second case forward selection procedure up to 5 descriptors, starting from the best single descriptor was used. This strategy was applied to a set of 387 DRAGON descriptors. It was found that only one of 33 models has necessary statistical parameters. This model was designed by means of the linear discriminant analysis on the basis of a single descriptor of H-bond (ΣC(ad)). The model has good statistical characteristics as evidenced by results to both internal cross-validation, and external validation with application of 44 new chemicals. This confirms an important role of hydrogen bond in the processes connected with penetration of chemical compounds through a blood-brain barrier. PMID:27143376

  7. Synthesis and bioevaluation of novel benzodipyranone derivatives as P-glycoprotein inhibitors for multidrug resistance reversal agents.

    PubMed

    Chen, Chien-Yu; Liu, Nai-Yu; Lin, Hui-Chang; Lee, Chih-Yu; Hung, Chin-Chuan; Chang, Chih-Shiang

    2016-08-01

    Multidrug resistance (MDR) is a phenomenon in which cells become resistant to structurally and mechanistically unrelated drugs, and it is one of the emerging problems in cancer therapy today. The relation between overexpression of the ABC transporter subfamily B member 1 (ABCB1/P-glycoprotein) and resistant cancers has been well characterized. In the present study, we successfully synthesized 52 novel benzodipyranone analogs and evaluated for their P-gp inhibitory activity in a P-gp transfected cell line, ABCB1/Flp-In™-293. Among these derivatives, 5a bearing on the 3-methylphenyl substituent, displayed the most potent P-gp inhibitory activity, which can enable the increase of the intracellular accumulation of P-gp substrate Calcein-AM. 5a exhibited more potency on promoted anticancer drugs cytotoxicity by reversing P-gp-mediated drug resistance in both ABCB1/Flp-In™-293 and KBvin cell lines. In particular, the compound 5a sensitized ABCB1/Flp-In™-293 cells toward paclitaxel, vincristine, and doxorubicin by 16.1, 21.0, and 1.6-fold at 10 μM, respectively. Further, 5a dramatically sensitized the resistant cell line KBvin toward paclitaxel and vincristine by 23.1 and 29.7-fold at 10 μM, respectively. It's possible that its mechanism of MDR inhibition can restore the intracellular accumulation of drugs and eventually chemosensitize cancer cells to anticancer drugs and reduce ABCB1 mRNA expression level. PMID:27131064

  8. Interaction of macrocyclic lactones with a Dirofilaria immitis P-glycoprotein.

    PubMed

    Mani, Thangadurai; Bourguinat, Catherine; Keller, Kathy; Ashraf, Shoaib; Blagburn, Byron; Prichard, Roger K

    2016-09-01

    Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of

  9. The human P-glycoprotein transporter enhances the type I interferon response to Listeria monocytogenes infection.

    PubMed

    Sigal, Nadejda; Kaplan Zeevi, Millie; Weinstein, Shiri; Peer, Dan; Herskovits, Anat A

    2015-06-01

    Human multidrug efflux transporters are known for their ability to extrude antibiotics and toxic compounds out of cells, yet accumulating data indicate they have additional functions in diverse physiological processes not related to drug efflux. Here, we show that the human multidrug transporter P-glycoprotein (P-gp) (also named MDR1 and ABCB1) is transcriptionally induced in the monocytic cell line THP-1 upon infection with the human intracellular bacterial pathogen Listeria monocytogenes. Notably, we found that P-gp is important for full activation of the type I interferon response elicited against L. monocytogenes bacteria. Both inhibition of P-gp function by verapamil and inhibition of its transcription using mRNA silencing led to a reduction in the magnitude of the type I response in infected cells. This function of P-gp was specific to type I interferon cytokines elicited against cytosolic replicating bacteria and was not observed in response to cyclic di-AMP (c-di-AMP), a molecule that was shown to be secreted by L. monocytogenes during infection and to trigger type I interferons. Moreover, P-gp was not involved in activation of other proinflammatory cytokines, such as those triggered by vacuolar-restricted L. monocytogenes or lipopolysaccharide (LPS). Taken together, these findings demonstrate a role for P-gp in proper development of an innate immune response against intracellular pathogens, highlighting the complexity in employing therapeutic strategies that involve inhibition of multidrug resistance (MDR) efflux pumps. PMID:25824830

  10. The Human P-Glycoprotein Transporter Enhances the Type I Interferon Response to Listeria monocytogenes Infection

    PubMed Central

    Sigal, Nadejda; Kaplan Zeevi, Millie; Weinstein, Shiri; Peer, Dan

    2015-01-01

    Human multidrug efflux transporters are known for their ability to extrude antibiotics and toxic compounds out of cells, yet accumulating data indicate they have additional functions in diverse physiological processes not related to drug efflux. Here, we show that the human multidrug transporter P-glycoprotein (P-gp) (also named MDR1 and ABCB1) is transcriptionally induced in the monocytic cell line THP-1 upon infection with the human intracellular bacterial pathogen Listeria monocytogenes. Notably, we found that P-gp is important for full activation of the type I interferon response elicited against L. monocytogenes bacteria. Both inhibition of P-gp function by verapamil and inhibition of its transcription using mRNA silencing led to a reduction in the magnitude of the type I response in infected cells. This function of P-gp was specific to type I interferon cytokines elicited against cytosolic replicating bacteria and was not observed in response to cyclic di-AMP (c-di-AMP), a molecule that was shown to be secreted by L. monocytogenes during infection and to trigger type I interferons. Moreover, P-gp was not involved in activation of other proinflammatory cytokines, such as those triggered by vacuolar-restricted L. monocytogenes or lipopolysaccharide (LPS). Taken together, these findings demonstrate a role for P-gp in proper development of an innate immune response against intracellular pathogens, highlighting the complexity in employing therapeutic strategies that involve inhibition of multidrug resistance (MDR) efflux pumps. PMID:25824830

  11. Synthesis and P-glycoprotein induction activity of colupulone analogs.

    PubMed

    Bharate, Jaideep B; Batarseh, Yazan S; Wani, Abubakar; Sharma, Sadhana; Vishwakarma, Ram A; Kaddoumi, Amal; Kumar, Ajay; Bharate, Sandip B

    2015-05-21

    Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 μM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 μM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol displayed a significant increase (29%) in Aβ transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent. PMID:25875530

  12. P-glycoprotein expression in normal and reactive bone marrows.

    PubMed Central

    Hegewisch-Becker, S.; Fliegner, M.; Tsuruo, T.; Zander, A.; Zeller, W.; Hossfeld, D. K.

    1993-01-01

    The expression of mdr1 gene product P-glycoprotein (P-gp) was investigated in 53 normal and reactive bone marrows by means of immunocytochemistry, using the monoclonal antibody (mAb) C219 and the alkaline phosphatase anti-alkaline phosphatase method. In a limited number of patients, data were confirmed by using the mAb MRK16 or a polymerase chain reaction assay for mdr1 gene expression. There was no history of prior chemotherapy or any malignancy in this group. Bone marrow aspirates were obtained as part of a routine diagnostic programme in bone marrow donors or in patients presenting with a variety of diagnoses such as unexplained gammopathy, fever, anaemia, other changes in peripheral blood smear, rheumatoid arthritis, vasculitis, or urticaria pigmentosa. Morphologically the bone marrow was normal in 23 patients, a megaloblastic erythropoiesis was seen in two patients and unspecific changes were seen in 28 patients. Twenty-seven of 53 samples were found to be positive for P-gp expression with the percentage of positive cells ranging from 2%-80% (mean = 24%). With a cutoff point of 10%, five of 23 normal (22%) and 13 of 28 reactive bone marrows (46%) were considered positive for P-gp expression. There was no obvious correlation between diagnosis or age and P-gp expression. Additional staining for the early surface marker CD-34 was performed in 12 samples, with none of them revealing more than 1% positivity. Since P-gp expression has so far been described only in CD-34 positive bone marrow cells, data suggest that P-gp expression may be reinduced in CD-34 negative cells under conditions which remain to be determined. Images Figure 1 Figure 2 PMID:8094974

  13. Molecular insight into conformational transmission of human P-glycoprotein

    NASA Astrophysics Data System (ADS)

    Chang, Shan-Yan; Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

    2013-12-01

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  14. Molecular insight into conformational transmission of human P-glycoprotein

    SciTech Connect

    Chang, Shan-Yan; Liu, Fu-Feng E-mail: ysun@tju.edu.cn; Dong, Xiao-Yan; Sun, Yan E-mail: ysun@tju.edu.cn

    2013-12-14

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  15. Interaction of the P-Glycoprotein Multidrug Transporter with Sterols.

    PubMed

    Clay, Adam T; Lu, Peihua; Sharom, Frances J

    2015-11-01

    The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3-9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains. PMID:26484739

  16. Phosphorylation of the multidrug resistant associated glycoprotein (p-glycoprotein): Preparation and characterization of 7-acetyltaxol

    SciTech Connect

    Mellado, W.

    1988-01-01

    To assess the role of phosphorylation in P-glycoprotein function, phosphorylation of P-glycoprotein in intact cells and in cell-free membrane fractions has been studied. Results obtained with cell-free membrane fractions indicate that P-glycoprotein is a substrate for a membrane-associated protein kinase A (PK-A). To assess whether P-glycoprotein was phosphorylated in vivo by PK-A, MDR cells were incubated with ({sup 32}P)Pi in the presence or absence of 100 uM 8Br-cAMP. The tryptic phosphopeptides of six P-glycoproteins from five independently derived MDR cell lines were analyzed by HPLC. A similar analysis carried out with two other P-glycoproteins (from J7.V3-1 and the lower band of J7.T1-50) demonstrated a major phosphopeptide with a retention time of 26 min. Fraction 26 was resolved as a single phosphopeptide by 2-D mapping. The phosphorylation of fraction 26 which was derived from P-glycoprotein in J7.V3-1 or the J7.T1-50 lower band was enhanced when the cells were treated with 8BrcAMP.

  17. 8-Prenylnaringenin is an inhibitor of multidrug resistance-associated transporters, P-glycoprotein and MRP1.

    PubMed

    Wesołowska, Olga; Wiśniewski, Jerzy; Sroda, Kamila; Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Paprocka, Maria; Duś, Danuta; Michalak, Krystyna

    2010-10-10

    Flavonoids with hydrophobic e.g. prenyl substituents might constitute the promising candidates for multidrug resistance (MDR) reversal agents. The interaction of 8-prenylnaringenin (8-isopentenylnaringenin), a potent phytoestrogen isolated from common hop (Humulus lupulus), with two multidrug resistance-associated ABC transporters of cancer cells, P-glycoprotein and MRP1, has been studied for the first time. Functional test based on the transport of fluorescent substrate BCECF revealed that the flavonoid strongly inhibited MRP1 transport activity in human erythrocytes (IC(50)=5.76+/-1.80muM). Expression of MDR-related transporters in drug-sensitive (LoVo) and doxorubicin-resistant (LoVo/Dx) human colon adenocarcinoma cell lines was characterized by RT-PCR and immunochemical methods and elevated expression of P-glycoprotein in resistant cells was found to be the main difference between these two cell lines. By means of flow cytometry it was shown that 8-prenylnaringenin significantly increased the accumulation of rhodamine 123 in LoVo/Dx cells. Doxorubicin accumulation in both LoVo and LoVo/Dx cells observed by confocal microscopy was also altered in the presence of 8-prenylnaringenin. However, the presence of the studied compound did not increase doxorubicin cytotoxicity to LoVo/Dx cells. It was concluded that 8-prenylnaringenin was not able to modulate MDR in human adenocarcinoma cell line in spite of the ability to inhibit both P-glycoprotein and MRP1 activities. To our best knowledge, this is the first report of 8-prenylnaringenin interaction with clinically important ABC transporters. PMID:20633549

  18. Generation of a drug resistance profile by quantitation of mdr-1/P-glycoprotein in the cell lines of the National Cancer Institute Anticancer Drug Screen.

    PubMed Central

    Alvarez, M; Paull, K; Monks, A; Hose, C; Lee, J S; Weinstein, J; Grever, M; Bates, S; Fojo, T

    1995-01-01

    Identifying new chemotherapeutic agents and characterizing mechanisms of resistance may improve cancer treatment. The Anticancer Drug Screen of the National Cancer Institute uses 60 cell lines to identify new agents. Expression of mdr-1/P-glycoprotein was measured by quantitative PCR. Expression was detected in 39 cell lines; the highest levels were in renal and colon carcinomas. Expression was also detected in all melanomas and central nervous system tumors, but in only one ovarian carcinoma and one leukemia cell line. Using a modified version of the COMPARE program, a high correlation was found between expression of mdr-1 and cellular resistance to a large number of compounds. Evidence that these compounds are P-glycoprotein substrates includes: (a) enhancement of cytotoxicity by verapamil; (b) demonstration of cross-resistance in a multidrug-resistant cell line, (c) ability to antagonize P-glycoprotein, increasing vinblastine accumulation by decreasing efflux; and (d) inhibition of photoaffinity labeling by azidopine. Identification of many heretofore unrecognized compounds as substrates indicates that P-glycoprotein has a broader substrate specificity than previously recognized. This study confirms the validity of this novel approach and provides the basis for similar studies examining a diverse group of gene products, including other resistance mechanisms, putative drug targets, and genes involved in the cell cycle and apoptosis. Images PMID:7738186

  19. Interaction of drugs of abuse and maintenance treatments with human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2).

    PubMed

    Tournier, Nicolas; Chevillard, Lucie; Megarbane, Bruno; Pirnay, Stéphane; Scherrmann, Jean-Michel; Declèves, Xavier

    2010-08-01

    Drug interaction with P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) may influence its tissue disposition including blood-brain barrier transport and result in potent drug-drug interactions. The limited data obtained using in-vitro models indicate that methadone, buprenorphine, and cannabinoids may interact with human P-gp; but almost nothing is known about drugs of abuse and BCRP. We used in vitro P-gp and BCRP inhibition flow cytometric assays with hMDR1- and hBCRP-transfected HEK293 cells to test 14 compounds or metabolites frequently involved in addiction, including buprenorphine, norbuprenorphine, methadone, ibogaine, cocaine, cocaethylene, amphetamine, N-methyl-3,4-methylenedioxyamphetamine, 3,4-methylenedioxyamphetamine, nicotine, ketamine, Delta9-tetrahydrocannabinol (THC), naloxone, and morphine. Drugs that in vitro inhibited P-gp or BCRP were tested in hMDR1- and hBCRP-MDCKII bidirectional transport studies. Human P-gp was significantly inhibited in a concentration-dependent manner by norbuprenorphine>buprenorphine>methadone>ibogaine and THC. Similarly, BCRP was inhibited by buprenorphine>norbuprenorphine>ibogaine and THC. None of the other tested compounds inhibited either transporter, even at high concentration (100 microm). Norbuprenorphine (transport efflux ratio approoximately 11) and methadone (transport efflux ratio approoximately 1.9) transport was P-gp-mediated; however, with no significant stereo-selectivity regarding methadone enantiomers. BCRP did not transport any of the tested compounds. However, the clinical significance of the interaction of norbuprenorphine with P-gp remains to be evaluated. PMID:19887017

  20. P-glycoprotein inhibitory activity of two phenolic compounds, (-)-syringaresinol and tricin from Sasa borealis.

    PubMed

    Jeong, Yeon Hee; Chung, Soo Yeon; Han, Ah-Reum; Sung, Min Kyung; Jang, Dae Sik; Lee, Jun; Kwon, Youngjoo; Lee, Hwa Jeong; Seo, Eun-Kyoung

    2007-01-01

    (-)-Syringaresinol and tricin, isolated from the AcOEt-soluble extract of the whole plants of Sasa borealis (Gramineae), showed inhibitory effects on the P-glycoprotein in adriamycin-resistant human breast cancer cells, MCF-7/ADR. PMID:17256728

  1. Localization of P-glycoprotein at the nuclear envelope of rat brain cells

    SciTech Connect

    Babakhanian, Karlo; Bendayan, Moise; Bendayan, Reina . E-mail: r.bendayan@utoronto.ca

    2007-09-21

    P-Glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.

  2. CEACAM1-Mediated Inhibition of Virus Production.

    PubMed

    Vitenshtein, Alon; Weisblum, Yiska; Hauka, Sebastian; Halenius, Anne; Oiknine-Djian, Esther; Tsukerman, Pinchas; Bauman, Yoav; Bar-On, Yotam; Stern-Ginossar, Noam; Enk, Jonatan; Ortenberg, Rona; Tai, Julie; Markel, Gal; Blumberg, Richard S; Hengel, Hartmut; Jonjic, Stipan; Wolf, Dana G; Adler, Heiko; Kammerer, Robert; Mandelboim, Ofer

    2016-06-14

    Cells in our body can induce hundreds of antiviral genes following virus sensing, many of which remain largely uncharacterized. CEACAM1 has been previously shown to be induced by various innate systems; however, the reason for such tight integration to innate sensing systems was not apparent. Here, we show that CEACAM1 is induced following detection of HCMV and influenza viruses by their respective DNA and RNA innate sensors, IFI16 and RIG-I. This induction is mediated by IRF3, which bound to an ISRE element present in the human, but not mouse, CEACAM1 promoter. Furthermore, we demonstrate that, upon induction, CEACAM1 suppresses both HCMV and influenza viruses in an SHP2-dependent process and achieves this broad antiviral efficacy by suppressing mTOR-mediated protein biosynthesis. Finally, we show that CEACAM1 also inhibits viral spread in ex vivo human decidua organ culture. PMID:27264178

  3. Pharmacokinetics and tolerability of NSC23925b, a novel P-glycoprotein inhibitor: preclinical study in mice and rats.

    PubMed

    Gao, Yan; Shen, Jacson K; Choy, Edwin; Zhang, Zhan; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

    2016-01-01

    Overexpression of P-glycoprotein (Pgp) increases multidrug resistance (MDR) in cancer, which greatly impedes satisfactory clinical treatment and outcomes of cancer patients. Due to unknown pharmacokinetics, the use of Pgp inhibitors to overcome MDR in the clinical setting remains elusive despite promising in vitro results. The purpose of our current preclinical study is to investigate the pharmacokinetics and tolerability of NSC23925b, a novel and potent P-glycoprotein inhibitor, in rodents. Plasma pharmacokinetic studies of single-dose NSC23925b alone or in combination with paclitaxel or doxorubicin were conducted in male BALB/c mice and Sprague-Dawley rats. Additionally, inhibition of human cytochrome P450 (CYP450) by NSC23925b was examined in vitro. Finally, the maximum tolerated dose (MTD) of NSC23925b was determined. NSC23925b displayed favorable pharmacokinetic profiles after intraperitoneal/intravenous (I.P./I.V.) injection alone or combined with chemotherapeutic drugs. The plasma pharmacokinetic characteristics of the chemotherapy drugs were not affected when co-administered with NSC23925b. All the animals tolerated the I.P./I.V. administration of NSC23925b. Moreover, the enzymatic activity of human CYP450 was not inhibited by NSC23925b. Our results demonstrated that Pgp inhibitor NSC23925b exhibits encouraging preclinical pharmacokinetic characteristics and limited toxicity in vivo. NSC23925b has the potential to treat cancer patients with MDR in the future. PMID:27157103

  4. Pharmacokinetics and tolerability of NSC23925b, a novel P-glycoprotein inhibitor: preclinical study in mice and rats

    PubMed Central

    Gao, Yan; Shen, Jacson K.; Choy, Edwin; Zhang, Zhan; Mankin, Henry J.; Hornicek, Francis J.; Duan, Zhenfeng

    2016-01-01

    Overexpression of P-glycoprotein (Pgp) increases multidrug resistance (MDR) in cancer, which greatly impedes satisfactory clinical treatment and outcomes of cancer patients. Due to unknown pharmacokinetics, the use of Pgp inhibitors to overcome MDR in the clinical setting remains elusive despite promising in vitro results. The purpose of our current preclinical study is to investigate the pharmacokinetics and tolerability of NSC23925b, a novel and potent P-glycoprotein inhibitor, in rodents. Plasma pharmacokinetic studies of single-dose NSC23925b alone or in combination with paclitaxel or doxorubicin were conducted in male BALB/c mice and Sprague-Dawley rats. Additionally, inhibition of human cytochrome P450 (CYP450) by NSC23925b was examined in vitro. Finally, the maximum tolerated dose (MTD) of NSC23925b was determined. NSC23925b displayed favorable pharmacokinetic profiles after intraperitoneal/intravenous (I.P./I.V.) injection alone or combined with chemotherapeutic drugs. The plasma pharmacokinetic characteristics of the chemotherapy drugs were not affected when co-administered with NSC23925b. All the animals tolerated the I.P./I.V. administration of NSC23925b. Moreover, the enzymatic activity of human CYP450 was not inhibited by NSC23925b. Our results demonstrated that Pgp inhibitor NSC23925b exhibits encouraging preclinical pharmacokinetic characteristics and limited toxicity in vivo. NSC23925b has the potential to treat cancer patients with MDR in the future. PMID:27157103

  5. Megakaryocyte-mediated inhibition of osteoclast development.

    PubMed

    Kacena, Melissa A; Nelson, Tracy; Clough, Mary E; Lee, Sun-Kyeong; Lorenzo, Joseph A; Gundberg, Caren M; Horowitz, Mark C

    2006-11-01

    A growing body of evidence indicates that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. We previously identified a novel regulatory pathway that controls bone formation, which is mediated by MK. In vivo megakaryocytosis resulted in massive bone formation. The co-culture of MK with osteoblasts (OB) resulted in increased OB proliferation in vitro, by a mechanism that required direct cell-to-cell contact. Here, we examined a second MK-mediated pathway that regulates osteoclast (OC) development. We have begun examining the unique inhibitory effect of MK on OC development. Spleen or bone marrow (BM) cells from C57BL/6 mice, as a source of OC precursors, were cultured with M-CSF and RANKL to induce OC development. MK were prepared by culturing fetal liver cells with thrombopoietin and separating cells into MK and non-MK populations. MK were titrated into spleen cell cultures and OC were identified as tartrate-resistant acid phosphatase-positive giant cells with >3 nuclei. There was a significant, P < 0.001, up to 10-fold reduction in OC formed when MK were added to the spleen cell cultures. We determined that 30% (vol:vol) MK conditioned media (CM) were able to completely block OC development from precursors, whereas 3% MK CM resulted in up to a 10-fold reduction in OC development, P < 0.001. These data indicate that a soluble factor(s) was responsible, at least in part, for the inhibition. We examined MK CM for known inhibitors of OC formation, using ELISAs. IL-4 was undetectable in MK CM, whereas IL-10 and IFN-gamma levels were similar in MK and non-MK CM. TGFbeta-1 levels were increased 2-fold in MK CM compared to control CM but were not responsible for the inhibition in OC development. Although, we found a significant increase in the levels of osteoprotegerin (OPG) in MK CM, antibody neutralization studies, MK derived from OPG-deficient mice, and tandem mass spectrophotometry, all confirm that OPG was not responsible for the MK-mediated

  6. P-glycoprotein ABCB1: a major player in drug handling by mammals.

    PubMed

    Borst, Piet; Schinkel, Alfred H

    2013-10-01

    Mammalian P-glycoproteins are active drug efflux transporters located in the plasma membrane. In the early nineties, we generated knockouts of the three P-glycoprotein genes of mice, the Mdr1a, Mdr1b, and Mdr2 P-glycoproteins, now known as Abcb1a, Abcb1b, and Abcb4, respectively. In the JCI papers that are the subject of this Hindsight, we showed that loss of Mdr1a (Abcb1a) had a profound effect on the tissue distribution and especially the brain accumulation of a range of drugs frequently used in humans, including dexamethasone, digoxin, cyclosporin A, ondansetron, domperidone, and loperamide. All drugs were shown to be excellent substrates of the murine ABCB1A P-glycoprotein and its human counterpart, the MDR1 P-glycoprotein, ABCB1. We found that the ability of ABCB1 to prevent accumulation of some drugs in the brain is a prerequisite for their clinical use, as absence of the transporter led to severe toxicity or undesired CNS pharmacodynamic effects. Subsequent work has fully confirmed the profound effect of the drug-transporting ABCB1 P-glycoprotein on the pharmacokinetics of drugs in humans. In fact, every new drug is now screened for transport by ABCB1, as this limits oral availability and penetration into sanctuaries protected by ABCB1, such as the brain. PMID:24084745

  7. Distinct P-glycoprotein precursors are overproduced in independently isolated drug-resistant cell lines.

    PubMed

    Greenberger, L M; Lothstein, L; Williams, S S; Horwitz, S B

    1988-06-01

    A family of P-glycoproteins are overproduced in multidrug-resistant cells derived from the murine macrophage-like line J774.2. To determine whether individual family members are overproduced in response to different drugs, the P-glycoprotein precursors in several independently isolated cell lines, which were selected for resistance to vinblastine or taxol, were compared. Individual cell lines selected with vinblastine overproduced P-glycoprotein precursors of either 120 or 125 kDa. Taxol-selected cell lines overproduced either the 125-kDa precursor or both precursors simultaneously. Two similar but distinct peptide maps for the mature P-glycoproteins were observed. These maps corresponded to each precursor regardless of the drug used for selection. One vinblastine-resistant cell line switched from the 125- to the 120-kDa precursor when grown in increasing concentrations of drug. This change coincided with the overexpression of a distinct subset of mRNA species that code for P-glycoprotein. It is concluded that precursor expression is not drug-specific. These data suggest that individual overproduced P-glycoprotein family members are translated as distinct polypeptides. The results may help to explain the diversity in the multidrug-resistant phenotype. PMID:2897689

  8. Sodium orthovanadate inhibits p53-mediated apoptosis.

    PubMed

    Morita, Akinori; Yamamoto, Shinichi; Wang, Bing; Tanaka, Kaoru; Suzuki, Norio; Aoki, Shin; Ito, Azusa; Nanao, Tomohisa; Ohya, Soichiro; Yoshino, Minako; Zhu, Jin; Enomoto, Atsushi; Matsumoto, Yoshihisa; Funatsu, Osamu; Hosoi, Yoshio; Ikekita, Masahiko

    2010-01-01

    Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. PMID:20048077

  9. A single active catalytic site is sufficient to promote transport in P-glycoprotein

    PubMed Central

    Bársony, Orsolya; Szalóki, Gábor; Türk, Dóra; Tarapcsák, Szabolcs; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Holb, Imre J.; Székvölgyi, Lóránt; Szabó, Gábor; Csanády, László; Szakács, Gergely; Goda, Katalin

    2016-01-01

    P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis. PMID:27117502

  10. A single active catalytic site is sufficient to promote transport in P-glycoprotein.

    PubMed

    Bársony, Orsolya; Szalóki, Gábor; Türk, Dóra; Tarapcsák, Szabolcs; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Holb, Imre J; Székvölgyi, Lóránt; Szabó, Gábor; Csanády, László; Szakács, Gergely; Goda, Katalin

    2016-01-01

    P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis. PMID:27117502

  11. Using Rhodamine 123 Accumulation in CD8+ Cells as a Surrogate Indicator to Study the P-Glycoprotein Modulating Effect of Cepharanthine Hydrochloride In Vivo

    PubMed Central

    Li, Han; Yan, Zhang; Ning, Wang; Xiao-Juan, Guo; Cai-Hong, Zang; Jin-Hua, Jiang; Fang, Ma; Qing-Duan, Wang

    2011-01-01

    The purpose of this study was the use of rhodamine 123 (Rho123) accumulation in peripheral blood CD8+cells as a surrogate indicator to evaluate the modulating effect of P-glycoprotein (P-gp) inhibitors in the multidrug resistance (MDR) tumor-bearing mouse model. Rho123 was administered to mice, and the fluorescence level in CD8+ cells was measured. Cepharanthine hydrochloride (CH) and verapamil (VER), two P-gp inhibitors, were administered to mice 1 hour prior to Rho123 administration in vivo or added to peripheral blood 1 hour prior to Rho123 addition ex vivo. The tumor inhibition effect of 5-fluorouracil/adriamycin/cisplatin (FAP) protocol plus CH was also investigated. A concentration- or dose-response relationship was shown between the concentration and dose of CH and Rho123 accumulation or the antitumor activity. In conclusion, the measurement of Rho123 accumulation in CD8+ cells provides a surrogate assay for the screening of candidate P-gp inhibitors in preclinical trials, and CH is effective in modulating P-gp-mediated MDR in vivo. PMID:21765632

  12. Classification of P-glycoprotein-interacting compounds using machine learning methods

    PubMed Central

    Prachayasittikul, Veda; Worachartcheewan, Apilak; Shoombuatong, Watshara; Prachayasittikul, Virapong; Nantasenamat, Chanin

    2015-01-01

    P-glycoprotein (Pgp) is a drug transporter that plays important roles in multidrug resistance and drug pharmacokinetics. The inhibition of Pgp has become a notable strategy for combating multidrug-resistant cancers and improving therapeutic outcomes. However, the polyspecific nature of Pgp, together with inconsistent results in experimental assays, renders the determination of endpoints for Pgp-interacting compounds a great challenge. In this study, the classification of a large set of 2,477 Pgp-interacting compounds (i.e., 1341 inhibitors, 913 non-inhibitors, 197 substrates and 26 non-substrates) was performed using several machine learning methods (i.e., decision tree induction, artificial neural network modelling and support vector machine) as a function of their physicochemical properties. The models provided good predictive performance, producing MCC values in the range of 0.739-1 for internal cross-validation and 0.665-1 for external validation. The study provided simple and interpretable models for important properties that influence the activity of Pgp-interacting compounds, which are potentially beneficial for screening and rational design of Pgp inhibitors that are of clinical importance. PMID:26862321

  13. Investigation of the Functional Role of P-Glycoprotein in Limiting the Oral Bioavailability of Lumefantrine

    PubMed Central

    Raju, Kanumuri S. R.; Singh, Sheelendra P.; Taneja, Isha

    2014-01-01

    In the quest to explore the reason for the low and variable bioavailability of lumefantrine, we investigated the possible role of P-glycoprotein (P-gp) in lumefantrine intestinal absorption. An in situ single-pass intestinal perfusion study in rats with the P-gp inhibitor verapamil or quinidine and an ATPase assay with human P-gp membranes indicated that lumefantrine is a substrate of P-gp which limits its intestinal absorption. To confirm these findings, an in vivo pharmacokinetic study was performed in rats. The oral administration of verapamil (10 mg/kg of body weight) along with lumefantrine caused a significant increase in its bioavailability with a concomitant decrease in clearance. The increase in bioavailability of lumefantrine could be due to inhibition of P-gp and/or cytochrome P450 3A in the intestine/liver by verapamil. However, in a rat intestinal microsomal stability study, lumefantrine was found to be resistant to oxidative metabolism. Further, an in situ permeation study clearly showed a significant role of P-gp in limiting the oral absorption of lumefantrine. Thus, the increase in lumefantrine bioavailability with verapamil is attributed in part to the P-gp-inhibitory ability of verapamil. In conclusion, lumefantrine is a substrate of P-gp, and active efflux by P-gp across the intestine partly contributed to the low/variable bioavailability of lumefantrine. PMID:24189249

  14. Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor.

    PubMed Central

    Raggers, R J; Vogels, I; van Meer, G

    2001-01-01

    The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [(14)C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [(14)C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth. PMID:11463358

  15. Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells

    PubMed Central

    Nieri, Paola; Romiti, Nadia; Adinolfi, Barbara; Chicca, Andrea; Massarelli, Ilaria; Chieli, Elisabetta

    2006-01-01

    Endogenous and synthetic cannabinoid molecules have been investigated as possible MDR-1/P-glycoprotein (P-gp) modulators in HK-2-immortalized renal cells, using calcein acetoxymethylester (calcein-AM) as a P-gp substrate. Among the endocannabinoid molecules tested, anandamide (AEA), but not 2-arachidonoyl-glycerol (2-AG) or palmitoyl-ethanolamide (PEA), increased the intracellular fluorescence emitted by calcein, a metabolic derivative of the P-gp substrate calcein-AM, indicative of a reduction in transport capacity. All the three synthetic cannabimimetics tested, that is, R-(+)-methanandamide (R(+)-MET), AM 251 and CP55,940 significantly increased calcein accumulation in the cytosol. RT–PCR demonstrated that HK-2 cells do not express CB1 or CB2 cannabinoid receptors. R(+)-MET, AM251 and CP55,940 were also evaluated as modulators of P-gp expression, by Western blot analysis. Only AM251 weakly enhanced the protein levels (by 1.2-fold) after a 4-day-long incubation with the noncytotoxic drug concentration 2 μM. The present data provide the first evidence that the endocannabinoid AEA and different synthetic cannabinoids may inhibit the P-gp activity in vitro via a cannabinoid receptor-independent mechanism. PMID:16715117

  16. Interactions of attention-deficit/hyperactivity disorder therapeutic agents with the efflux transporter P-glycoprotein

    PubMed Central

    Zhu, Hao-Jie; Wang, Jun-Sheng; Donovan, Jennifer L.; Jiang, Yan; Gibson, Bryan B.; DeVane, C. Lindsay; Markowitz, John S.

    2009-01-01

    The objective of this study was to assess the potential interactions of the drug transporter P-glycoprotein with attention-deficit/hyperactivity disorder (ADHD) therapeutic agents atomoxetine — and the individual isomers of methylphenidate, amphetamine, and modafinil utilizing established in vitro assay. An initial ATPase assay indicated that both d- and l-methylphenidate have weak affinity for P-glycoprotein. The intracellular accumulation of P-glycoprotein substrates doxorubicin and rhodamine123 in the P-glycoprotein overexpressing cell line LLC-PK1/MDR1 was determined to evaluate potential inhibitory effects on P-glycoprotein. The results demonstrated that all compounds, except both modafinil isomers, significantly increased doxorubicin and rhodamine123 accumulation in LLC-PK1/MDR1 cells at higher concentrations. To investigate the P-glycoprotein substrate properties, the intracellular concentrations of the tested compounds in LLC-PK1/MDR1 and P-glycoprotein negative LLC-PK1 cells were measured in the presence and absence of the P-glycoprotein inhibitor PSC833. The results indicate that the accumulation of d-methylphenidate in LLC-PK1 cells was 32.0% higher than in LLC-PK1/MDR1 cells. Additionally, coadministration of PSC833 leads to 52.9% and 45.6% increases in d-modafinil and l-modafinil accumulation, respectively, in LLC-PK1/MDR1 cells. Further studies demonstrated that l-modafinil transport across LLC-PK1/MDR1 cell monolayers in the basolateral-to-apical (B–A) direction was significantly higher than in the apical-to-basolateral (A–B) direction. PSC833 treatment significantly decreased the transport of l-modafinil in B–A direction. In conclusion, our results suggest that all tested agents with the exception of modafinil isomers are relatively weak P-glycoprotein inhibitors. Furthermore, P-glycoprotein may play a minor role in the transport of d-methylphenidate, d-modafinil, and l-modafinil. PMID:17963743

  17. Characterization of Haemonchus contortus P-glycoprotein-16 and its interaction with the macrocyclic lactone anthelmintics.

    PubMed

    Godoy, P; Che, H; Beech, R N; Prichard, R K

    2015-11-01

    Anthelmintic resistance in veterinary nematodes, including Haemonchus contortus, has become a limitation to maintaining high standards of animal health. Resistance in this parasite, to all drug families including the macrocyclic lactones (MLs) is a serious issue worldwide. Mechanisms of resistance to the MLs appear to be complex and to include the elimination of these compounds by ABC transporter-like proteins present in nematodes. In order to investigate the potential involvement of ABC transporters in ML resistance in H. contortus, we have characterized the functionality of the ABC transporter H. contortus P-glycoprotein-16 (Hco-PGP-16) expressed in mammalian cells. This has included a study of its interaction with different MLs, including the avermectins, abamectin (ABA) and ivermectin (IVM), and the milbemycin, moxidectin (MOX). Hco-PGP-16 transport activity was studied using the fluorophore Rhodamine 123 (Rho 123). Transfected cells expressing Hco-PGP-16 accumulated less than 50% of Rho 123 than control cells, suggesting an active transport of this tracer dye by Hco-PGP-16. The influence of the MLs on the Rho123 transport by Hco-PGP-16 was then investigated. A marked inhibition of Rho123 transport by ABA and IVM was observed. In contrast, MOX showed less effect on inhibition of Rho123 transport by Hco-PGP-16, and the inhibition was not saturable. The difference in the interaction of the avermectins and MOX with Hco-PGP-16 may help explain the slower rate of development of resistance to MOX compared with the avermectins in H. contortus. PMID:26657092

  18. Reversal of P-glycoprotein-medicated multidrug resistance by LBM-A5 in vitro and a study of its pharmacokinetics in vivo.

    PubMed

    Zhao, Tianxiao; Song, Yun; Liu, Baomin; Qiu, Qianqian; Jiao, Lei; Li, Yunman; Huang, Wenlong; Qian, Hai

    2015-01-01

    The overexpression of P-glycoprotein (P-gp) in tumors leads to multidrug resistance (MDR), which is a significant obstacle in clinical cancer chemotherapy. The co-administration of anticancer drugs and MDR modulators is a promising strategy for overcoming this problem. Our study aimed to explore the reversal mechanism and safety of the MDR modulator LBM-A5 in vitro, and evaluate its pharmacokinetics and effects on doxorubicin metabolism in vivo. We evaluated an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of anticancer agents mediated by LBM-A5, the effect of LBM-A5 on rhodamine123 intracellular accumulation, and the efflux in K562/DOX cells to investigate the reversal mechanisms of LBM-A5. The results showed that LBM-A5 inhibits rhodamine123 efflux and increases intracellular accumulation by inhibiting the efflux pump function of P-gp. Furthermore, the therapeutic index and CYP3A4 activity analysis in vitro suggested that LBM-A5 is reasonably safe to use. Also, LBM-A5 (10 mg/kg body mass) achieved the required plasma concentration in sufficient time to reverse MDR in vivo. Importantly, the LBM-A5 treatment group shared similar doxorubicin (DOX) pharmacokinetics with the free DOX group. Our results suggest that LBM-A5 effectively reverses MDR (EC50 = 483.6 ± 81.7 nmol·L(-1)) by inhibiting the function of P-gp, with relatively ideal pharmacokinetics and in a safe manner, and so may be a promising candidate for cancer chemotherapy research. PMID:25427107

  19. Pharmacokinetic Compatibility of Ginsenosides and Schisandra Lignans in Shengmai-san: From the Perspective of P-Glycoprotein

    PubMed Central

    Liang, Yan; Zhou, Yuanyuan; Zhang, Jingwei; Rao, Tai; Zhou, Lijun; Xing, Rong; Wang, Qian; Fu, Hanxu; Hao, Kun; Xie, Lin; Wang, Guangji

    2014-01-01

    Background Phytochemical-mediated alterations in P-glycoprotein (P-gp) activity may result in herb-drug interactions by altering drug pharmacokinetics. Shengmai-san, a traditional Chinese herbal medicine composed by Panax Ginseng, Ophiopogon Japonicus, and Schisandra Chinensis, is routinely being used for treating various coronary heart diseases. In our previous studies, Schisandra Lignans Extract (SLE) was proved as a strong P-gp inhibitor, and herein, the compatibility of Shengmai-san was studied by investigating the influence of SLE on the pharmacokinetics of the ginsenosides from the perspective of P-gp. Methodology Pharmacokinetic experiments were firstly performed based on in vitro uptake, efflux and transport experiments in Caco-2, LLC-PK1 wild-type and MDR1-overexpressing L-MDR1 cells. During the whole experiment, digoxin, a classical P-gp substrate, was used as a positive control drug to verify the cells used are the valid models. Meanwhile, the effects of SLE on the pharmacokinetics of ginsenosides were further investigated in rats after single-dose and multi-dose of SLE. Results and Conclusions The efflux ratios of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 were found more than 3.5 in L-MDR1 cells and can be decreased significantly by verapamil (a classical P-gp inhibitor). Contrarily, the efflux ratios of other ginsenosides (Rh1, F1, Re, and Rg1) were lower than 2.0 and not affected by verapamil. Then, the effects of SLE on the uptake and transport of ginsenosides were investigated, and SLE was found can significantly enhance the uptake and inhibit the efflux ratio of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 in Caco-2 and L-MDR1 cells. Besides, In vivo experiments showed that single-dose and multi-dose of SLE at 500 mg/kg could increase the area under the plasma concentration time curve of Rb2, Rc and Rd significantly without affecting terminal elimination half-time. In conclusion, SLE could enhance the exposure of ginsenosides Rb2, Rc, Rg2, Rg3, Rd and

  20. Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

    PubMed

    Mushtaq, Gohar; Greig, Nigel H; Anwar, Firoz; Al-Abbasi, Fahad A; Zamzami, Mazin A; Al-Talhi, Hasan A; Kamal, Mohammad A

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  1. Neuroprotective Mechanisms Mediated by CDK5 Inhibition

    PubMed Central

    Mushtaq, Gohar; Greig, Nigel H.; Anwar, Firoz; Al-Abbasi, Fahad A.; Zamzami, Mazin A.; Al-Talhi, Hasan A.; Kamal, Mohammad A.

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not well-understood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine-induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  2. Biochemical mechanism of modulation of human P-glycoprotein by stemofoline.

    PubMed

    Chanmahasathien, Wisinee; Ohnuma, Shinobu; Ambudkar, Suresh V; Limtrakul, Pornngarm

    2011-12-01

    The resistance to chemotherapeutic drugs by cancer cells is considered to be one of the major obstacles for success in the treatment of cancer. A major mechanism underlying this multidrug resistance is the overexpression of P-glycoprotein (P-gp), resulting in insufficient drug delivery to the tumor sites. A previous study has shown that stemofoline, an alkaloid isolated from Stemona burkillii, could enhance the sensitivity of chemotherapeutics in a synergistic fashion. In the present study, we have focused on the effect of stemofoline on the modulation of P-gp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The effects of stemofoline on a radiolabeled drug, [(3)H]-vinblastine, and fluorescent P-gp substrates, rhodamine 123 and calcein-AM accumulation or retention were investigated to confirm this finding. Stemofoline could increase the accumulation or retention of radiolabeled drugs or fluorescent P-gp substrates in a dose-dependent manner. For additional studies on drug-P-gp binding, P-gp ATPase activity was stimulated by stemofoline in a concentration-dependent manner. More evidence was offered that stemofoline inhibits the effect on photoaffinity labeling of P-gp with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. These data indicate that stemofoline may interact directly with P-gp and inhibit P-gp activity, whereas stemofoline has no effect on P-gp expression. Taken together, the results exhibit that stemofoline possesses an effective MDR modulator, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells. PMID:21786221

  3. Several major antiepileptic drugs are substrates for human P-glycoprotein.

    PubMed

    Luna-Tortós, Carlos; Fedrowitz, Maren; Löscher, Wolfgang

    2008-12-01

    One of the current hypotheses of pharmacoresistant epilepsy proposes that transport of antiepileptic drugs (AEDs) by drug efflux transporters such as P-glycoprotein (Pgp) at the blood-brain barrier may play a significant role in pharmacoresistance in epilepsy by extruding AEDs from their intended site of action. However, several recent in vitro studies using cell lines that overexpress efflux transporters indicate that human Pgp may not transport AEDs to any relevant extent. In this respect it has to be considered that most AEDs are highly permeable, so that conventional bi-directional transport assays as used in these previous studies may fail to identify AEDs as Pgp substrates, particularly if these drugs are not high-affinity substrates for Pgp. In the present study, we used a modified transport assay that allows evaluating active transport independently of the passive permeability component. In this concentration equilibrium transport assay (CETA), the drug is initially added at identical concentration to both sides of a polarized, Pgp-overexpressing cell monolayer instead of applying the drug to either the apical or basolateral side for studying bi-directional transport. Direct comparison of the conventional bi-directional (concentration gradient) assay with the CETA, using MDR1-transfected LLC cells, demonstrated that CETA, but not the conventional assay, identified phenytoin and phenobarbital as substrates of human Pgp. Furthermore, directional transport was determined for lamotrigine and levetiracetam, but not carbamazepine. Transport of AEDs could be completely or partially (>50%) inhibited by the selective Pgp inhibitor, tariquidar. However, transport of phenobarbital and levetiracetam was also inhibited by MK571, which preferentially blocks transport by multidrug resistance transporters (MRPs), indicating that, in addition to Pgp, these AEDs are substrates of MRPs. The present study provides the first direct evidence that several AEDS are substrates of

  4. The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids

    PubMed Central

    2005-01-01

    The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a ‘flippase’ for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD–GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD–GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD–GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD–lactosylceramide drastically reduced the rate of flipping compared with NBD–PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids. PMID:15799713

  5. The Effects of Cetirizine on P-glycoprotein Expression and Function In vitro and In situ

    PubMed Central

    Mesgari Abbasi, Mehran; Valizadeh, Hadi; Hamishekar, Hamed; Mohammadnejad, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Purpose: P-glycoprotein (P-gp) plays a major role in oral absorption of drugs. Induction or inhibition of P-gp by drugs contributes to variability of its transport activity and often results in clinically relevant drug-drug interactions. The purpose of this study was to investigate the effect of cetirizine, a second generation H1 antihistamine, on P-gp function and expression in vitro and in situ. Methods: The in-vitro rhodamin-123 (Rho123) efflux assay in Caco-2 cells was used to study the effect of cetirizine on P-gp function. Western blot analysis was used for surveying the effect of cetirizine on expression of P-gp in Caco-2 cells. Rat in situ single-pass intestinal permeability technique was used to calculate the intestinal permeability of a known P-gp substrate (digoxin) in the presence of cetirizine. The amounts of digoxin and cetirizine in intestinal perfusion samples were analyzed using a HPLC method. Results: The results showed significant increase in Rho123 uptake (P < 0.05) and also P-gp band intensity decrease in cetirizine-treated cells in vitro. Furthermore the intestinal permeability of digoxin was also increased significantly in the presence of cetirizine (P < 0.01). Conclusion: Therefore it is concluded that cetirizine is a P-gp inhibitor and this should be considered in co administration of cetrizine with other P-gp substrate drugs. Further investigations are required to confirm our results and to determine the mechanism underlying P-gp inhibition by cetirizine. PMID:27123426

  6. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  7. Enhancing Activity of Anticancer Drugs in Multidrug Resistant Tumors by Modulating P-Glycoprotein through Dietary Nutraceuticals.

    PubMed

    Khan, Muhammad; Maryam, Amara; Mehmood, Tahir; Zhang, Yaofang; Ma, Tonghui

    2015-01-01

    Multidrug resistance is a principal mechanism by which tumors become resistant to structurally and functionally unrelated anticancer drugs. Resistance to chemotherapy has been correlated with overexpression of p-glycoprotein (p-gp), a member of the ATP-binding cassette (ABC) superfamily of membrane transporters. P-gp mediates resistance to a broad-spectrum of anticancer drugs including doxorubicin, taxol, and vinca alkaloids by actively expelling the drugs from cells. Use of specific inhibitors/blocker of p-gp in combination with clinically important anticancer drugs has emerged as a new paradigm for overcoming multidrug resistance. The aim of this paper is to review p-gp regulation by dietary nutraceuticals and to correlate this dietary nutraceutical induced-modulation of p-gp with activity of anticancer drugs. PMID:26514453

  8. Tariquidar Is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein

    PubMed Central

    Weidner, Lora D.; Fung, King Leung; Kannan, Pavitra; Moen, Janna K.; Kumar, Jeyan S.; Mulder, Jan; Innis, Robert B.; Gottesman, Michael M.

    2016-01-01

    Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [11C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes. PMID:26658428

  9. High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

    PubMed

    Guertin, Amy D; O'Neil, Jennifer; Stoeck, Alexander; Reddy, Joseph A; Cristescu, Razvan; Haines, Brian B; Hinton, Marlene C; Dorton, Ryan; Bloomfield, Alicia; Nelson, Melissa; Vetzel, Marilynn; Lejnine, Serguei; Nebozhyn, Michael; Zhang, Theresa; Loboda, Andrey; Picard, Kristen L; Schmidt, Emmett V; Dussault, Isabelle; Leamon, Christopher P

    2016-08-01

    Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR. PMID:27256377

  10. The role of P-glycoprotein in the bioactivation of raloxifene.

    PubMed

    Chang, Jae H; Kochansky, Christopher J; Shou, Magang

    2006-12-01

    Drug transporters have been shown to alter drug metabolism. Similarly, bioactivation of drugs may also be altered by drug transporters. The aim of this work was to examine the role of P-glycoprotein (Pgp) in the bioactivation of a Pgp substrate, raloxifene, and a non-Pgp substrate, naphthalene. To evaluate the extent of bioactivation, covalent binding was measured. In both freshly isolated and cryopreserved hepatocytes, the extent of raloxifene covalent binding increased significantly (p < 0.05) in the presence of verapamil, whereas no change was observed with the covalent binding of naphthalene. To ascertain that the change was a Pgp effect, covalent binding was examined in microsomes in which raloxifene and naphthalene covalent binding was not altered in the presence of verapamil. In addition, the measure of raloxifene-glutathione adducts in the cryopreserved hepatocytes showed that the formation of the adducts increased in the presence of verapamil, which supports the idea that blocking Pgp in the liver increases metabolism and, therefore, the bioactivation of raloxifene. Because raloxifene and naphthalene are known to undergo bioactivation mediated by CYP3A4, covalent binding in the presence of ketoconazole was examined. In both hepatocytes and microsomes, raloxifene covalent binding decreased significantly (p < 0.01). It is interesting that naphthalene covalent binding was not affected. In the presence of the CYP2E inhibitor 4-methylpyrazole, a decrease in naphthalene covalent binding was observed, suggesting that the formation of the 1,2-epoxide may be the main culprit contributing to naphthalene covalent binding. In conclusion, these data suggest that in addition to other "protective" mechanisms, Pgp may attenuate bioactivation of drugs. PMID:16959878

  11. Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

    PubMed

    Pluchino, Kristen M; Hall, Matthew D; Moen, Janna K; Chufan, Eduardo E; Fetsch, Patricia A; Shukla, Suneet; Gill, Deborah R; Hyde, Stephen C; Xia, Di; Ambudkar, Suresh V; Gottesman, Michael M

    2016-02-23

    The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes. PMID:26820614

  12. In Silico Screening for Inhibitors of P-Glycoprotein That Target the Nucleotide Binding Domains

    PubMed Central

    Brewer, Frances K.; Follit, Courtney A.; Vogel, Pia D.

    2014-01-01

    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. PMID:25270578

  13. Eletriptan metabolism by human hepatic CYP450 enzymes and transport by human P-glycoprotein.

    PubMed

    Evans, David C; O'Connor, Desmond; Lake, Brian G; Evers, Raymond; Allen, Christopher; Hargreaves, Richard

    2003-07-01

    "Reaction phenotyping" studies were performed with eletriptan (ETT) to determine its propensity to interact with coadministered medications. Its ability to serve as a substrate for human P-glycoprotein (P-gp) was also investigated since a central mechanism of action has been proposed for this "triptan" class of drug. In studies with a characterized bank of human liver microsome preparations, a good correlation (r2 = 0.932) was obtained between formation of N-desmethyl eletriptan (DETT) and CYP3A4-catalyzed testosterone 6 beta-hydroxylation. DETT was selected to be monitored in our studies since it represents a significant ETT metabolite in humans, circulating at concentrations 10 to 20% of those observed for parent drug. ETT was metabolized to DETT by recombinant CYP2D6 (rCYP2D6) and rCYP3A4, and to a lesser extent by rCYP2C9 and rCYP2C19. The metabolism of ETT to DETT in human liver microsomes was markedly inhibited by troleandomycin, erythromycin, miconazole, and an inhibitory antibody to CYP3A4, but not by inhibitors of other major P450 enzymes. ETT had little inhibitory effect on any of the P450 enzymes investigated. ETT was determined to be a good substrate for human P-gp in vitro. In bidirectional transport studies across LLC-MDR1 and LLC-Mdr1a cell monolayers, ETT had a BA/AB transport ratio in the range 9 to 11. This finding had significance in vivo since brain exposure to ETT was reduced 40-fold in Mdr1a+/+ relative to Mdr1a-/- mice. ETT metabolism to DETT is therefore catalyzed primarily by CYP3A4, and plasma concentrations are expected to be increased when coadministered with inhibitors of CYP3A4 and P-gp activity. PMID:12814962

  14. Calcium and P-glycoprotein independent synergism between schweinfurthins and verapamil

    PubMed Central

    Sheehy, Ryan M; Kuder, Craig H; Bachman, Zoe; Hohl, Raymond J

    2015-01-01

    Schweinfurthins are intriguing natural products with anti-cancer activities and as yet incompletely understood mechanisms of action. We investigated whether inhibitors of P-glycoprotein (Pgp), in a manner analogous to other natural products, might enhance schweinfurthins' growth inhibitory actions by increasing intracellular schweinfurthin levels. Both the schweinfurthin-sensitive glioblastoma multiforme cell line SF-295 and relatively insensitive lung carcinoma cell line A549 were treated with 2 schweinfurthin analogs: 3-deoxyschweinfurthin B-p-nitro bis-stilbene (3dSB-PNBS) and 5′-methylschweinfurthin G (methyl-G). There was a synergistic enhancement of growth inhibition with the combination of the Pgp inhibitor verapamil and both analogs in SF-295 cells. Methyl-G, verapamil, and the combination did not result in alterations to intracellular calcium concentration. Verapamil increased the intracellular concentration of 3dSB-PNBS in both SF-295 and A549 cells in a Pgp-independent manner. Methyl-G, verapamil, and the combination do not result in increased ER stress. Methyl-G increased the intracellular concentration of a known Pgp substrate, Rhodamine 123 in SF-295 cells. Reduction of cellular cholesterol leads to the accumulation of Pgp substrates, as Pgp requires cholesterol for proper function. Since 3dSB enhances lovastatin-induced upregulation of the cholesterol efflux pump ABCA1, it is intriguing that co-treatment with cholesterol rescued the methyl-G-induced increase in Rhodamine 123 intracellular concentration. These studies support the hypothesis that verapamil potentiates the schweinfurthin growth inhibitory effect by increasing its intracellular concentration. PMID:26046259

  15. Simotinib as a modulator of P-glycoprotein: substrate, inhibitor, or inducer?

    PubMed

    Huang, Lingling; Shen, Cheng; Chen, Yanfen; Yan, Huiwen; Cheng, Zeneng; Zhu, Qubo

    2016-04-01

    As a new antitumor drug, simotinib hydrochloride is prescribed for prolonged periods, often to patients with comorbidities. Therefore, the risk for developing drug resistance and drug-drug interactions between simotinib and other agents has to be taken into consideration. As P-glycoprotein (P-gp) is an efflux transporter, which plays a significant role in drug resistance and influences the pharmacological properties and toxicities of the drugs it interacts with, the interactions between simotinib and P-gp were investigated. Cytotoxicity was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Intracellular drug concentrations were detected by high-performance liquid chromatography, fluorescence-activated cell sorting and using a fluorescence reader. P-gp ATPase activity was measured using the Pgp-Glo assay, and intracellular pH was assessed using the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl. The expression and transcription of P-gp were detected by western blotting and the luciferase assay. Simotinib has no cross-resistance to P-gp substrates, and its efflux rate was independent of either the P-gp expression or the coadministered P-gp substrate. Simotinib reversed chemotherapeutic agent resistance in a short time by increasing the intracellular concentration of the chemotherapeutic agent and blocked rhodamine 123 efflux. Further studies demonstrated that simotinib inhibited P-gp activity by modulating its ATPase activity and the intracellular pH. Although simotinib induced P-gp expression after extended treatment, the induced expression of P-gp had little impact on drug resistance. Simotinib is not a substrate of P-gp. As a modulator, it functions mainly as an inhibitor of P-gp by modulating the intracellular pH and ATPase activity, although it also induces P-gp expression after extended treatment. PMID:26766493

  16. A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

    PubMed Central

    Protasevich, Irina I.; Brouillette, Christie G.; Harrell, Patina M.; Hildebrandt, Ellen; Gasser, Brigitte; Mattanovich, Diethard; Ward, Andrew; Chang, Geoffrey; Urbatsch, Ina L.

    2011-01-01

    Background Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers. Methodology/Principal Findings Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from Tm ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids. Conclusion The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins. PMID:21826197

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

    PubMed

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

    2010-01-01

    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. PMID:20957096

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

    PubMed Central

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

    2010-01-01

    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. PMID:20957096

  19. Effects of steroids and verapamil on P-glycoprotein ATPase activity: progesterone, desoxycorticosterone, corticosterone and verapamil are mutually non-exclusive modulators.

    PubMed Central

    Orlowski, S; Mir, L M; Belehradek, J; Garrigos, M

    1996-01-01

    P-glycoprotein (P-gp) is a membranous ATPase responsible for the multidrug resistance (MDR) phenotype. Using membrane vesicles prepared from the highly resistant cell line DC-3F/ADX we studied the influence of P-gp ATPase activity of four progesterone derivatives which specifically bind to P-gp and reverse MDR. Progesterone and desoxycorticosterone stimulate P-gp ATPase activity with, respectively, apparent concentrations giving half-maximal activation of 20-25 microM and 40-50 microM, and activation factors of 2.3 (at 100 microM progesterone) and 1.8 (at 170 microM desoxycorticosterone). Hydrocortisone above 100 microM stimulates P-gp ATPase activity while corticosterone has no apparent stimulating effect. Our data are consistent with the location of the binding sites for the progesterone derivatives on the P-gp membranous domain. The effects of these steroids on verapamil-stimulated P-gp ATPase activity support a non-competitive mechanism, i.e. the binding sites for verapamil and steroids are mutually non-exclusive for P-gp ATPase modulation. A similar non-competitive inhibition of progesterone-stimulated P-gp ATPase activity by desoxycorticosterone or by corticosterone leads to the conclusion that these steroids, although sharing related structures, have distinct modulating sites on P-gp. As expected from their mutually non-exclusive interactions on P-gp, progesterone and verapamil when mixed induce a synergistic modulation of P-gp ATPase activity. Since drug transport by P-gp is believed to be coupled to its ATPase activity, a corresponding synergistic effect of these two modulators for the inhibition of P-gp-mediated drug resistance can be expected. PMID:8713080

  20. Effects of brain IKKβ gene silencing by small interfering RNA on P-glycoprotein expression and brain damage in the rat kainic acid-induced seizure model.

    PubMed

    Yu, Nian; Liu, Hao; Zhang, Yan-Fang; Su, Ling-Ying; Liu, Xin-Hong; Li, Le-Chao; Hao, Jin-Bo; Huang, Xian-Jing; Di, Qing

    2014-01-01

    Multidrug resistance mediated by over-expression of P-glycoprotein (P-gp) in brain is an important mechanism accounting for the drug-therapy failure in epilepsy. Over-expression of P-gp in epilepsy rat brain may be regulated by inflammation and nuclear factor-kappa B (NF-κB) activation. Inhibitory κ B kinase subunit β (IKKβ) is an up-stream molecular controlling NF-κB activation. With the small interfering RNA (siRNA) technique and kainic acid (KA)-induced rat epileptic seizure model, the present study was aimed to further evaluate the role of NF-κB inhibition, via blocking IKKβ gene transcription, in the epileptic brain P-gp over-expression, seizure susceptibility, and post-seizure brain damage. siRNA targeting IKKβ was administered to rats via intracerebroventricular injection before seizure induction by KA microinjection; scrambled siRNA was used as control. Brain mRNA and protein levels of IKKβ and P-gp were detected by RT-PCR and immunohistochemistry. NF-κB activity was measured by electrophoretic mobility shift assay. Latency to grade III or V seizure onset was recorded, brain damage was evaluated by neuronal cell counting and epileptiform activity was monitored by electroencephalography. IKKβ siRNA pre-treatment inhibited NF-κB activation and abolished P-gp over-expression in KA-induced epileptic rat brain, accompanied by decreased seizure susceptibility. These findings suggested that epileptogenic-induced P-gp over-expression could be regulated by IKKβ through the NF-κB pathway. PMID:24040792

  1. IMMUNOHISTOCHEMICAL DETECTION OF P-GLYCOPROTEIN IN TELEOST TISSUES USING MAMMALIAN POLYCLONAL AND MONOCLONAL ANTIBODIES

    EPA Science Inventory

    Mammalian P-glycoprotein is a highly conserved 170 kD integral plasma membrane protein functioning as an energy dependent efflux pump of exogenous and endogenous lipophilic aromatic compounds entering the cell by diffusion. n this study, the tissue specificity of one polyclonal (...

  2. In silico Analysis for Predicting Fatty Acids of Black Cumin Oil as Inhibitors of P-Glycoprotein

    PubMed Central

    Ali, Babar; Jamal, Qazi Mohd. Sajid; Mir, Showkat R.; Shams, Saiba; Al-Wabel, Naser A.; Kamal, Mohammad A.

    2015-01-01

    Background: Black cumin oil is obtained from the seeds of Nigella sativa L. which belongs to family Ranunculaceae. The seed oil has been reported to possess antitumor, antioxidant, antibacterial, anti-inflammatory, hypoglycemic, central nervous system depressant, antioxidant, and immunostimulatory activities. These bioactivities have been attributed to the fixed oil, volatile oil, or their components. Seed oil consisted of 15 saturated fatty acids (17%) and 17 unsaturated fatty acids (82.9%). Long chain fatty acids and medium chain fatty acids have been reported to increase oral bioavailability of peptides, antibiotics, and other important therapeutic agents. In earlier studies, permeation enhancement and bioenhancement of drugs has been done with black cumin oil. Objective: In order to recognize the mechanism of binding of fatty acids to P-glycoprotein (P-gp), linoleic acid, oleic acid, margaric acid, cis-11, 14-eicosadienoic acid, and stearic acid were selected for in silico studies, which were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. Materials and Methods: Template search with BLAST and HHblits has been performed against the SWISS-MODEL template library. The target sequence was searched with BLAST against the primary amino acid sequence of P-gp from Rattus norvegicus. Results: The amount of energy needed by linoleic acid, oleic acid, eicosadienoic acid, margaric acid, and stearic acid to bind with P-gp were found to be − 10.60, −10.48, −9.95, −11.92, and − 10.37 kcal/mol, respectively. The obtained data support that all the selected fatty acids have contributed to inhibit P-gp activity thereby enhances the bioavailability of drugs. Conclusion: This study plays a significant role in finding hot spots in P-gp and may offer the further scope of designing potent and specific inhibitors of P-gp. SUMMARY Generation of 3D structure of fatty acid compounds from Black cumin oil and 3D homology modeling of Rat P

  3. Absence of P-glycoprotein transport in the pharmacokinetics and toxicity of the herbicide paraquat.

    PubMed

    Lacher, Sarah E; Gremaud, Julia N; Skagen, Kasse; Steed, Emily; Dalton, Rachel; Sugden, Kent D; Cardozo-Pelaez, Fernando; Sherwin, Catherine M T; Woodahl, Erica L

    2014-02-01

    Genetic variation in the multidrug resistance gene ABCB1, which encodes the efflux transporter P-glycoprotein (P-gp), has been associated with Parkinson disease. Our goal was to investigate P-gp transport of paraquat, a Parkinson-associated neurotoxicant. We used in vitro transport models of ATPase activity, xenobiotic-induced cytotoxicity, transepithelial permeability, and rhodamine-123 inhibition. We also measured paraquat pharmacokinetics and brain distribution in Friend leukemia virus B-type (FVB) wild-type and P-gp-deficient (mdr1a(-/-)/mdr1b(-/-)) mice following 10, 25, 50, and 100 mg/kg oral doses. In vitro data showed that: 1) paraquat failed to stimulate ATPase activity; 2) resistance to paraquat-induced cytotoxicity was unchanged in P-gp-expressing cells in the absence or presence of P-gp inhibitors GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] and verapamil-37.0 [95% confidence interval (CI): 33.2-41.4], 46.2 (42.5-50.2), and 34.1 µM (31.2-37.2)-respectively; 3) transepithelial permeability ratios of paraquat were the same in P-gp-expressing and nonexpressing cells (1.55 ± 0.39 and 1.39 ± 0.43, respectively); and 4) paraquat did not inhibit rhodamine-123 transport. Population pharmacokinetic modeling revealed minor differences between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice: clearances of 0.47 [95% confidence interval (CI): 0.42-0.52] and 0.78 l/h (0.58-0.98), respectively, and volume of distributions of 1.77 (95% CI: 1.50-2.04) and 3.36 liters (2.39-4.33), respectively; however, the change in clearance was in the opposite direction of what would be expected. It is noteworthy that paraquat brain-to-plasma partitioning ratios and total brain accumulation were the same across doses between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice. These studies indicate that paraquat is not a P-gp substrate. Therefore, the association between ABCB1 pharmacogenomics and

  4. Absence of P-Glycoprotein Transport in the Pharmacokinetics and Toxicity of the Herbicide Paraquat

    PubMed Central

    Lacher, Sarah E.; Gremaud, Julia N.; Skagen, Kasse; Steed, Emily; Dalton, Rachel; Sugden, Kent D.; Cardozo-Pelaez, Fernando; Sherwin, Catherine M. T.

    2014-01-01

    Genetic variation in the multidrug resistance gene ABCB1, which encodes the efflux transporter P-glycoprotein (P-gp), has been associated with Parkinson disease. Our goal was to investigate P-gp transport of paraquat, a Parkinson-associated neurotoxicant. We used in vitro transport models of ATPase activity, xenobiotic-induced cytotoxicity, transepithelial permeability, and rhodamine-123 inhibition. We also measured paraquat pharmacokinetics and brain distribution in Friend leukemia virus B-type (FVB) wild-type and P-gp-deficient (mdr1a−/−/mdr1b−/−) mice following 10, 25, 50, and 100 mg/kg oral doses. In vitro data showed that: 1) paraquat failed to stimulate ATPase activity; 2) resistance to paraquat-induced cytotoxicity was unchanged in P-gp-expressing cells in the absence or presence of P-gp inhibitors GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] and verapamil—37.0 [95% confidence interval (CI): 33.2–41.4], 46.2 (42.5–50.2), and 34.1 µM (31.2–37.2)—respectively; 3) transepithelial permeability ratios of paraquat were the same in P-gp-expressing and nonexpressing cells (1.55 ± 0.39 and 1.39 ± 0.43, respectively); and 4) paraquat did not inhibit rhodamine-123 transport. Population pharmacokinetic modeling revealed minor differences between FVB wild-type and mdr1a−/−/mdr1b−/− mice: clearances of 0.47 [95% confidence interval (CI): 0.42–0.52] and 0.78 l/h (0.58–0.98), respectively, and volume of distributions of 1.77 (95% CI: 1.50–2.04) and 3.36 liters (2.39–4.33), respectively; however, the change in clearance was in the opposite direction of what would be expected. It is noteworthy that paraquat brain-to-plasma partitioning ratios and total brain accumulation were the same across doses between FVB wild-type and mdr1a−/−/mdr1b−/− mice. These studies indicate that paraquat is not a P-gp substrate. Therefore, the association between

  5. Interaction potential of Carmegliptin with P-glycoprotein (Pgp) transporter in healthy volunteers

    PubMed Central

    Kuhlmann, Olaf; Carlile, David; Noe, Johannes; Bentley, Darren

    2014-01-01

    Objective The primary objective of this study was to investigate the interaction potential of carmegliptin with P-glycoprotein transporter in vitro and in vivo. A secondary objective was to investigate the safety and tolerability of carmegliptin alone or co-administered with verapamil. Research design and methods The inhibition potential of carmegliptin was tested in vitro and in a non-randomized open-label study in 16 healthy male volunteers. On day 1 a single dose of carmegliptin (150 mg) was given, followed by a single dose of verapamil (80 mg) on day 7, on day 10 a single dose of carmegliptin (150 mg) together with verapamil (80 mg t.i.d.), and verapamil (80 mg t.i.d.) on days 11–14. Finally, on day 15 a single dose of 150 mg carmegliptin together with 80 mg t.i.d. verapamil was administered. Pharmacokinetic and safety parameters were assessed. Results Carmegliptin showed in vitro a low cell permeability and was a good substrate for human MDR1 cells. When carmegliptin was taken with verapamil, the mean exposure and Cmax to carmegliptin increased by 29% and 53%, respectively. Increases in exposure were slightly greater on the sixth day of verapamil dosing than on the first day. Verapamil Cmax was 17% lower on average when given with carmegliptin than when verapamil was taken alone, and similar trends were apparent in corresponding norverapamil pharmacokinetics. All reported adverse events (n = 28) were mild in intensity, and verapamil had no apparent effect on the pattern or incidence of events. Conclusions In vitro, carmegliptin is a substrate but not an inhibitor of human Pgp. Consistently, the co-administration of carmegliptin with verapamil altered the pharmacokinetics of carmegliptin slightly and moderately increased the exposure. Peak exposure of verapamil and its metabolite norverapamil tended to be lower when co-administered with carmegliptin. The combination of carmegliptin and verapamil was generally well tolerated. Although the

  6. Progesterone interacts with P-glycoprotein in multidrug-resistant cells and in the endometrium of gravid uterus.

    PubMed

    Yang, C P; DePinho, S G; Greenberger, L M; Arceci, R J; Horwitz, S B

    1989-01-15

    P-Glycoprotein (P-GP) plays a pivotal role in maintaining the multidrug-resistant (MDR) phenotype. This membrane glycoprotein is overproduced in MDR cells and the endometrium of the mouse gravid uterus (Arceci, R.J., Croop, J.M., Horwitz, S.B., and Housman, D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4350-4354). This latter observation and an interest in endogenous substrates for P-GP led to a study of the interaction of steroids with P-GP found in the endometrium of the mouse gravid uterus and in MDR cells derived from the murine macrophage-like cell J774.2. [3H]Azidopine labeling of P-GP from these two sources was inhibited by various steroids, particularly progesterone. Progesterone also markedly inhibited [3H]vinblastine binding to membrane vesicles prepared from MDR cells, enhanced vinblastine accumulation in MDR cells, and increased the sensitivity of MDR cells to vinblastine. In addition, we have demonstrated that the hydrophobicity of a steroid is important in determining its effect on inhibition of drug binding to P-GP. It is concluded that progesterone, a relatively nontoxic endogenous steroid, interacts with P-GP and is capable of reversing drug resistance in MDR cells. PMID:2562956

  7. Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein.

    PubMed Central

    Schuurhuis, G. J.; Broxterman, H. J.; de Lange, J. H.; Pinedo, H. M.; van Heijningen, T. H.; Kuiper, C. M.; Scheffer, G. L.; Scheper, R. J.; van Kalken, C. K.; Baak, J. P.

    1991-01-01

    Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance. Images Figure 1 PMID:1681887

  8. Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL.

    PubMed

    Souza, Paloma S; Madigan, James P; Gillet, Jean-Pierre; Kapoor, Khyati; Ambudkar, Suresh V; Maia, Raquel C; Gottesman, Michael M; Fung, King Leung

    2015-08-15

    Multidrug resistance (MDR) has been associated with expression of ABC transporter genes including P-glycoprotein (Pgp, MDR1, ABCB1). However, deregulation of apoptotic pathways also renders cells resistant to chemotherapy. To discover apoptosis-related genes affected by Pgp expression, we used the HeLa MDR-off system. We found that using doxycycline to control Pgp expression has a significant advantage over tetracycline, in that doxycycline caused less endogenous gene expression modification/perturbation, and was more potent than tetracycline in suppressing Pgp expression. Cells overexpressing Pgp have lower TNFSF10 (TRAIL) expression than their parental cells. Controlled downregulation of Pgp increased endogenous TRAIL protein expression. Also, ectopic overexpression of TRAIL in Pgp-positive cells was associated with a reduction in Pgp levels. However, cells expressing a functionally defective mutant Pgp showed an increase in TRAIL expression, suggesting that Pgp function is required for TRAIL suppression. Cells in which Pgp is knocked down by upregulation of TRAIL expression are less susceptible to TRAIL ligand (sTRAIL)-induced apoptosis. Our findings reveal an inverse correlation between functional Pgp and endogenous TRAIL expression. Pgp function plays an important role in the TRAIL-mediated apoptosis pathway by regulating endogenous TRAIL expression and the TRAIL-mediated apoptosis pathway in MDR cancer cells. PMID:26101157

  9. Influence of combinations of digitonin with selected phenolics, terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells.

    PubMed

    Eid, Safaa Yehia; El-Readi, Mahmoud Zaki; Eldin, Essam Eldin Mohamed Nour; Fatani, Sameer Hassan; Wink, Michael

    2013-12-15

    P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of β-carotene (40μM) and sanguinarine (1μM) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5μM), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p<0.01) and 46% (p<0.01) in Caco-2, and to 61% (p<0.05) and 1% (p<0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells. PMID:23999162

  10. Cyclosporine inhibits macrophage-mediated antigen presentation

    SciTech Connect

    Ziegler, H.K.; Palay, D.; Wentworth, P.; Cluff, C.

    1986-03-01

    The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin-2. Pretreatment (2 hrs., 37/sup 0/C) of macrophages with cyclosporine resulted in a population of macrophages with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of antigen and untreated T cells, the dose of cyclosporine which produced 50% inhibition was 1.5 ..mu..g/ml. Appropriate control experiments indicated that cyclosporine was indeed inhibiting at the macrophage level. The addition of interleukin-1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions which produced >90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabelled antigen was normal. Thus, cyclosporine inhibits antigen presentation by a mechanism which appears unrelated to changes in Il-1 elaboration, prostaglandin production, Ia expression, or antigen uptake and catabolism.

  11. In silico structure-based screening of versatile P-glycoprotein inhibitors using polynomial empirical scoring functions.

    PubMed

    Shityakov, Sergey; Förster, Carola

    2014-01-01

    P-glycoprotein (P-gp) is an ATP (adenosine triphosphate)-binding cassette transporter that causes multidrug resistance of various chemotherapeutic substances by active efflux from mammalian cells. P-gp plays a pivotal role in limiting drug absorption and distribution in different organs, including the intestines and brain. Thus, the prediction of P-gp-drug interactions is of vital importance in assessing drug pharmacokinetic and pharmacodynamic properties. To find the strongest P-gp blockers, we performed an in silico structure-based screening of P-gp inhibitor library (1,300 molecules) by the gradient optimization method, using polynomial empirical scoring (POLSCORE) functions. We report a strong correlation (r (2)=0.80, F=16.27, n=6, P<0.0157) of inhibition constants (Kiexp or pKiexp; experimental Ki or negative decimal logarithm of Kiexp) converted from experimental IC50 (half maximal inhibitory concentration) values with POLSCORE-predicted constants (KiPOLSCORE or pKiPOLSCORE), using a linear regression fitting technique. The hydrophobic interactions between P-gp and selected drug substances were detected as the main forces responsible for the inhibition effect. The results showed that this scoring technique might be useful in the virtual screening and filtering of databases of drug-like compounds at the early stage of drug development processes. PMID:24711707

  12. In silico structure-based screening of versatile P-glycoprotein inhibitors using polynomial empirical scoring functions

    PubMed Central

    Shityakov, Sergey; Förster, Carola

    2014-01-01

    P-glycoprotein (P-gp) is an ATP (adenosine triphosphate)-binding cassette transporter that causes multidrug resistance of various chemotherapeutic substances by active efflux from mammalian cells. P-gp plays a pivotal role in limiting drug absorption and distribution in different organs, including the intestines and brain. Thus, the prediction of P-gp–drug interactions is of vital importance in assessing drug pharmacokinetic and pharmacodynamic properties. To find the strongest P-gp blockers, we performed an in silico structure-based screening of P-gp inhibitor library (1,300 molecules) by the gradient optimization method, using polynomial empirical scoring (POLSCORE) functions. We report a strong correlation (r2=0.80, F=16.27, n=6, P<0.0157) of inhibition constants (Kiexp or pKiexp; experimental Ki or negative decimal logarithm of Kiexp) converted from experimental IC50 (half maximal inhibitory concentration) values with POLSCORE-predicted constants (KiPOLSCORE or pKiPOLSCORE), using a linear regression fitting technique. The hydrophobic interactions between P-gp and selected drug substances were detected as the main forces responsible for the inhibition effect. The results showed that this scoring technique might be useful in the virtual screening and filtering of databases of drug-like compounds at the early stage of drug development processes. PMID:24711707

  13. Doxycycline-mediated Inhibition of Choroidal Neovascularization

    PubMed Central

    Samtani, S.; Amaral, J.; Campos, M.; Fariss, R. N.; Becerra, S. P.

    2010-01-01

    Purpose Doxycycline, a broad spectrum antibiotic, has certain anti-angiogenic properties and can inhibit matrix metalloproteinases (MMPs/gelatinases). We investigated the effects of doxycycline on choroidal neovascularization (CNV), and regulation of MMP-2/-9 and pigment epithelium-derived factor (PEDF). Methods Doxycycline was orally administered to rats at 500, 50, 5, and 0.5 mg/kg/day, using non-treated animals as controls. Experimental CNV was induced with laser 7 days after doxycycline treatment started. At seven days post-induction, animals were euthanized, and eyes collected. RPE/choroid flat-mounts were labeled with isolectin IB4 to determine CNV lesion volumes using confocal microscopy and Volocity® software. MMP-2, MMP-9 and PEDF protein levels were determined by ELISA. MMP catalytic activity was determined in solution using fluorogenic gelatin and peptide substrates, by gelatin zymography in SDS-PAGE and by in situ DQ-gelatin zymography in RPE/choroid sections. Results CNV complex lesion volumes decreased with doxycycline in a dose-response relationship. A dosage of 500 mg/kg/day caused a 70% inhibition of CNV complex volume compared to control animals. Doxycycline elevated PEDF levels in plasma, and did not affect the plasma pro- and active MMP-2 and MMP-9 levels. However, the in vitro enzymatic activities of purified MMP-2 and MMP-9 declined significantly with doxycycline. MMP-2, MMP-9 and gelatinolytic activities in situ increased early in CNV lesion development. Doxycycline treatments and exogenous additions inhibited gelatinolytic activities in CNV lesions. Conclusions Doxycycline effectively hampered the progression of experimental CNV. The results suggest that orally administrated doxycycline can reach the choroid to attenuate proteolytic enzymes that remodel Bruch's membrane and promote the anti-angiogenic PEDF to inhibit neovascularization. PMID:19516001

  14. Evaluation of immunohistochemical expression of P-glycoprotein in neoplasms of the mammary gland in bitches.

    PubMed

    Badowska-Kozakiewicz, A M; Malicka, E

    2010-01-01

    The aim of the study was to investigate the P-glycoprotein expression in correlation with other neoplasm traits such as: histological type, the differentiation grade, proliferative activity, expression of the cyclooxygenase-2. Material for the investigation comprised 50 tumours of the mammary gland collected from bitches during surgical procedures performed in Warsaw Veterinary Clinics and Small Animal Clinic of the Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - SGGW. All together 8 adenomas, 22 complex carcinomas, 15 simple carcinomas and 5 solid carcinomas. In case of cancers, the degree of histological malignancy was established: 1st degree of histological malignancy--18 neoplasms, 2nd degree of histological malignancy--14 neoplasms and 3rd degree of histological malignancy--10 neoplasms. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Proliferative activity was highly dependent on type of the neoplasm and the degree of histological malignancy. The highest value of the mitotic index was characteristic for solid and simple cancers and neoplasms with the highest degree of histological malignancy. Results of expression of the nuclear antigen Ki-67 were similar. Expression of P-glycoprotein was revealed in all types of neoplasms. The expression of P-glycoprotein was identified in cytoplasm and cell membranes of neoplastic cells. Positive expression of P-gp was observed in 76% of cancers. Complex carcinomas were the biggest group among the cancer types which demonstrated positive reaction of P-gp. High expression of P-gp was also established in cancers with the highest degree of malignancy. In bitches aged 9 through 12 years, the cancers featuring a positive reaction of P-gp constituted the most numerous group (63.2%); on the other hand, this cancer type barely appeared in the oldest bitches (10.5%). PMID:20731191

  15. A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2016-01-01

    The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology. PMID:27043281

  16. Clofazimine and B4121 sensitize an intrinsically resistant human colon cancer cell line to P-glycoprotein substrates.

    PubMed

    van Rensburg, C E; Joone, G K; O'Sullivan, J F

    2000-01-01

    The potential of B4121 to sensitize three intrinsically resistant human colon cancer cell lines (CaCo2, ATCC HTB 37; COLO 32 DM, ATCC CCL 220; HT-29, ATCC HTB 38) to vinblastine, doxorubicin, daunorubicin, paclitaxel, taxotere and cisplatin at a non-toxic, therapeutically relevant concentration of 0.25 microg/ml was compared with that of clofazimine at a similar concentration. The cell line expressing high levels of P-glycoprotein (P-gp), COLO 320 DM, was susceptible to chemosensitization by the experimental agents for the P-gp substrates (paclitaxel, taxotere, daunorubicin, vinblastine and doxorubicin) but not for cisplatin. CaCo2 cells expressed lower levels of P-gp and were only marginally susceptible to sensitization by any one of these drugs, except in the case of sensitization by B4121 for doxorubicin and taxotere, whereas the HT-29, a P-gp negative cell line, was unaffected. The riminophenazines, especially B4121, might prove useful as combination treatment in circumventing P-gp mediated resistance of colon cancers. PMID:10601617

  17. Interactions between the flavonoid biochanin A and P-glycoprotein substrates in rats: in vitro and in vivo.

    PubMed

    Zhang, Shuzhong; Sagawa, Kazuko; Arnold, Robert D; Tseng, Elaine; Wang, Xiaodong; Morris, Marilyn E

    2010-01-01

    The purpose of this study was to investigate the in vitro and in vivo interactions between flavonoids and P-glycoprotein (P-gp) substrates. The inhibitory effects of flavonoids on P-gp were determined by accumulation studies in P-gp-overexpressing MCF-7/ADR cells using daunomycin (DNM) as a model substrate. Morin, phloretin, biochanin A, chalcone, and silymarin significantly increased DNM accumulation by greater than 2.5-fold, suggesting they are P-gp inhibitors. To explore potential in vivo interactions of flavonoids with P-gp, the effect of biochanin A on the pharmacokinetics of the P-gp substrates doxorubicin, cyclosporine A, and paclitaxel was investigated. In contrast to the in vitro results, intraperitoneal or oral administration of biochanin A did not significantly change the pharmacokinetics of doxorubicin and cyclosporine A. Moderate interaction was observed between biochanin A and paclitaxel, resulting in lower AUC values after both i.v. and oral administration of paclitaxel. The disconnect between the in vitro and in vivo data suggests that P-gp interactions mediated by biochanin A may be limited due to its poor bioavailability and rapid clearance. It is also possible that other transporters or metabolizing enzymes are more important in the in vivo disposition of doxorubicin, cyclosporine A, and paclitaxel than P-gp. PMID:19499569

  18. Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle

    PubMed Central

    Frank, Gabriel A.; Shukla, Suneet; Rao, Prashant; Borgnia, Mario J.; Bartesaghi, Alberto; Merk, Alan; Mobin, Aerfa; Esser, Lothar; Earl, Lesley A.; Gottesman, Michael M.; Xia, Di

    2016-01-01

    The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP. PMID:27190212

  19. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    PubMed Central

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake of verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-to-apical transepithelial efflux across the MDCK II MDR1 monolayers with a permeability of 5.7 × 10−5 cm sec−1 compared to an apical to basolateral permeability of 1.3 × 10−5 cm sec-1. The efflux could be inhibited with the P-gp inhibitor zosuquidar. Zosuquidar (0.4 μmol/L) reduced the efflux ratio (PB-A/PA-B) for verapamil 4.6–1.6. The presence of telmisartan, however, only caused a slight reduction in P-gp-mediated verapamil transport to an efflux ratio of 3.4. Overall, the results of the present in vitro approach indicate, that clinical use of telmisartan as a P-gp inhibitor may not be an effective strategy for increasing brain uptake of verapamil by co-administration with telmisartan. PMID:26171231

  20. Do drugs have access to the P-glycoprotein drug-binding pocket through gates?

    PubMed

    Ferreira, Ricardo J; Ferreira, Maria-José U; Dos Santos, Daniel J V A

    2015-10-13

    The P-glycoprotein efflux mechanism is being studied since its identification as a leading protagonist in multidrug resistance. Recently, it was suggested that drugs enter the drug-binding pocket (DBP) through gates located between the transmembrane domains. For both a substrate and a modulator, the potential of mean force curves along the reaction coordinate obtained with the WHAM approach were similar, with no activation energy required for crossing the gate. Moreover, drug transit from bulk water into the DBP was characterized as an overall free-energy downhill process. PMID:26574244

  1. Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation.

    PubMed

    Omote, Hiroshi; Figler, Robert A; Polar, Mark K; Al-Shawi, Marwan K

    2004-04-01

    A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K(m) for colchicine and a lower K(m) for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this

  2. The effect of quinidine, a strong P-glycoprotein inhibitor, on the pharmacokinetics and central nervous system distribution of naloxegol.

    PubMed

    Bui, Khanh; She, Fahua; Zhou, Diansong; Butler, Kathleen; Al-Huniti, Nidal; Sostek, Mark

    2016-04-01

    Naloxegol is a PEGylated, oral, peripherally acting μ-opioid receptor antagonist approved in the United States for treatment of opioid-induced constipation in patients with noncancer pain. Naloxegol is metabolized by CYP3A, and its properties as a substrate for the P-glycoprotein (PGP) transporter limit its central nervous system (CNS) permeability. This double-blind, randomized, 2-part, crossover study in healthy volunteers evaluated the effect of quinidine (600 mg PO), a CYP3A/PGP transporter inhibitor, on the pharmacokinetics and CNS distribution of naloxegol (25 mg PO). In addition, the effects of quinidine on morphine (5 mg/70 kg IV)-induced miosis and exposure to naloxegol were assessed. Coadministration of quinidine and naloxegol increased naloxegol's AUC 1.4-fold and Cmax 2.5-fold but did not antagonize morphine-induced miosis, suggesting that PGP inhibition does not increase the CNS penetration of naloxegol. Naloxegol pharmacokinetics was unaltered by coadministration of morphine and either quinidine or placebo; conversely, pharmacokinetics of morphine and its metabolites (in the presence of quinidine) were unaltered by coadministration of naloxegol. Naloxegol was safe and well tolerated, alone or in combination with quinidine, morphine, or both. The observed increase in exposure to naloxegol in the presence of quinidine is primarily attributed to quinidine's properties as a weak CYP3A inhibitor. PMID:26248047

  3. Synthesis, biological evaluation and 3D-QSAR studies of new chalcone derivatives as inhibitors of human P-glycoprotein.

    PubMed

    Parveen, Zahida; Brunhofer, Gerda; Jabeen, Ishrat; Erker, Thomas; Chiba, Peter; Ecker, Gerhard F

    2014-04-01

    P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an important role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed the highest activity (IC50=42nM), which is one order of magnitude higher than the activity for an equilipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors, methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric features influencing P-gp inhibitory activity. PMID:24613626

  4. Human hepatoma cells rich in P-glycoprotein are sensitive to aclarubicin and resistant to three other anthracyclines.

    PubMed Central

    Lehne, G.; De Angelis, P.; Clausen, O. P.; Rugstad, H. E.

    1996-01-01

    Drug resistance is a major obstacle to successful chemotherapy of primary liver cancer, which is associated with high expression of the multidrug resistance (MDR) gene product P-glycoprotein (Pgp), a multidrug efflux transporter. The most effective single agents in treatment of primary liver carcinoma belong to the anthracycline family, yet several anthracyclines are known to be substrates for Pgp. In the present study, we compared four anthracyclines with respect to cell growth inhibition, intracellular accumulation and cellular efflux using the HB8065/R human hepatoma cell line which is rich in Pgp, and the Pgp-poor parental line HB8065/S. The anthracyclines were also administered in conjunction with the Pgp-modifying agents verapamil and SDZ PSC 833 to assess modulation of resistance. The HB8065/R cells were sensitive to aclarubicin (ACL) and highly resistant to epirubicin (EPI), doxorubicin (DOX) and daunorubicin (DNR). SDZ PSC 833 enhanced accumulation, decreased efflux and increased cytotoxicity of EPI, DOX and DNR in the HB8065/R cells, but none of these effects was seen with ACL. In conclusion, ACL is apparently not transported by Pgp and retains its activity in a multidrug-resistant human hepatoma cell line; such properties can be exploited for clinical purposes. Images Figure 5 PMID:8956784

  5. Reversal of P-glycoprotein-dependent resistance to vinblastine by newly synthesized bisbenzylisoquinoline alkaloids in mouse leukemia P388 cells.

    PubMed

    Wang, Feng-Peng; Wang, Li; Yang, Jin-Song; Nomura, Masaaki; Miyamoto, Ken-Ichi

    2005-10-01

    We examined the ability of partially synthesized new compounds from fangchinoline and tetrandrine to reverse P-glycoprotein (P-gp)-dependent multidrug resistance (MDR) in vitro and in vivo. All compound enhanced the in vitro cyctotoxic effect of vinblastin (VBL) at 0.1 microM as potent as 10 microM verapamil against the resistant cell line P388/ADR. The combination effect tended to be strong by substitution of bulky group, resulting 5,14-dibromotetrandrine (compound #9) showed the strongest effect. Compound #9 increased intracellular VBL accumulation in P388/ADR cells, much stronger than verapamil, as well as cytotoxic combined effect. This mechanism seems to inhibit the function of P-gp, but not the expression of P-gp. In combination with VBL, this compound also synergistically prolonged the life-span of P388/ADR-bearing mice. Bisbenzylisoquinoline alkaloids and their derivatives are possible to be good candidates as modifier of MDR in cancer chemotherapy. PMID:16204959

  6. The P-glycoprotein inhibitor cyclosporin A differentially influences behavioural and neurochemical responses to the antidepressant escitalopram.

    PubMed

    O'Brien, Fionn E; O'Connor, Richard M; Clarke, Gerard; Donovan, Maria D; Dinan, Timothy G; Griffin, Brendan T; Cryan, John F

    2014-03-15

    Recent studies have raised the possibility that P-glycoprotein (P-gp) inhibition may represent a putative augmentation strategy for treatment with certain antidepressants. Indeed, we have previously shown that administration of the P-gp inhibitor verapamil increased the brain distribution and behavioural effects of the antidepressant escitalopram. The aim of the current study was to investigate if similar effects occur with another P-gp inhibitor, cyclosporin A (CsA). CsA pre-treatment exacerbated the severity of behaviours in an escitalopram-induced mouse model of serotonin syndrome, a potentially life-threatening adverse drug reaction associated with serotonergic drugs. P-gp inhibition by CsA enhanced the brain distribution of escitalopram by 70-80%. Serotonin (5-HT) turnover in the prefrontal cortex was reduced by escitalopram, and this effect was augmented by CsA. However, CsA pre-treatment did not augment the effect of escitalopram in the tail suspension test (TST) of antidepressant-like activity. Microdialysis experiments revealed that pre-treatment with CsA failed to augment, but blunted, the increase in extracellular 5-HT in response to escitalopram administration. This blunting effect may contribute to the lack of augmentation in the TST. Taken together, the present studies demonstrate that co-administration of CsA and escitalopram produces differential effects depending on the behavioural and neurochemical assays employed. Thus, the results highlight the need for further studies involving more selective pharmacological tools to specifically evaluate the impact of P-gp inhibition on behavioural responses to antidepressants which are subject to efflux by P-gp. PMID:24280122

  7. Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

    PubMed Central

    Ledwitch, Kaitlyn V.; Barnes, Robert W.; Roberts, Arthur G.

    2016-01-01

    Drug–drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug–Pgp interactions, verapamil was found to have little effect on digoxin–Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin–Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil–digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes. PMID:26823559

  8. An electrically mediated inhibition in goldfish medulla.

    PubMed

    Korn, H; Faber, D S

    1975-03-01

    1. Passive hyperpolarizing potentials (PHPs) have been recorded intracellularly from goldfish medullary neurons in the vicinity of the Mauthner cell (M-cell). They are evoked when this cell is activated antidromically by stimulation of the spinal cord, and orthodromically via the ipsilateral eighth nerve; when appropriately timed they block or delay spikes induced both directly and transsynaptically. 2. Since the PHPs and the M-cell spike have the same latency, time course, and all-or-none character, they cannot be generated by chemically mediated synaptic transmission. This conclusion is further supported by the evidence that PHP amplitude and time course are independent of membrane potential. 3. The analysis of the mechanism underlying PHP generation has been based on the hypothesis that they are brought about by the extracellular currents flowing to the axon cap during an M-cell action potential. Specifically, it was postulated that some of this current is channeled back to the axon cap region intracellularly through processes of PHP-exhibiting neurons, and that these cells are passively hyperpolarized by the associated inward transmembrane current flow. This model would require that PHP-exhibiting neurons send processes into the axon cap. This hypothesis is confirmed by the following: a) When the PHP is timed to occur during the conductance increase associated with a spike after hyperpolarization, it is reduced, as would be expected for a passive current flow across a membrane resistance. b) PHPs are not found in all medullary neurons in the vicinity of the M-cell, but rather in a specific neuronal population. c) PHP-exhibiting neurons have been identified following Procion yellow injections; as predicted, they issue one process, presumably the axon, which projects toward the M-cell axon cap area. d) The PHP can be stimulated by passing a cathodal current from a microelectrode located in the axon cap; it is not mimicked when the cathodal electrode is moved

  9. Tumor endothelial expression of P-glycoprotein upon microvesicular transfer of TrpC5 derived from adriamycin-resistant breast cancer cells

    SciTech Connect

    Dong, YePing; Pan, QiongXi; Jiang, Li; Chen, Zhen; Zhang, FangFang; Liu, YanJun; Xing, Hui; Shi, Mei; Li, Jiao; Li, XiYuan; Zhu, YaoDan; Chen, Yun; Bruce, Iain C.; Jin, Jian Ma, Xin

    2014-03-28

    Highlights: • TrpC5 was mainly accumulated in microvesicles of drug-resistant MCF-7/ADM cells. • Microvesicles from MCF-7/ADM transferred TrpC5 to endothelial cells. • TrpC5 inhibition reduced P-glycoprotein accumulation on tumor blood vessels in vivo. - Abstract: Treatment of carcinoma commonly fails due to chemoresistance. Studies have shown that endothelial cells acquire resistance via the tumor microenvironment. Microvesicle (MV) shedding from the cell membrane to the microenvironment plays an important role in communication between cells. The aim of the present study was to determine whether MCF-7 adriamycin-resistant cells (MCF-7/ADM) shed MVs that alter the characteristics of human microvessel endothelial cells (HMECs). MVs from tumor cells transferred a Ca{sup 2+}-permeable channel TrpC5 to HMECs, inducing the expression of P-glycoprotein (P-gp) by activation of the transcription factor NFATc3 (nuclear factor of activated T cells isoform c3). Expression of the mdr1 gene was blocked by the TrpC5-blocking antibody T5E3, and the production of P-gp in HMECs was reduced by blockade of TrpC5. Thus, we postulate that endothelial cells acquire the resistant protein upon exposure to TrpC5-containg MVs in the microenvironment, and express P-gp in the TrpC5–NFATc3 signal pathway.

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

    NASA Technical Reports Server (NTRS)

    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.

  11. Impact of the changes in P-glycoprotein activity on domperidone pharmacokinetics in rat plasma.

    PubMed

    Bamburowicz-Klimkowska, Magdalena; Zywiec, Katarzyna; Potentas, Agata; Szutowski, Mirosław

    2007-01-01

    The effect of quinidine (QD) and grapefruit juice (GFJ) extract, P-glycoprotein inhibitors, on the domperidone (DOM) concentration in rat plasma was investigated. DOM, a dopamine D(2)-receptor antagonist is a substrate for P-glycoprotein. DOM(10 mg/kg) was administered orally 2 h after GFJ extract (0.2 ml/kg) or QD (25 mg/kg). DOM concentration in plasma samples was determined by HPLC with fluorescence detection. The GFJ extract and QD administration significantly increased c(max) of DOM by 19% and 36%, respectively, and the AUC(0-0.25) (area under the concentration-time curve from time zero to 15 min) by 29% and 44%, respectively. In addition, QD significantly increased the DOM AUC(0-2) (32%), whereas 19% increase was observed after GFJ extract administration. In conclusion, GFJ and QD significantly influenced DOM rat plasma concentration during the first two hours after DOM administration indicating that interaction takes place during absorption phase. PMID:18195466

  12. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

    PubMed

    Pasquier, Jennifer; Rioult, Damien; Abu-Kaoud, Nadine; Hoarau-Véchot, Jessica; Marin, Matthieu; Le Foll, Frank

    2015-01-01

    The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation. PMID:26114386

  13. The influence of a caveolin-1 mutant on the function of P-glycoprotein

    PubMed Central

    Lee, Chih-Yuan; Lai, Ting-Yu; Tsai, Meng-Kun; Ou-Yang, Pu; Tsai, Ching-Yi; Wu, Shu-Wei; Hsu, Li-Chung; Chen, Jin-Shing

    2016-01-01

    The genetic heterogeneity in cancer cells has an increased chance in the acquisition of new mutant such as drug-resistant phenotype in cancer cells. The phenotype of drug resistance in cancer cells could be evaluated by the number or function of drug transporters on cell membranes, which would lead to decreased intracellular anti-cancer drugs concentration. Caveolae are flask-shaped invaginations on cell membrane that function in membrane trafficking, endocytosis, and as a compartment where receptors and signaling proteins are concentrated. Caveolin-1 (CAV1) is the principal structural protein of caveolae and closely correlates with multidrug resistance in cancer cells. In a systematic study of the ubiquitin-modified proteome, lysine 176 of CAV1 was identified as a potential post-translational modification site for ubiquitination. In this article, we identified a mutation at lysine 176 to arginine (K176R) on CAV1 would interfere with the biogenesis of caveolae and broke the interaction of CAV1 with P-glycoprotein. Functional assays further revealed that K176R mutant of CAV1 in cancer cells increased the transport activity of P-glycoprotein and decreased the killing ability of anti-cancer drugs in non-small-cell lung cancer cell lines. PMID:26843476

  14. Drug-selected coexpression of human glucocerebrosidase and P-glycoprotein using a bicistronic vector.

    PubMed Central

    Aran, J M; Gottesman, M M; Pastan, I

    1994-01-01

    Bicistronic cassettes under control of a single promoter have recently been suggested as useful tools for coordinate expression of two different foreign proteins in mammalian cells. Using the long 5' untranslated region of encephalomyocarditis virus as translational enhancer of the second gene, a bicistronic unit composed of cDNA for human P-glycoprotein [the product of the multidrug resistance gene, MDR1 (also called PGY1)] as selectable marker and cDNA for human glucocerebrosidase (GC; EC 3.2.1.45) (a membrane-associated lysosomal hydrolase) was constructed. NIH 3T3 cells transfected with a Harvey murine sarcoma virus retroviral vector carrying this bicistronic cassette (pHaMCG) express active P-glycoprotein and GC and expression of both proteins augments coordinately with selection for increased colchicine resistance. Percoll gradient analysis of homogenates showed that GC was targeted to the lysosomal fraction. The ability to select for expression of GC with natural product drugs after introduction of the pHaMCG retroviral vector may be useful in gene therapy strategies for Gaucher disease. Images PMID:7909160

  15. Inhibitory effects of pomelo on the metabolism of tacrolimus and the activities of CYP3A4 and P-glycoprotein.

    PubMed

    Egashira, Kanoko; Ohtani, Hisakazu; Itoh, Suwako; Koyabu, Noriko; Tsujimoto, Masayuki; Murakami, Hideyasu; Sawada, Yasufumi

    2004-08-01

    We recently reported a case of increase in the blood level of tacrolimus following intake of pomelo in a renal transplant recipient. To clarify the mechanism of this increase in the blood level of tacrolimus, we investigated the effect of pomelo juice extract on the activities of CYP3A4 and P-glycoprotein, in comparison with that of extract of grapefruit juice (GFJ). The 10% ethyl acetate extracts of the juice of three pomelos of different origins (Banpeiyu, pomelo I; Hirado Buntan, pomelo II; and Tosa Buntan, pomelo III) and GFJ significantly inhibited 6beta-hydroxylation of testosterone in human liver microsomes by 76.4, 67.2, 37.5, and 83.9%, respectively. The extract of pomelo I was as potent as that of GFJ. The metabolism of tacrolimus itself was also inhibited by the extract of pomelo I, as well as that of GFJ. Furthermore, the inhibition of both 6beta-hydroxylation of testosterone and metabolism of tacrolimus by pomelo I and GFJ was preincubation time-dependent. On the other hand, the extract of pomelo I had little effect on the transcellular transport of tacrolimus or [(3)H]digoxin across a monolayer of LLC-GA5-COL150 cells (a porcine kidney epithelial cell line, LLC-PK1, transfected with human MDR1 cDNA and overexpressing human P-glycoprotein). In conclusion, pomelo constituents inhibit the activity of CYP3A4 and may thereby produce an increase in the blood level of tacrolimus. PMID:15258108

  16. Is P-glycoprotein (ABCB1) a phase 0 or a phase 3 colchicine transporter depending on colchicine exposure conditions?

    SciTech Connect

    Decleves, Xavier. E-mail: xavier.decleves@univ-paris5.fr; Niel, Elisabeth; Debray, Marcel; Scherrmann, Jean-Michel

    2006-12-01

    This study investigates the P-glycoprotein (Pgp)-mediated transport of its substrates in accumulation or efflux modes under steady-state conditions. The kinetics of colchicine uptake and efflux, a substrate of both Pgp and intracellular tubulin, were studied in HL60 and HL60/DNR cells; HL60/DNR cells contain 25 times more Pgp than do HL60 cells. HL60/DNR cells in a medium containing 6.25 nM colchicine, which mimics therapeutic conditions, reached steady-state twice as rapidly as did HL60 cells, and accumulated 24-times less colchicine than did HL60 cells. The Pgp inhibitor GF120918, increased colchicine uptake by HL60 cells 1.2-fold and that of HL60/DNR cells 17-fold, while it had no effect on colchicine efflux from either cell line that had been incubated with colchicine for 24 h. Colchicine kinetics fitted well a two closed-compartment model, showing that the low intracellular accumulation of colchicine in HL60/DNR cells resulted from a 11-fold decrease in colchicine uptake and a 2.3-fold increase in colchicine efflux, that could be attributed to Pgp-mediated efflux activity in HL60/DNR cells. Intracellular colchicine was mainly and similarly distributed in the cytosol in both cell lines. These data demonstrate that the kinetics of the intracellular colchicine accumulation depend on the density of Pgp and that Pgp is more a phase 0 (preventing cellular uptake) than a phase 3 (effluxing intracellular substrate) transporter under steady-state conditions, although the situation is reversed after a short incubation time (30 min), when intracellular free colchicine concentration is probably high enough for it to be removed from the cell by Pgp.

  17. Differential roles of P-glycoprotein, multidrug resistance-associated protein 2, and CYP3A on saquinavir oral absorption in Sprague-Dawley rats.

    PubMed

    Usansky, Helen H; Hu, Peidi; Sinko, Patrick J

    2008-05-01

    The objective of this investigation was to differentiate the roles of P-glycoprotein (Pgp), multidrug resistance-associated protein 2 (Mrp2), and CYP3A on saquinavir (SQV) oral absorption. With use of single-pass jejunal perfusion (in situ) and portal vein-cannulated rats (in vivo), SQV absorption was studied under chemical inhibition of Pgp [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2 isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918)], Mrp2 [(3-(((3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl) ((3-(dimethylamino-3-oxopropyl)thio)methyl)-thio) propanoic acid (MK571)], and/or CYP3A (midazolam). Plasma concentrations of SQV and related metabolites were analyzed by liquid chromatography-tandem mass spectrometry. When given alone, SQV absorption was extremely low both in situ (F(a) = 0.07%) and in vivo [C(max) = 0.068 microg/ml; area under the curve (AUC) = 6.8 microg x min/ml]. Coadministration of GF120918 boosted SQV absorption by more than 20-fold with decreased variation in AUCs (percent coefficient of variation = 30% versus 100%). In contrast, coadministration of MK571 or midazolam increased SQV absorption only 2- to 3-fold without improving the variation in AUCs. SQV oral absorption was not further improved when it was given with GF120918 and midazolam or with GF120918 and MK571. The current results provide, for the first time, direct and explicit evidence that the low oral absorption of SQV is controlled by a secretory transporter, Pgp, and not by limited passive diffusion owing to its poor physicochemical properties. Pgp-mediated transport is also responsible for the highly variable oral bioavailability of SQV. In contrast, intestinal Mrp2 and intestinal CYP3A appear to play minor roles in SQV oral bioavailability. Given the differential and complex roles of Pgp and CYP3A in SQV oral absorption, the optimization of AIDS boosting regimens requires careful consideration to avoid therapy-limiting drug-drug transporter

  18. Effect of pluronic P123 and F127 block copolymer on P-glycoprotein transport and CYP3A metabolism.

    PubMed

    Guan, Yanbin; Huang, Jiangeng; Zuo, Lan; Xu, Jiaqiang; Si, Luqin; Qiu, Jun; Li, Gao

    2011-10-01

    The aim of the present study was to evaluate the effect of pluronic P123 (P123) and pluronic F127 (F127) on intestinal P-glycoprotein (P-gp) and cytochrome P450 3A using the specific substrates rhodamine-123 (R-123) and midazolam, respectively. Caco-2 cells and everted gut sacs were used as models of intestinal mucosa to assess intestinal absorption of R-123, while rat intestinal microsomes were utilized to examine the effect of P123 and F127 on in vitro midazolam metabolism. P123 and F127 were observed to increase the intracellular accumulation of R-123 in Caco-2 cells in a dose-dependent manner. P123 significantly lowered the efflux ratio of R-123 at two concentrations in Caco-2 monolayers, whereas F127 lowered the efflux ratio only at 1%. Moreover, both pluronics markedly enhanced mucosal to serosal absorption of R-123 in excised ileum of rats. However, no significant difference in relative enzyme activity were observed between P123- or F127-treated and control groups, regardless of the concentrations of P123 and F127 studied. Collectively, these results obtained from the present study demonstrated that P123 and F127 were capable of inhibiting the intestinal P-gp activity, but had little or no effect on intestinal cytochrome P450 3A activity, indicating that P123 and F127 can potentially be used as pharmaceutical ingredients to improve the oral bioavailability of coadministered P-gp substrates via P-gp efflux pump inhibition. PMID:22076772

  19. Inhibitory effects of furanocoumarin derivatives in Kampo extract medicines on P-glycoprotein at the blood-brain barrier.

    PubMed

    Iwanaga, Kazunori; Yoneda, Shinji; Hamahata, Yukimi; Miyazaki, Makoto; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Kakemi, Masawo

    2011-01-01

    Furanocoumarin derivatives, known as components of grapefruit juice, showing inhibitory effects against P-glycoprotein (P-gp) in the intestine are also contained in the plants of rutaceae and umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of byakangelicol and rivulobirin A, known as furanocoumarins showing P-gp inhibitory effect using Caco-2 monolayer, against P-gp at the blood-brain barrier (BBB) under both in vitro and in vivo conditions. First we studied the membrane permeability of furanocoumarins to clarify whether they can be absorbed from the intestine. Both furanocoumarins showed high permeability through the Caco-2 monolayer, suggesting that they can easily reach the systemic circulation after oral administration. Then, we evaluated the effect of these furanocoumarins on the uptake of calcein acetoxymethyl ester (calcein-AM), a P-gp substrate, into bovine brain microvascular endothelial cells (BBMEC). Both furanocoumarins significantly increased the uptake amount of calcein-AM into BBMEC by the inhibition of P-gp at the BBB in vitro. Next we also investigated the P-gp inhibitory effect of these furanocoumarins at the rat BBB in vivo using verapamil as a P-gp substrate. Both furanocoumarins increased the B/P ratio of verapamil compared to the control, even under in vivo conditions; however, the extent of the inhibitory effect was much lower than in vitro condition. In conclusion, byakangelicol and rivulobirin A may inhibit P-gp expressed at the BBB even under in vivo conditions. Further studies using Kampo extract medicines under in vivo condition are necessary for safe drug therapy. PMID:21804213

  20. Inhibitory effect of clemastine on P-glycoprotein expression and function: an in vitro and in situ study

    PubMed Central

    Abbasi, Mehran Mesgari; Valizadeh, Hadi; Hamishekar, Hamed; Mohammadnejad, Leila; Zakeri-Milani, Parvin

    2016-01-01

    Objective(s): Transporters have an important role in pharmacokinetics of drugs. Inhibition or induction of drug transporters activity can affect drug absorption, safety, and efficacy. P-glycoprotein (P-gp) is the most important membrane transporter that is responsible for active efflux of drugs. It is important to understand which drugs are substrates, inhibitors, or inducers of P-gp to minimize or avoid unwanted interactions. The aim of this study was to investigate the effects of clemastine on the expression and function of P-gp. Materials and Methods: The effect of clemastine on P-gp function and expression was evaluated in vitro byrhodamine-123 (Rho123) efflux assay in Caco-2 cells and Western blot analysis. Rat in situ single pass intestinal permeability model was used to investigate the clemastine effect on digoxin Peff, as a known P-gp substrate. Digoxin levels in intestinal perfusates were assayed by high performance liquid chromatography (HPLC) method. Results: The Caco-2 intracellular accumulation of Rho123 in clemastine and verapamil treated cells was 90.8 ± 9.8 and 420.6±25.4 pg/mg protein, respectively which was significantly higher than that in control cells (50.2±6.0; P<0.05). Immunoblotting results indicated that clemastine decreased expression of P-gp in Caco-2 cells in vitro. More over effective intestinal permeability (Peff) of digoxin in the presence of clemastine, was significantly increased compare to control group. Conclusion: Findings of our study suggested dose dependent P-gp inhibition activity for clemastine in vitro and in situ. Therefore co-administration of clemastine with P-gp substrates may result in unwanted interactions and side effects. PMID:27279987

  1. Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.

    PubMed

    Loo, Tip W; Clarke, David M

    2015-12-01

    ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease. PMID:26507655

  2. Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2015-01-01

    ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease. PMID:26507655

  3. Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P4503A4.

    PubMed

    Achira, M; Suzuki, H; Ito, K; Sugiyama, Y

    1999-01-01

    It has been suggested that cytochrome P450 3A4 (CYP3A4) and MDR1 P-glycoprotein (P-gp) act synergistically to limit the bioavailability of orally administered agents. In order to determine the relative role of these proteins, it is essential to identify a selective inhibitor for either P-gp or CYP3A4. In the present investigation, comparative studies were performed to examine the effect of inhibitors on the function of these proteins. The IC50of P-gp function, determined by examining the inhibition of the transcellular transport of vinblastine across Caco-2 monolayers, was in the order PSC833 < ketoconazole, verapamil < N-(2(R)-hydroxy-1(S)-indanyl)-5-(2(S)-(1,1-dimethylethylaminocarbonyl)-4-(furo(2,3-b)pyridin-5-yl)methyl)piperazin-1-yl)-4(S)-hydroxy-2(R)-phenylmethylpentanamide (L-754,394). In contrast, the IC50of CYP3A4 function, determined by examining the inhibition of the metabolism of midazolam by intestinal and liver microsomes, was in the order L-754,384 < ketoconazole < PSC 833 and verapamil. The ratio of IC50for P-gp to that for CYP3A4 was more than 200 for L-754,394, 60 ~ 150 for ketoconazole, 1.5 for verapamil, and 0.05 for PSC 833. Collectively, it was demonstrated that PSC 833 and L-754,394 can be used as selective inhibitors of P-gp and CYP3A4, respectively. PMID:11741214

  4. Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

    PubMed Central

    Rees, Martin D.; Bottle, Steven E.; Fairfull-Smith, Kathryn E.; Malle, Ernst; Whitelock, John M.; Davies, Michael J.

    2014-01-01

    Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases. PMID:19379130

  5. Spillover-mediated feedforward-inhibition functionally segregates interneuron activity

    PubMed Central

    Coddington, Luke T.; Rudolph, Stephanie; Lune, Patrick Vande; Overstreet-Wadiche, Linda; Wadiche, Jacques I.

    2013-01-01

    Summary Neurotransmitter spillover represents a form of neural transmission not restricted to morphologically defined synaptic connections. Communication between climbing fibers (CFs) and molecular layer interneurons (MLIs) in the cerebellum is mediated exclusively by glutamate spillover. Here, we show how CF stimulation functionally segregates MLIs based on their location relative to glutamate release. Excitation of MLIs that reside within the domain of spillover diffusion coordinates inhibition of MLIs outside the diffusion limit. CF excitation of MLIs is dependent on extrasynaptic NMDA receptors that enhance the spatial and temporal spread of CF signaling. Activity mediated by functionally segregated MLIs converges onto neighboring Purkinje cells (PCs) to generate a long-lasting biphasic change in inhibition. These data demonstrate how glutamate release from single CFs modulates excitability of neighboring PCs, thus expanding the influence of CFs on cerebellar cortical activity in a manner not predicted by anatomical connectivity. PMID:23707614

  6. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites

    SciTech Connect

    Cordon-Cardo, C.; O'Brien, J.P.; Casals, D.; Biedler, J.L.; Melamed, M.R.; Bertino, J.R. ); Rittman-Grauer, L. )

    1989-01-01

    Endothelial cells of human capillary blood vessels at the blood-brain and other blood-tissue barrier sites express P-glycoprotein as detected by mouse monoclonal antibodies against the human multidrug-resistance gene product. This pattern of endothelial cell expression may indicate a physiological role for P-glycoprotein in regulating the entry of certain molecules into the central nervous system and other anatomic compartments, such as the testes. These tissues, which limit the access of systemic drugs, are known pharmacologic sanctuaries for metastatic cancer. P-glycoprotein expression in capillary endothelium of brain and testes and not other tissues (i.e., kidney and placenta) may in part explain this phenomenon and could have important implications in cancer chemotherapy.

  7. Inhibition of telomerase activity enhances hyperthermia-mediated radiosensitization.

    PubMed

    Agarwal, Manjula; Pandita, Shruti; Hunt, Clayton R; Gupta, Arun; Yue, Xuan; Khan, Saira; Pandita, Raj K; Pratt, David; Shay, Jerry W; Taylor, John-Stephen A; Pandita, Tej K

    2008-05-01

    Hyperthermia is a potent sensitizer of cell killing by ionizing radiation (IR); however, hyperthermia also induces heat shock protein 70 (HSP70) synthesis and HSP70 expression is associated with radioresistance. Because HSP70 interacts with the telomerase complex and expression of the telomerase catalytic unit (hTERT) extends the life span of the human cells, we determined if heat shock influences telomerase activity and whether telomerase inhibition enhances heat-mediated IR-induced cell killing. In the present study, we show that moderate hyperthermia (43 degrees C) enhances telomerase activity. Inhibition of telomerase activity with human telomerase RNA-targeted antisense agents, and in particular GRN163L, results in enhanced hyperthermia-mediated IR-induced cell killing, and ectopic expression of catalytic unit of telomerase (TERT) decreased hyperthermia-mediated IR-induced cell killing. The increased cell killing by heat and IR exposure in telomerase-inhibited cells correlates with delayed appearance and disappearance of gamma-H2AX foci as well as decreased chromosome repair. These results suggest that inactivation of telomerase before combined hyperthermia and radiotherapy could improve tumor killing. PMID:18451164

  8. Structural and dynamic perspectives on the promiscuous transport activity of P-glycoprotein.

    PubMed

    Subramanian, Nandhitha; Condic-Jurkic, Karmen; O'Mara, Megan L

    2016-09-01

    The multidrug transporter P-glycoprotein (P-gp) is expressed in the blood-brain barrier endothelium where it effluxes a range of drug substrates, preventing their accumulation within the brain. P-gp has been studied extensively for 40 years because of its crucial role in the absorption, distribution, metabolism and elimination of a range of pharmaceutical compounds. Despite this, many aspects of the structure-function mechanism of P-gp are unresolved. Here we review the emerging role of molecular dynamics simulation techniques in our understanding of the membrane-embedded conformation of P-gp. We discuss its conformational plasticity in the presence and absence of ATP, and recent efforts to characterize the drug binding sites and uptake pathways. PMID:27180050

  9. Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain

    PubMed Central

    Cannon, Ronald E.; Peart, John C.; Hawkins, Brian T.; Campos, Christopher R.; Miller, David S.

    2012-01-01

    P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-α) and sphingolipid-based signaling. Critical to this pathway was signaling through sphingosine-1-phosphate receptor 1 (S1PR1). In brain capillaries, S1P acted through S1PR1 to rapidly and reversibly reduce P-glycoprotein transport activity. Sphingosine reduced transport by a sphingosine kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S1P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, 3H-verapamil (threefold increase), 3H-loperamide (fivefold increase), and 3H-paclitaxel (fivefold increase); blocking S1PR1 abolished this effect. Tight junctional permeability, measured as brain 14C-sucrose accumulation, was not altered. Therefore, targeting signaling through S1PR1 at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain. PMID:22949658

  10. P-glycoprotein and its inducible expression in three bivalve species after exposure to Prorocentrum lima.

    PubMed

    Huang, Lu; Liu, Su-Li; Zheng, Jian-Wei; Li, Hong-Ye; Liu, Jie-Sheng; Yang, Wei-Dong

    2015-12-01

    P-glycoprotein (P-gp or ABCB1) belongs to the family of ATP-binding cassette (ABC) transporters responsible for multixenobiotic resistance (MXR) in aquatic organisms. To provide more information of P-gp in shellfish, in this study, complete cDNA of P-gp in three bivalve species including Ruditapes philippinarum, Scapharca subcrenata and Tegillarca granosa were cloned and its expressions in gill, digestive gland, adductor muscle and mantle of the three bivalves were detected after exposure to Prorocentrum lima, a toxogenic dinoflagellate. The complete sequences of R. philippinarum, S. subcrenata and T. granosa P-gp showed high homology with MDR/P-gp/ABCB proteins from other species, having a typical sequence organization as full transporters from the ABCB family. Phylogenetic analyses revealed that the amino acid sequences of P-gp from S. subcrenata and T. granosa had a closest relationship, forming an independent branch, then grouping into the other branch with Mytilus californianus, Mytilus galloprovincialis and Crassostrea gigas. However, P-gp sequences from R. philippinarum were more similar to the homologs from the more distantly related Aplysia californica than to homologs from S. subcrenata and T. granosa, suggesting that bivalves P-gp might have different paralogs. P-glycoprotein expressed in all detected tissues but there were large differences between them. After exposure to P. lima, the expression of P-gp changed in the four tissues in varying degrees within the same species and between different species, but the changes in mRNA and protein level were not always synchronous. PMID:26539802

  11. Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

    PubMed Central

    O’Mara, Megan L.; Mark, Alan E.

    2014-01-01

    ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg2+ with each NBD indicates that the coordination of ATP and Mg2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations

  12. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    PubMed

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins. PMID:19961540

  13. 2'[(18)F]-fluoroethylrhodamine B is a promising radiotracer to measure P-glycoprotein function.

    PubMed

    Trencsényi, György; Kertész, István; Krasznai, Zoárd T; Máté, Gábor; Szalóki, Gábor; Szabó Judit, P; Kárpáti, Levente; Krasznai, Zoltán; Márián, Teréz; Goda, Katalin

    2015-07-10

    In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp. PMID:25857708

  14. P-glycoprotein expression in Perna viridis after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins.

    PubMed

    Huang, Lu; Wang, Jie; Chen, Wen-Chang; Li, Hong-Ye; Liu, Jie-Sheng; Tao Jiang; Yang, Wei-Dong

    2014-08-01

    Bivalves naturally exposed to toxic algae have mechanisms to prevent from harmful effects of diarrhetic shellfish poisoning (DSP) toxins. However, quite few studies have examined the mechanisms associated, and the information currently available is still insufficient. Multixenobiotic resistance (MXR) is ubiquitous in aquatic invertebrates and plays an important role in defense against xenobiotics. Here, to explore the roles of P-glycoprotein (P-gp) in the DSP toxins resistance in shellfish, complete cDNA of P-gp gene in the mussel Perna viridis was cloned and analyzed. The accumulation of okadaic acid (OA), a main component of DSP toxins, MXR activity and expression of P-gp in gills of P. viridis were detected after exposure to Prorocentrum lima, a dinoflagellate producing DSP toxins in the presence or absence of P-gp inhibitors PGP-4008, verapamil (VER) and cyclosporin A (CsA). The mussel P. viridis P-gp closely matches MDR/P-gp/ABCB protein from various organisms, having a typical sequence organization as full transporters from the ABCB family. After exposure to P. lima, OA accumulation, MXR activity and P-gp expression significantly increased in gills of P. viridis. The addition of P-gp-specific inhibitors PGP-4008 and VER decreased MXR activity induced by P. lima, but had no effect on the OA accumulation in gills of P. viridis. However, CsA, a broad-spectrum inhibitor of ABC transporter not only decreased MXR activity, but also increased OA accumulation in gills of P. viridis. Together with the ubiquitous presence of other ABC transporters such as MRP/ABCC in bivalves and potential compensatory mechanism in P-gp and MRP-mediated resistance, we speculated that besides P-gp, other ABC transporters, especially MRP might be involved in the resistance mechanisms to DSP toxins. PMID:24811006

  15. Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein.

    PubMed

    Ohura, Kayoko; Nakada, Yuichiro; Kotani, Shunsuke; Imai, Teruko

    2015-09-01

    The aim of this study was to develop a suitable prodrug for fexofenadine (FXD), a model parent drug, that is resistant to intestinal esterase but converted to FXD by hepatic esterase. Carboxylesterases (CESs), human carboxylesterase 1 (hCE1) and human carboxylesterase 2 (hCE2), are the major esterases in human liver and intestine, respectively. These two CESs show quite different substrate specificities, and especially, hCE2 poorly hydrolyzes prodrugs with large acyl groups. FXD contains a carboxyl group and is poorly absorbed because of low membrane permeability and efflux by P-glycoprotein (P-gp). Therefore, two potential FXD prodrugs, ethyl-FXD and 2-hydroxyethyl-FXD, were synthesized by substitution of the carboxyl group in FXD. Both derivatives were resistant to intestinal hydrolysis, indicating their absorption as intact prodrugs. Ethyl-FXD was hydrolyzed by hepatic hCE1, but 2-hydroxyethyl-FXD was not. Both derivatives showed high membrane permeability in human P-gp-negative LLC-PK1 cells. In LLC-GA5-COL300 cells overexpressing human P-gp, ethyl-FXD was transported by P-gp, but its efflux was easily saturated. Whereas 2-hydroxyethyl-FXD showed more efficient P-gp-mediated transport than FXD. Although the structure of 2-hydroxyethyl-FXD only differs from ethyl-FXD by substitution of a hydroxyl group, 2-hydroxyethyl-FXD is unsuitable as a prodrug. However, ethyl-FXD is a good candidate prodrug because of good intestinal absorption and hepatic conversion by hCE1. PMID:25953731

  16. Role of P-glycoprotein and permeability upon the brain distribution and pharmacodynamics of etamicastat: a comparison with nepicastat.

    PubMed

    Loureiro, Ana I; Bonifácio, Maria João; Fernandes-Lopes, Carlos; Pires, Nuno; Igreja, Bruno; Wright, Lyndon C; Soares-da-Silva, Patrício

    2015-01-01

    1. This study explores the impact of permeability and P-glycoprotein (P-gp) efflux, upon brain exposure to etamicastat, a new dopamine-β-hydroxylase (DBH) inhibitor and consequently brain levels of catecholamines. 2. Brain exposure to etamicastat (10 mg/kg), following intravenous administration to mice, was residual and upon oral administration of the same dose no compound was detected, concurring with the absence of effects upon brain catecholamines. The intravenous co-administration of elacridar (1.0 mg/kg), a known P-gp/BCRP dual modulator, significantly increased brain etamicastat exposure, but the levels attained were very low when compared to those of nepicastat, a centrally active DBH inhibitor. 3. In vitro permeability studies from apical-to-basal direction conducted in Caco-2 cells and MDCK-II cells showed that etamicastat apparent permeability was 1.2 × 10(-5) and 1.1 × 10(-6 )cm/s, respectively, 5- and 50-fold lower as compared to nepicastat. The secretory efflux ratio in MDCK-II cells overexpressing human P-gp showed an efflux ratio greater than 2, for both compounds, which was significantly decreased by elacridar. Despite its lower bioavailability and higher clearance, as compared to nepicastat, etamicastat showed preferential distribution to peripheral tissues and high plasma free fraction (15.5%), which may explain its effects upon peripheral DBH and catecholamine levels. 4. Though P-gp-mediated efflux may contribute to the limited brain penetration of etamicastat, the low permeability along with the pharmacokinetic properties of etamicastat may be perceived as the main contributors for its peripheral selectivity, which is advantageous for a cardiovascular drug candidate. PMID:25915108

  17. P-glycoprotein antagonists confer synergistic sensitivity to short-chain ceramide in human multidrug resistant cancer cells

    PubMed Central

    Chapman, Jacqueline V.; Gouazé-Andersson, Valérie; Karimi, Ramin; Messner, Maria C; Cabot, Myles C.

    2011-01-01

    P-glycoprotein (P-gp) antagonists inhibit ceramide metabolism at the juncture of glycosylation. The purpose of this study was to test whether targeting P-gp would be a viable alternative to targeting glucosylceramide synthase (GCS) for enhancing ceramide cytotoxicity. A2780 wild-type, and multidrug resistant 2780AD and NCI/ADR-RES human ovarian cancer cell lines and the cell-permeable ceramide analog, C6-ceramide (C6-cer), were employed. Compared to P-gp-poor A2780 cells, P-gp-rich 2780AD cells converted 3.7-fold more C6-cer to nontoxic C6-glucosylceramide (C6-GC), whereas cell-free GCS activities were equal. 2780AD cells displayed resistance to C6-cer (10 μM) that was reversed by inclusion of the P-gp antagonist tamoxifen (5 μM) but not by inclusion of a GCS inhibitor. Co-administration of C6-cer and P-gp antagonists was also effective in NCI/ADR-RES cells. For example, C6-cer, VX-710 (Biricodar), and cyclosporin A (cyc A) exposure resulted in viabilities of ~90% of control; however, C6-cer/VX-710 and C6-cer/cyc A additions were synergistic and resulted in viabilities of 22 and 17%, respectively. Further, whereas C6-ceramide and cyc A imparted 1.5- and zero-fold increases in caspase 3/7 activity, the combination produced a 3.5-fold increase. Although the upstream elements of cell death have not been elucidated, the novel C6-ceramide/P-gp antagonist combination merits further study and assessment of clinical translational potential. PMID:21396934

  18. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein

    PubMed Central

    Davis, Sterenn; Davis, Benjamin M.; Richens, Joanna L.; Vere, Kelly-Ann; Petrov, Peter G.; Winlove, C. Peter; O’Shea, Paul

    2015-01-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir’s affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers. PMID:26026069

  19. Avermectin induces P-glycoprotein expression in S2 cells via the calcium/calmodulin/NF-κB pathway.

    PubMed

    Luo, Liang; Sun, Yin-Jian; Yang, Lin; Huang, Shile; Wu, Yi-Jun

    2013-04-25

    Avermectin (AVM) is a macrocyclic lactone agent widely used as a nematicide, acaricide and insecticide in veterinary medicine and plant protection. P-glycoprotein (P-gp) is an ATP-dependent drug efflux pump for xenobiotic compounds, and is involved in multidrug resistance. To understand the development of AVM resistance in invertebrates, we investigated the mechanisms by which AVM affected P-gp expression in Drosophila S2 cells. We found that AVM induced upregulation of P-gp protein expression, increased P-gp ATPase activity and enhanced cellular efflux of the P-gp substrate rhodamine 123 from cells. Furthermore, we observed that AVM-induced expression of P-gp was due to elevation of intracellular calcium concentration ([Ca(2+)](i)). This occurred both directly, by activating calcium ion channels, and indirectly, by activating chloride ion channels. These results are supported by our observations that verapamil, a Ca(2+) channel blocker, and niflumic acid, a chloride channel antagonist, significantly attenuated AVM-induced [Ca(2+)](i) elevation, thereby reducing P-gp expression. Inhibition of P-gp with anti-P-gp antibody or cyclosporine A (a P-gp inhibitor) reduced the AVM-induced elevation of [Ca(2+)](i), implying that P-gp and [Ca(2+)](i) regulate each other. Finally, we found that trifluoperazine, a calmodulin inhibitor, and pyrrolidine dithiocarbamic acid, an NF-κB inhibitor, attenuated the AVM-induced expression of P-gp, suggesting that AVM induces P-gp protein expression via the calmodulin/Relish (NF-κB) signaling pathway. PMID:23523950

  20. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.

    PubMed

    Davis, Sterenn; Davis, Benjamin M; Richens, Joanna L; Vere, Kelly-Ann; Petrov, Peter G; Winlove, C Peter; O'Shea, Paul

    2015-08-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir's affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers. PMID:26026069

  1. Mechanism of ritonavir changes in methadone pharmacokinetics and pharmacodynamics: II. Ritonavir effects on CYP3A and P-glycoprotein activities.

    PubMed

    Kharasch, E D; Bedynek, P S; Walker, A; Whittington, D; Hoffer, C

    2008-10-01

    Ritonavir diminishes methadone plasma concentrations, an effect attributed to CYP3A induction, but the actual mechanisms are unknown. We determined short-term (2-day) and steady-state (2-week) ritonavir effects on intestinal and hepatic CYP3A4/5 (probed with intravenous (IV) and oral alfentanil (ALF) and with miosis) and P-glycoprotein (P-gp) (fexofenadine), and on methadone pharmacokinetics and pharmacodynamics in healthy volunteers. Acute ritonavir increased the area under the concentration-time curve (AUC)(0-infinity)/dose ratio (ritonavir/control) for oral ALF 25-fold. Steady-state ritonavir increased the AUC(0-Infinity)/dose ratio for IV and oral ALF 4- and 10-fold, respectively; reduced hepatic extraction (from 0.26 to 0.07) and intestinal extraction (from 0.51 to 0); and increased bioavailability (from 37 to 95%). Acute ritonavir inhibits first-pass CYP3A > 96%. Chronic ritonavir inhibits hepatic CYP3A (> 70%) and first-pass CYP3A (> 90%). Acute and steady-state ritonavir increased the fexofenadine AUC(0-infinity) 2.8- and 1.4-fold, respectively, suggesting P-gp inhibition. Steady-state compared with acute ritonavir caused mild apparent induction of P-gp and hepatic CYP3A, but net inhibition still predominated. Ritonavir inhibited both intestinal and hepatic CYP3A and drug transport. ALF miosis noninvasively determined CYP3A inhibition by ritonavir. PMID:19238656

  2. RNA interference (RNAi) mediated stable knockdown of protein casein kinase 2-alpha (CK2α) inhibits migration and invasion and enhances cisplatin-induced apoptosis in HEp-2 laryngeal carcinoma cells.

    PubMed

    Zhang, Fang; Yang, Bo; Shi, Shengli; Jiang, Xuejun

    2014-07-01

    Laryngeal carcinoma is a common malignant neoplasm occurring in the head and neck, threatening human health. Protein casein kinase 2-alpha (CK2α) has been indicated to participate in the pathogenesis of this cancer; however, the underlying mechanisms still need to be elucidated. In this study, short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology was utilized to inhibit the CK2α expression in HEp-2 laryngeal carcinoma cells. Results showed that both mRNA and protein expression levels of endogenous CK2α were markedly decreased in HEp-2 cells transfected with CK2α specific shRNA. Transwell assays revealed that stable knockdown of CK2α significantly inhibited the migration and invasion of HEp-2 cells. As compared with cells treated with negative control shRNA, epithelial cadherin (E-cadherin) expression was increased, but snail, slug and vimentin were decreased in cells transfected with CK2α shRNA, indicating that inhibition of CK2α expression may suppress the epithelial-mesenchymal transition (EMT) process of laryngeal carcinoma in vitro. Moreover, suppression of CK2α was found to enhance the apoptosis induced by cisplatin in laryngeal carcinoma cells, probably through inhibition of permeability glycoprotein (P-glycoprotein) and multidrug-resistance protein (MRP1). In conclusion, our study may provide a promising therapeutic strategy for human laryngeal carcinoma by targeting CK2α. PMID:24831064

  3. Interaction of HM30181 with P-glycoprotein at the murine blood-brain barrier assessed with positron emission tomography

    PubMed Central

    Bauer, Florian; Wanek, Thomas; Mairinger, Severin; Stanek, Johann; Sauberer, Michael; Kuntner, Claudia; Parveen, Zahida; Chiba, Peter; Müller, Markus; Langer, Oliver; Erker, Thomas

    2013-01-01

    HM30181, a potent and selective inhibitor of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp), was shown to enhance oral bioavailability and improve antitumour efficacy of paclitaxel in mouse tumour models. In search for a positron emission tomography (PET) radiotracer to visualise Pgp expression levels at the blood-brain barrier (BBB), we examined the ability of HM30181 to inhibit Pgp at the murine BBB. HM30181 was shown to be approximately equipotent with the reference Pgp inhibitor tariquidar in inhibiting rhodamine 123 efflux from CCRF-CEM T cells (IC50, tariquidar: 8.2±2.0 nM, HM30181: 13.1±2.3 nM). PET scans with the Pgp substrate (R)-[11C]verapamil in FVB wild-type mice pretreated i.v. with HM30181 (10 or 21 mg/kg) failed to show significant increases in (R)-[11C]verapamil brain uptake compared with vehicle treated animals. PET scans with [11C]HM30181 showed low and not significantly different brain uptake of [11C]HM30181 in wild-type, Mdr1a/b(−/−) and Bcrp1(−/−) mice and significantly, i.e. 4.7-fold (P<0.01), higher brain uptake, relative to wild-type animals, in Mdr1a/b(−/−)Bcrp1(−/−) mice. This was consistent with HM30181 being at microdoses a dual substrate of Pgp and breast cancer resistance protein (Bcrp). In vitro autoradiography on low (EMT6) and high (EMT6Ar1.0) Pgp expressing murine breast tumour sections showed 1.9 times higher binding of [11C]HM30181 in EMT6Ar1.0 tumours (P<0.001) which was displaceable with unlabelled tariquidar, elacridar or HM30181 (1 μM). Our data suggest that HM30181 is not able to inhibit Pgp at the murine BBB at clinically feasible doses and that [11C]HM30181 is not suitable as a PET tracer to visualise cerebral Pgp expression levels. PMID:23022332

  4. Oncogenic microtubule hyperacetylation through BEX4-mediated sirtuin 2 inhibition.

    PubMed

    Lee, Jin-Kwan; Lee, Janet; Go, Heounjeong; Lee, Chang Geun; Kim, Suhyeon; Kim, Hyun-Soo; Cho, Hyeseong; Choi, Kyeong Sook; Ha, Geun-Hyoung; Lee, Chang-Woo

    2016-01-01

    Five brain-expressed X-linked (BEX) gene members (BEX1-5) are arranged in tandem on chromosome X, and are highly conserved across diverse species. However, little is known about the function and role of BEX. This study represents a first attempt to demonstrate the molecular details of a novel oncogene BEX4. Among BEX proteins, BEX4 localizes to microtubules and spindle poles, and interacts with α-tubulin (α-TUB) and sirtuin 2 (SIRT2). The overexpression of BEX4 leads to the hyperacetylation of α-TUB by inhibiting SIRT2-mediated deacetylation. Furthermore, we found BEX4 expression conferred resistance to apoptotic cell death but led to acquisition of aneuploidy, and also increased the proliferating potential and growth of tumors. These results suggest that BEX4 overexpression causes an imbalance between TUB acetylation and deacetylation by SIRT2 inhibition and induces oncogenic aneuploidy transformation. PMID:27512957

  5. P-glycoprotein expression in Ehrlich ascites tumour cells after in vitro and in vivo selection with daunorubicin.

    PubMed Central

    Nielsen, D.; Eriksen, J.; Maare, C.; Jakobsen, A. H.; Skovsgaard, T.

    1998-01-01

    Fluctuation analysis experiments were performed to assess whether selection or induction determines expression of P-glycoprotein and resistance in the murine Ehrlich ascites tumour cell line (EHR2) after exposure to daunorubicin. Thirteen expanded populations of EHR2 cells were exposed to daunorubicin 7.5 x 10(-9) M or 10(-8) M for 2 weeks. Surviving clones were scored and propagated. Only clones exposed to daunorubicin 7.5 x 10(-9) M could be expanded for investigation. Drug resistance was assessed by the tetrazolium dye (MTT) cytotoxicity assay. Western blot was used for determination of P-glycoprotein. Compared with EHR2, the variant cells were 2.5- to 5.2-fold resistant to daunorubicin (mean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spontaneous mutations conferred drug resistance in EHR2 cells exposed to daunorubicin 7.5 x 10(-9) M. At this level (5 log cell killing) of drug exposure, the mutation rate was estimated at 4.1 x 10(-6) per cell generation. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-8) M. The revertant EHR2/0.8/R was treated in vivo with daunorubicin for 24 h. After treatment, P-glycoprotein increased in EHR2/0.8/R (sevenfold) and the cell line developed resistance to daunorubicin (12-fold), suggesting that in EHR2/0.8/R the mdr1 gene was activated by induction. In conclusion, our study demonstrates that P-glycoprotein expression and daunorubicin resistance are primarily acquired by selection of spontaneously arising mutants. However, under certain conditions the mdr1 gene may be activated by induction. PMID:9820176

  6. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

    PubMed

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. PMID:26212258

  7. Thunbergia laurifolia extract minimizes the adverse effects of toxicants by regulating P-glycoprotein activity, CYP450, and lipid metabolism gene expression in HepG2 cells.

    PubMed

    Rocejanasaroj, A; Tencomnao, T; Sangkitikomol, W

    2014-01-01

    Thunbergia laurifolia (TL) is widely used as an antidote in Thai traditional medicine against toxic substances such as alcohol, pesticides, arsenic, and strychnine. We found that the lyophilized form of TL in 80% ethanol possessed the antioxidant levels within the range 23,163.9 ± 1457.4 Trolox equivalents mM/kg dry mass and 899.8 ± 14.5 gallic acid equivalents mM/kg dry mass using the oxygen radical absorbance capacity assay and the Folin Ciocalteu phenol assay, respectively. TL extract (TLE) at a high dose (3000 mg/L) induced cytotoxicity according to the neutral red assay and the MTT assay. However, TLE doses of 800-3000 mg/L could reduce intracellular oxidative stress in a dose-dependent manner (P < 0.05) using the dichlorodihydrofluorescein diacetate assay. TLE significantly enhanced the mRNA expression of CYP1A1, CYP1A2, CYP2B6, CYP3A4, and PPARg, but it significantly inhibited the mRNA expression of CYP3A7, CYP2D6, and CYP2E1 (P < 0.05) by reverse transcription-polymerase chain reaction. Moreover, TLE could increase the activity of a multidrug transporter, P-glycoprotein, which accelerated the excretion of toxic substances from HepG2 cells. It is suggested that TLE may be beneficial for detoxification by reducing oxidative stress, minimizing toxicity by regulating the expression CYP450 mRNAs for suitable production of CYP450 isoenzymes, and increasing PPARγ mRNA expression and P-glycoprotein activity in HepG2 cells, thereby maintaining xenobiotic biotransformation balance. PMID:24446304

  8. Effect of HM30181 mesylate salt-loaded microcapsules on the oral absorption of paclitaxel as a novel P-glycoprotein inhibitor.

    PubMed

    Kim, Jin Cheul; Kim, Kyeong Soo; Kim, Dong Shik; Jin, Sung Giu; Kim, Dong Wuk; Kim, Yong Il; Park, Jae-Hyun; Kim, Jong Oh; Yong, Chul Soon; Youn, Yu Seok; Woo, Jong Soo; Choi, Han-Gon

    2016-06-15

    The purpose of this study was to develop HM30181 mesylate salt (HM30181M)-loaded microcapsules as a novel P-glycoprotein inhibitor for enhancing the oral absorption of paclitaxel. The effect of various carriers including hydrophilic polymers and solvents on the solubility of HM30181M were evaluated. Among the hydrophilic polymers and solvents tested, HPMC and methylene chloride (and ethanol) provided the highest HM30181M solubility. Numerous HM30181M-loaded microcapsules were prepared with HPMC, silicon dioxide and acidifying agents using a spray-drying technique, and their solubility, dissolution and physicochemical properties were evaluated. Furthermore, a pharmacokinetic study was performed after oral administration of paclitaxel alone, simultaneously with HM30181M powder or HM30181M-loaded microcapsules to rats. Among the acidifying agents investigated, phosphoric acid provided the best improvement in the solubility and dissolution of HM30181M. Moreover, the microcapsule composed of HM30181M, HPMC, silicon dioxide and phosphoric acid at a weight ratio of 3:6:3:2 remarkably enhanced the solubility and dissolution of HM30181M compared with the HM30181M powder alone. The microcapsules were spherical in shape, had a reduced particle size of about 7μm, and contained HM30181M in an amorphous state. Furthermore, this microcapsule significantly enhanced HM30181M absorption, making it about 1.7-fold faster and 1.6-fold greater after simultaneous administration, leading to about 70- and 2-fold improved oral bioavailability of paclitaxel compared with paclitaxel alone and the simultaneous administration with HM30181M powder, respectively. Thus, this novel microcapsule could be a potential candidate for effective P-glycoprotein inhibition during oral administration of paclitaxel. PMID:27106527

  9. Synergism between ivermectin and the tyrosine kinase/P-glycoprotein inhibitor crizotinib against Haemonchus contortus larvae in vitro.

    PubMed

    Raza, Ali; Kopp, Steven R; Kotze, Andrew C

    2016-08-30

    Anthelmintic resistance is a major problem in parasitic nematodes of livestock worldwide. One means to counter resistance is to use synergists that specifically inhibit resistance mechanisms in order to restore the toxicity, and hence preserve the usefulness, of currently available anthelmintics. P-glycoproteins (P-gps) eliminate a wide variety of structurally unrelated xenobiotics from cells, and have been implicated in anthelmintic resistance. Crizotinib is a tyrosine kinase inhibitor under development as a cancer therapeutic. The compound also inhibits P-gps, and has been shown to reverse multidrug resistance in cancer cells. We were therefore interested in determining if the compound was able to increase the sensitivity of Haemonchus contortus larvae to ivermectin, as measured by in vitro larval development and migration assays with a drug-resistant and a -susceptible isolate. In migration assays, co-administration of crizotinib increased the toxicity of ivermectin to resistant larvae (up to 5.7-fold decrease in ivermectin IC50), and rendered the resistant larvae equally or more sensitive to ivermectin than the susceptible isolate. On the other hand, co-administration of crizotinib had no effect on ivermectin sensitivity in the susceptible isolate. In development assays, significant increases in the sensitivity of both the resistant (up to 1.9-fold) and susceptible (up to 1.6-fold) larvae to ivermectin were observed, although the magnitude of the observed synergism was less than seen in migration assays, and the resistant larvae retained significant levels of ivermectin resistance. By highlighting the ability of the P-gp inhibitor crizotinib to increase the sensitivity of H. contortus larvae to ivermectin, this study provides further evidence that P-gp inhibitors are potential tools for modulating the efficacy of anthelmintics. In addition, the differences in the outcomes of the two assays, with 'resistance-breaking' effects being much more marked in migration

  10. Snapshots of ligand entry, malleable binding and induced helical movement in P-glycoprotein

    PubMed Central

    Szewczyk, Paul; Tao, Houchao; McGrath, Aaron P.; Villaluz, Mark; Rees, Steven D.; Lee, Sung Chang; Doshi, Rupak; Urbatsch, Ina L.; Zhang, Qinghai; Chang, Geoffrey

    2015-01-01

    P-glycoprotein (P-gp) is a transporter of great clinical and pharmacological significance. Several structural studies of P-gp and its homologs have provided insights into its transport cycle, but questions remain regarding how P-gp recognizes diverse substrates and how substrate binding is coupled to ATP hydrolysis. Here, four new P-gp co-crystal structures with a series of rationally designed ligands are presented. It is observed that the binding of certain ligands, including an ATP-hydrolysis stimulator, produces a large conformational change in the fourth transmembrane helix, which is positioned to potentially transmit a signal to the nucleotide-binding domains. A new ligand-binding site on the surface of P-gp facing the inner leaflet of the membrane is also described, providing vital insights regarding the entry mechanism of hydrophobic drugs and lipids into P-gp. These results represent significant advances in the understanding of how P-gp and related transporters bind and export a plethora of metabolites, antibiotics and clinically approved and pipeline drugs. PMID:25760620

  11. Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Ernst, S.; Verhalen, B.; Zarrabi, N.; Wilkens, S.; Börsch, M.

    2011-03-01

    In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.

  12. Verapamil and Rifampin Effect on P-Glycoprotein Expression in Hepatocellular Carcinoma

    PubMed Central

    Jalali, Amir; Ghasemian, Sepideh; Najafzadeh, Hossein; Galehdari, Hamid; Seifi, Masoud Reza; Zangene, Fateme; Dehdardargahi, Shaiesteh

    2014-01-01

    Background: High expression of p-glycoprotein (P-gp) has been associated with a poor prognosis in patients with hepatocellular carcinoma (HCC). It is likely that P-gp overexpression is responsible for multidrug resistance in HCC. Objectives: The aim of this study was to elucidate the effect of potent carcinogen nitrosamine with and without verapamil and rifampin drugs on P-gp expression at the mRNA level in HCC. Materials and Methods: Four groups of rats (n = 5) were selected with different treatments and one group as control. mRNA concentration changes were monitored using quantitative PCR (QPCR). Results: A significant difference was found between verapamil treated group and the control regarding the mRNA level. The mdr1a mRNA was significantly decreased in the verapamil group (P ≤ 0.001). Rifampin administrated group had a decreased level of the mdr1a mRNA compared to the control group (P ≤ 0.006). No significant changes were observed in HCC induced rats regarding the mdr1a mRNA level when treated with verapamil and rifampin. An enhanced expression of the mdr1a gene was found In the HCC induced animals when treated with drugs. Conclusions: Verapamil and rifampin were found specific and effective against P-gp expression in HCC. In conclusion, treatment efficacy of most anticancer drugs is increased in combination with verapamil and rifampin against most advanced HCC. PMID:25625052

  13. Molecular docking characterizes substrate-binding sites and efflux modulation mechanisms within P-glycoprotein.

    PubMed

    Ferreira, Ricardo J; Ferreira, Maria-José U; dos Santos, Daniel J V A

    2013-07-22

    P-Glycoprotein (Pgp) is one of the best characterized ABC transporters, often involved in the multidrug-resistance phenotype overexpressed by several cancer cell lines. Experimental studies contributed to important knowledge concerning substrate polyspecificity, efflux mechanism, and drug-binding sites. This information is, however, scattered through different perspectives, not existing a unifying model for the knowledge available for this transporter. Using a previously refined structure of murine Pgp, three putative drug-binding sites were hereby characterized by means of molecular docking. The modulator site (M-site) is characterized by cross interactions between both Pgp halves herein defined for the first time, having an important role in impairing conformational changes leading to substrate efflux. Two other binding sites, located next to the inner leaflet of the lipid bilayer, were identified as the substrate-binding H and R sites by matching docking and experimental results. A new classification model with the ability to discriminate substrates from modulators is also proposed, integrating a vast number of theoretical and experimental data. PMID:23802684

  14. Selenorhodamine Photosensitizers for Photodynamic Therapy of P-Glycoprotein-Expressing Cancer Cells

    PubMed Central

    2015-01-01

    We examined a series of selenorhodamines with amide and thioamide functionality at the 5-position of a 9-(2-thienyl) substituent on the selenorhodamine core for their potential as photosensitizers for photodynamic therapy (PDT) in P-glycoprotein (P-gp) expressing cells. These compounds were examined for their photophysical properties (absorption, fluorescence, and ability to generate singlet oxygen), for their uptake into Colo-26 cells in the absence or presence of verapamil, for their dark and phototoxicity toward Colo-26 cells, for their rates of transport in monolayers of multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells, and for their colocalization with mitochondrial specific agents in Colo-26 cells. Thioamide derivatives 16b and 18b were more effective photosensitizers than amide derivatives 15b and 17b. Selenorhodamine thioamides 16b and 18b were useful in a combination therapy to treat Colo-26 cells in vitro: a synergistic therapeutic effect was observed when Colo-26 cells were exposed to PDT and treatment with the cancer drug doxorubicin. PMID:25250825

  15. Interaction of mouse intestinal P-glycoprotein with oral antidiabetic drugs and its inhibitors.

    PubMed

    Kalsi, Harman; Grewal, Ravneet K

    2015-09-01

    Type 2 diabetes (T2DM) is a progressive insulin secretory defect accompanied by resistance to insulin, and thereby making glycemic control a major concern in the treatment of these patients. Oral drug administration, though a popular option for its non-invasiveness, suffer from poor bioavailability. It could be related to the efflux transport of intestinal P-glycoprotein (Pgp). In the present study, we explored the binding interactions of antidiabetic drugs i.e., sulfonylurea drugs (glimepiride, glipizide, glyburide) and rapid acting insulin secretagogues viz., nateglinide, repaglinide and rosiglitazone; and Pgp inhibitors i.e., Generation I (verapamil and tamoxifen), III (tetradrine and tariquidar), and natural inhibitors (fumagillin and piperine) in mouse Pgp model. Our results revealed that fumagillin piperine and verapamil possess maximum interaction energies with Pgp compared to antidiabetic drugs. These observations elucidate the role of fumagillin and piperine as potential natural compounds which could intervene in the efflux action of Pgp in extruding the antidiabetic drugs and may have implications for increasing efficacy of oral antidiabetic therapy. PMID:26548081

  16. Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding

    SciTech Connect

    Aller, Stephen G.; Yu, Jodie; Ward, Andrew; Weng, Yue; Chittaboina, Srinivas; Zhuo, Rupeng; Harrell, Patina M.; Trinh, Yenphuong T.; Zhang, Qinghai; Urbatsch, Ina L.; Chang, Geoffrey

    2009-04-22

    P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of -6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

  17. Providing a molecular mechanism for P-glycoprotein; why would I bother?

    PubMed Central

    Callaghan, Richard

    2015-01-01

    It is almost 40 years since the drug efflux pump P-glycoprotein (permeability glycoprotein or P-gp) was shown to confer multi-drug resistance in cancer cells. This protein has been one of the most extensively investigated transport proteins due to its intriguing mechanism and its affect in oncology. P-gp is known to interact with over 300 compounds and the ability to achieve this has not yet been revealed. Following the binding of substrate and nucleotide, a complex series of conformational changes in the membrane and cytosolic domains translocates substrate across the membrane. Despite over 30 years of biochemical investigation, the availability of structural data and a plethora of chemical tools to modulate its function, the molecular mechanism remains a mystery. In addition, overcoming its activity in resistant cancer cells has not been achieved in the clinic, thereby garnering some degree of pessimism in the field. This review highlights the progress that has been achieved in understanding this complex protein and the value of undertaking molecular studies. PMID:26517914

  18. Fas signaling promotes chemoresistance in gastrointestinal cancer by up-regulating P-glycoprotein

    PubMed Central

    Wang, Yadong; Lin, Shiyong; Chen, Jinmin; Wang, Jing; Wang, Zhiqing; Jiang, Bo

    2014-01-01

    Fas signaling promotes metastasis of gastrointestinal (GI) cancer cells by inducing epithelial-mesenchymal transition (EMT), and EMT acquisition has been found to cause cancer chemoresistance. Here, we demonstrated that the response to chemotherapy of GI cancer patients with higher expression of FasL was significantly worse than patients with lower expression. Fas-induced activation of the ERK1/2-MAPK pathway decreased the sensitivity of GI cancer cells to chemotherapeutic agents and promoted the expression of P-glycoprotein (P-gp). FasL promoted chemoresistance of GI cancer cell via upregulation of P-gp by increasing β-catenin and decreasing miR-145. β-catenin promoted P-gp gene transcription by binding with P-gp promoter while miR-145 suppressed P-gp expression by interacting with the mRNA 3′UTR of P-gp. Immunostaining and qRT-PCR analysis of human GI cancer samples revealed a positive association among FasL, β-catenin, and P-gp, but a negative correlation between miR-145 and FasL or P-gp. Altogether, our results showed Fas signaling could promote chemoresistance in GI cancer through modulation of P-gp expression by β-catenin and miR-145. Our findings suggest that Fas signaling-based cancer therapies should be administered cautiously, as activation of this pathway may not only lead to apoptosis but also induce chemoresistance. PMID:25333257

  19. Beneficial effect of tetrandrine on refractory epilepsy via suppressing P-glycoprotein.

    PubMed

    Chen, Yinghui; Xiao, Xia; Wang, Cuicui; Jiang, Huiyuan; Hong, Zhen; Xu, Guoxiong

    2015-01-01

    Patients with refractory epilepsy are resistance to antiepileptic drugs (AEDs). The mechanisms of drug resistance are varied, but one of them is the overexpression of multidrug transporters, such as P-glycoprotein (P-gp), in the brain. Tetrandrine (TTD) is a bis-benzylisoquinoline alkaloid isolated from the root of Stephania tetrandra (S, Moore) and is found to have a favorable effect against multidrug resistance (MDR) in chemotherapy. However, whether TTD affects AEDs in refractory epilepsy is unknown. In this study, we investigated the change in AED treatment efficacy in doxorubicin-induced drug resistant cells after TTD administration. We also examined the effect of TTD on seizure behaviors in the refractory epileptic rats, specifically the expression of MDR1 mRNA and P-gp protein in the cortex and hippocampus of the refractory epileptic rats. Our results demonstrated that TTD decreased cell resistance to phenytoin and valproate. TTD decreased seizure rate and increased the treatment efficacy of AEDs by reducing the expression of P-gp at mRNA and protein levels in vivo. These data support the use of TTD as an adjuvant drug for treating refractory epilepsy. PMID:25233150

  20. P-glycoprotein inhibitors of natural origin as potential tumor chemo-sensitizers: A review.

    PubMed

    Abdallah, Hossam M; Al-Abd, Ahmed M; El-Dine, Riham Salah; El-Halawany, Ali M

    2015-01-01

    Resistance of solid tumors to treatment is significantly attributed to pharmacokinetic reasons at both cellular and multi-cellular levels. Anticancer agent must be bio-available at the site of action in a cytotoxic concentration to exert its proposed activity. P-glycoprotein (P-gp) is a member of the ATP-dependent membrane transport proteins; it is known to pump substrates out of cells in ATP-dependent mechanism. The over-expression of P-gp in tumor cells reduces the intracellular drug concentrations, which decreases the cytotoxicity of a broad spectrum of antitumor drugs. Accordingly, P-gp inhibitors/blockers are potential enhancer for the cellular bioavailability of several clinically important anticancer drugs such as, anthracyclines, taxanes, vinca alkaloids, and podophyllotoxins. Besides several chemically synthesized P-gp inhibitors/blockers, some naturally occurring compounds and plant extracts were reported for their modulation of multidrug resistance; however, this review will focus only on major classes of naturally occurring inhibitors viz., flavonoids, coumarins, terpenoids, alkaloids and saponins. PMID:25685543

  1. P-glycoprotein inhibitors of natural origin as potential tumor chemo-sensitizers: A review

    PubMed Central

    Abdallah, Hossam M.; Al-Abd, Ahmed M.; El-Dine, Riham Salah; El-Halawany, Ali M.

    2014-01-01

    Resistance of solid tumors to treatment is significantly attributed to pharmacokinetic reasons at both cellular and multi-cellular levels. Anticancer agent must be bio-available at the site of action in a cytotoxic concentration to exert its proposed activity. P-glycoprotein (P-gp) is a member of the ATP-dependent membrane transport proteins; it is known to pump substrates out of cells in ATP-dependent mechanism. The over-expression of P-gp in tumor cells reduces the intracellular drug concentrations, which decreases the cytotoxicity of a broad spectrum of antitumor drugs. Accordingly, P-gp inhibitors/blockers are potential enhancer for the cellular bioavailability of several clinically important anticancer drugs such as, anthracyclines, taxanes, vinca alkaloids, and podophyllotoxins. Besides several chemically synthesized P-gp inhibitors/blockers, some naturally occurring compounds and plant extracts were reported for their modulation of multidrug resistance; however, this review will focus only on major classes of naturally occurring inhibitors viz., flavonoids, coumarins, terpenoids, alkaloids and saponins. PMID:25685543

  2. Heterogeneous transport of digitalis-like compounds by P-glycoprotein in vesicular and cellular assays.

    PubMed

    Gozalpour, Elnaz; Wilmer, Martijn J; Bilos, Albert; Masereeuw, Rosalinde; Russel, Frans G M; Koenderink, Jan B

    2016-04-01

    Digitalis-like compounds (DLCs), the ancient medication of heart failure and Na,K-ATPase inhibitors, are characterized by their toxicity. Drug-drug interactions (DDIs) at absorption and excretion levels play a key role in their toxicity, hence, knowledge about the transporters involved might prevent these unwanted interactions. In the present study, the transport of fourteen DLCs with human P-glycoprotein (P-gp; ABCB1) was studied using a liquid chromatography-mass spectrometry (LC-MS) quantification method. DLC transport by P-gp overexpressing Madin-Darby canine kidney (MDCK) and immortalized human renal cells (ciPTEC) was compared to vesicular DLC transport. Previously, we identified convallatoxin as a substrate using membrane vesicles overexpressing P-gp; however, we could not measure transport of other DLCs in this assay (Gozalpour et al., 2014a). Here, we showed that lipophilic digitoxin, digoxigenin, strophanthidin and proscillaridin A are P-gp substrates in cellular accumulation assays, whereas the less lipophilic convallatoxin was not. P-gp function in the cellular accumulation assays depends on the entrance of lipophilic compounds by passive diffusion, whereas the vesicular transport assay is more appropriate for hydrophilic substrates. In conclusion, we identified digitoxin, digoxigenin, strophanthidin and proscillaridin A as P-gp substrates using cellular accumulation assays and recognized lipophilicity as an important factor in selecting a suitable transport assay. PMID:26708294

  3. Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling.

    PubMed

    Jones, P M; George, A M

    2005-04-30

    Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein. PMID:15826647

  4. Rhodamine-123: a p-glycoprotein marker complex with sodium lauryl sulfate.

    PubMed

    Al-Mohizea, Abdullah M; Al-Jenoobi, Fahad Ibrahim; Alam, Mohd Aftab

    2015-03-01

    Aim of this study was to investigate the role of sodium lauryl sulfate (SLS) as P-glycoprotein inhibitor. The everted rat gut sac model was used to study in-vitro mucosal to serosal transport of Rhodamine-123 (Rho-123). Surprisingly, SLS decreases the serosal absorption of Rho-123 at all investigated concentrations. Investigation reveals complex formation between Rhodamine-123 and sodium lauryl sulfate. Interaction profile of SLS & Rho-123 was studied at variable SLS concentrations. The SLS concentration higher than critical micelle concentration (CMC) increases the solubility of Rho-123 but could not help in serosal absorption, on the contrary the absorption of Rho-123 decreased. Rho-123 and SLS form pink color complex at sub-CMC. The SLS concentrations below CMC decrease the solubility of Rho-123. For further studies, Rho-123 & SLS complex was prepared by using solvent evaporation technique and characterized by using differential scanning calorimeter (DSC). Thermal analysis also proved the formation of complex between SLS & Rho-123. The P values were found to be significant (<0.05) except group comprising 0.0001% SLS, and that is because 0.0001% SLS is seems to be very low to affect the solubility or complexation of Rho-123. PMID:25730814

  5. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

    PubMed

    Bosilkovska, M; Samer, C F; Déglon, J; Rebsamen, M; Staub, C; Dayer, P; Walder, B; Desmeules, J A; Daali, Y

    2014-09-01

    The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session. PMID:24722393

  6. Analysis of the relationship between P-glycoprotein and abamectin resistance in Tetranychus cinnabarinus (Boisduval).

    PubMed

    Xu, Zhifeng; Shi, Li; Peng, Jianfang; Shen, Guangmao; Wei, Peng; Wu, Qiong; He, Lin

    2016-05-01

    Abamectin is an effective acaricide and widely used in the control of Tetranychus cinnabarinus. With the increase of control failures, it is however important to clarify the resistance mechanism to improve the control of this mite. P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump for xenobiotic compounds and is involved in multidrug resistance. In this study, the results showed that verapamil, the specific inhibitor of Pgp, could enhance the lethal effect of abamectin on mites, and this effect is more enhanced in abamectin-resistant strain (AbR, mortality increased 74.51%) than that in susceptible strain (SS, 19.91%). Further analysis showed that the activity of Pgp ATPase in AbR was significantly higher (1.65-fold) than that in SS. After exposure to sublethal concentration of abamectin, the ATPase activity in AbR was significantly increased 1.43-fold to that in control; but there was no significant difference in SS after treatment. Two Pgp gene sequences (TcPgp1 and TcPgp2) from ABCB subfamily were characterized, and their expressions were much more sensitive to abamectin's stimulation in AbR strain than SS. These findings indicate a direct relationship between Pgp and abamectin resistance, and abamectin-induced Pgp expression may be involved in the modulation of abamectin efflux in T. cinnabarinus. PMID:27017885

  7. Tissue-specific regulation of expression and activity of P-glycoprotein in adjuvant arthritis rats.

    PubMed

    Achira, Meguru; Totsuka, Ryuichi; Fujimura, Hisako; Kume, Toshiyuki

    2002-07-01

    Cyclosporine A and steroids are effective against rheumatoid arthritis and also known as substrates of P-glycoprotein (P-gp). We investigated the effect of arthritis on the hepatic and intestinal P-gp activity in rats, and substantiated the expression level of the hepatic P-gp. Doxorubicin was used as a P-gp substrate. Cumulative biliary excretion and intestinal exsorption of doxorubicin following intravenous administration were compared between adjuvant arthritis (AA) and normal rats. Intestinal P-gp activity was also investigated by intestinal everted sac method, and hepatic P-gp was detected by FITC-labeled antibody and visualized using a confocal laser microscope system. Biliary clearance of doxorubicin in AA rats was significantly decreased from that in normal rats. The expression level of the hepatic P-gp in AA rats was very low compared to normal rats, indicating down-regulation. Intestinal exsorption clearance was not different between AA and normal rats. Permeability of doxorubicin across intestinal everted sac was comparable between AA and normal rats, corresponding to in vivo study. In AA rats, hepatic P-gp activity was decreased due to the reduction of expression level, but intestinal P-gp activity was not changed. Different regulation systems may be involved in liver and intestine. PMID:12113888

  8. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.

    PubMed Central

    Oude Elferink, R P; Ottenhoff, R; van Wijland, M; Smit, J J; Schinkel, A H; Groen, A K

    1995-01-01

    Disruption of the mdr2 gene in mice leads to a complete absence of phospholipid from bile (Smit, J. J. M., et al. 1993. Cell. 75:451-462). We have investigated the control of both mdr2 P-glycoprotein (Pgp) expression and bile salt secretion on biliary lipid secretion in the mouse. Lipid secretion was monitored at various bile salt output rates in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. In (-/-) mice, phospholipid secretion was negligible at all bile salt output rates. In (+/-) mice, a curvilinear relation between bile salt and phospholipid secretion was observed similar to that in (+/+) mice; however, at all bile salt secretion rates phospholipid secretion was reduced compared to (+/+) mice, indicating that mdr2 Pgp exerts a strong control over secretion. Infusion of increasing amounts of taurocholate up to maximal secretory rate led to a decline in the phospholipid and cholesterol secretion in both (+/+) and (+/-) mice in accordance to what has been observed in other species. In contrast, in (-/-) mice cholesterol secretion increased under these conditions while phospholipid output remained extremely low. The increased cholesterol secretion may represent extraction of cholesterol from the canalicular plasma membrane by taurocholate micelles as opposed to the concomitant secretion of both phospholipid and cholesterol in the presence of a functional mdr2 Pgp. Increased bile flow in (-/-) mice could be attributed completely to an increase in the bile salt-independent fraction and may therefore be caused by the bile duct proliferation in these mice. Images PMID:7814632

  9. Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii*

    PubMed Central

    Bottova, Iveta; Hehl, Adrian B.; Štefanić, Saša; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-01-01

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  10. Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii.

    PubMed

    Bottova, Iveta; Hehl, Adrian B; Stefanić, Sasa; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-06-26

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  11. Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

    PubMed

    Rosenberg, M F; Velarde, G; Ford, R C; Martin, C; Berridge, G; Kerr, I D; Callaghan, R; Schmidlin, A; Wooding, C; Linton, K J; Higgins, C F

    2001-10-15

    P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested. PMID:11598005

  12. Interaction of P-glycoprotein with anti-tumor drugs: the site, gate and pathway.

    PubMed

    Zhang, Junqiao; Li, Debing; Sun, Tianyang; Liang, Lijun; Wang, Qi

    2015-09-01

    Understanding the mechanism and pathway of anti-cancer drugs to be pumped out by P-glycoprotein (P-gp) in cancer cell is very important for the successful chemotherapy. P-gp is a member of ATP-binding cassette (ABC) transporters. In this study, random accelerated molecular dynamics (RAMD) simulation was used to explore the potential egress pathway of ligands from the binding pocket. This could be considered as a reverse process of drug binding. The most possible portal of drugs to dissociate is TM4/TM6, which is almost the same for different drugs, such as paclitaxel and doxorubicin. The interactions in the binding site are found to be remarkably stronger than that outside of the binding site. The results were suggested by the free energy calculation between P-gp and different drugs from metadynamics simulation. All the results indicate that the flexibility of inner residues, especially the residue Phe339, is very important for the drugs to access the binding site. PMID:26205623

  13. Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

    PubMed

    Pawagi, A B; Wang, J; Silverman, M; Reithmeier, R A; Deber, C M

    1994-01-14

    Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization. PMID:7904655

  14. Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2

    PubMed Central

    LI, QIONGYU; WANG, XIANGFENG; SHEN, ALING; ZHANG, YUCHEN; CHEN, YOUQIN; SFERRA, THOMAS J.; LIN, JIUMAO; PENG, JUN

    2015-01-01

    Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC. PMID:26640560

  15. Inducibility of the P-glycoprotein transport activity in the marine mussel Mytilus galloprovincialis and the freshwater mussel Dreissena polymorpha.

    PubMed

    Smital, Tvrtko; Sauerborn, Roberta; Hackenberger, Branimir K

    2003-12-10

    Previous investigations directed to the determination of the P-glycoprotein (Pgp) expression in aquatic organisms have indicated the possibility of the multixenobiotic resistance mechanism (MXR) induction as a response to organic pollution. However, in numerous cases no significant and/or no clear relationship between Pgp contents and pollution level was detected. Concerning these discrepancies the results of an extensive, 3-year study of the Pgp mediated MXR induction in the selected freshwater (Dreissena polymorpha) and marine (Mytilus galloprovincialis) bivalves are presented here. The main goals of the study were to ascertain the rate-dynamic, level, as well as the possible usability of MXR in environmental biomonitoring. Since the primary result of MXR induction should be the decreased intracellular accumulation of xenobiotics, the determination of MXR induction was performed using the measurement of Pgp transport activity. We measured the accumulation or the efflux rate of the model Pgp substrate rhodamine B (RB) in gills of the mussels previously exposed to pollution. The study was performed in several steps: from the exposure experiments in laboratory, using model inducers rhodamine 123 (R123) and water extract of Diesel-2 oil (D2), to the final in situ testing in real environmental conditions. Our results confirmed that Pgp activity is induced/induces according to the level of pollution, and that 4-days period was already long enough for the significant induction and deinduction of MXR activity. However, the inducibility of Pgp transport activity was significantly limited--the maximal level of induction obtained in this study resulted in 50-60% lower RB accumulation in the gills of induced specimens (laboratory or in situ exposed to pollution), when compared to control, non-induced animals. The obtained level of Pgp related MXR induction, resulting in halfway lesser accumulation of a model pollutant (RB), extrapolated to the similar scenario with toxic

  16. Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).

    PubMed

    Chufan, Eduardo E; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T; Durell, Stewart R; Ambudkar, Suresh V

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each

  17. QSAR studies of macrocyclic diterpenes with P-glycoprotein inhibitory activity.

    PubMed

    Sousa, Inês J; Ferreira, Maria-José U; Molnár, Joseph; Fernandes, Miguel X

    2013-02-14

    Multidrug resistance (MDR) represents a major limitation for cancer chemotherapy. There are several mechanisms of MDR but the most important is associated with P-glycoprotein (P-gp) overexpression. The development of modulators of P-gp that are able to re-establish drug sensitivity of resistant cells has been considered a promising approach for overcoming MDR. Macrocyclic lathyrane and jatrophane-type diterpenes from Euphorbia species were found to be strong MDR reversing agents. In this study we applied quantitative structure-activity relationship (QSAR) methodology in order to identify the most relevant molecular features of macrocyclic diterpenes with P-gp inhibitory activity and to determine which structural modifications can be performed to improve their activity. Using experimental biological data at two concentrations (4 and 40 μg/ml), we developed a QSAR model for a set of 51 bioactive diterpenic compounds which includes lathyrane and jatrophane-type diterpenes and another model just for jatrophanes. The cross-validation correlation values for all diterpenes QSAR models developed for biological activities at compound concentrations of 4 and 40 μg/ml were 0.758 and 0.729, respectively. Regarding the prediction ability, we get R²(pred) values of 0.765 and 0.534 for biological activities at compound concentrations of 4 and 40 μg/ml, respectively. Applying the cross-validation test to jatrophanes QSAR models, we obtained 0.680 and 0.787 for biological activities at compound concentrations of 4 and 40 μg/ml concentrations, respectively. For the same concentrations, the obtained R²(pred) values for jatrophanes models were 0.541 and 0.534, respectively. The obtained models were statistically valid and showed high prediction ability. PMID:23228414

  18. P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway

    PubMed Central

    Galski, Hanan; Oved-Gelber, Tamar; Simanovsky, Masha; Lazarovici, Philip; Gottesman, Michael M.; Nagler, Arnon

    2014-01-01

    The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. PMID:23774624

  19. P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway.

    PubMed

    Galski, Hanan; Oved-Gelber, Tamar; Simanovsky, Masha; Lazarovici, Philip; Gottesman, Michael M; Nagler, Arnon

    2013-09-01

    The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. PMID:23774624

  20. Modulation of P-glycoprotein function and multidrug resistance in cancer cells by Thai plant extracts.

    PubMed

    Takano, M; Kakizoe, S; Kawami, M; Nagai, J; Patanasethnont, D; Sripanidkulchai, B; Yumoto, R

    2014-11-01

    The effects of ethanol extracts from Thai plants belonging to the families of Annonaceae, Rutaceae, and Zingiberaceae on P-glycoprotein (P-gp) function and multidrug resistance were examined in paclitaxel-resistant HepG2 (PR-HepG2) cells. All the extracts tested, significantly increased the accumulation of [3H]paclitaxel, a P-gp substrate, in the cells. Among nine extracts, Z01 and Z02, extracts from Curcuma comosa and Kaempferia marginata (Zingiberaceae family), respectively, potently increased the accumulation. In addition, Z01 and Z02 increased the accumulation of other P-gp substrates, rhodamine 123 and doxorubicin, in PR-HepG2 cells in a concentration-dependent manner. Increased accumulation of rhodamine 123 and doxorubicin by Z01 and Z02 was also confirmed by confocal laser scanning microscopy. The effect of Z01 and Z02 pretreatment on the expression of MDR1 mRNA was also examined. The expression of MDR1 mRNA was not affected by the treatment of PR-HepG2 cells with these extracts for 48 hours. Cytotoxicity of paclitaxel was examined by XTT and protein assays in the absence and presence of Z02. Z02 potentiated the cytotoxicity of paclitaxel in PR-HepG2 cells. These results suggest that Curcuma comosa and Kaempferia marginata belonging to Zingiberaceae are useful sources to search for new P-gp modulator(s) that can be used to overcome multidrug resistance of cancer cells. PMID:25985578

  1. P-glycoprotein expression and localization in the rat uterus throughout gestation and labor.

    PubMed

    Huang, Qi-Tao; Shynlova, Oksana; Kibschull, Mark; Zhong, Mei; Yu, Yan-Hong; Matthews, Stephen G; Lye, Stephen J

    2016-09-01

    Uterine tissues contain the efflux transporter P-glycoprotein (P-gp, encoded by Abcb1a/1b gene), but little is known about how it changes through gestation. Our aim was to investigate the expression profile and cellular localization of P-gp in the pregnant, laboring and post-partum (PP) rat uterus. We propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial Abcb1a/1b/P-gp. Samples from bilaterally and unilaterally pregnant rats were collected throughout gestation, during labor, and PP (n=4-6/gestational day). RNA and protein were isolated and subjected to quantitative PCR and immunoblotting; P-gp transcript and protein were localized by in situ hybridization and immunohistochemistry. Expression of Abcb1a/1b gene and membrane P-gp protein in uterine tissue (1) increased throughout gestation, peaked at term (GD19-21) and dropped during labor (GD23L); and (2) was upregulated only in gravid but not in empty horn of unilaterally pregnant rats. (3) The drop of Abcb1a/1b mRNA on GD23 was prevented by artificial maintenance of elevated progesterone (P4) levels in late gestation; (4) injection of the P4 receptor antagonist RU486 on GD19 caused a significant decrease in Abcb1 mRNA levels. (5) In situ hybridization and immunohistochemistry indicated that Abcb1/P-gp is absent from myometrium throughout gestation; (6) was expressed exclusively by uterine microvascular endothelium (at early gestation) and luminal epithelium (at mid and late gestation), but was undetectable during labor. In conclusion, ABC transporter protein P-gp in pregnant uterus is hormonally and mechanically regulated. However, its substrate(s) and precise function in these tissues during pregnancy remains to be determined. PMID:27335130

  2. Increased P-glycoprotein messenger RNA stability in rat liver tumors in vivo.

    PubMed

    Lee, C H; Bradley, G; Ling, V

    1998-10-01

    P-glycoproteins (Pgp) are comprised of a small family of plasma membrane proteins whose abundance in cultured cells is often associated with the multidrug resistance phenotype. Overexpression of Pgp has been observed in many types of human cancers, but the molecular basis for this overexpression has not been established. We have used primary monolayer cultures of adult rat hepatocytes and a stepwise model of rat liver carcinogenesis to study the regulation of Pgp gene expression. We observed a marked overexpression of Pgp, specifically the class II Pgp, in both systems. In addition, we observed that a number of unrelated genes including alpha-tubulin, beta-actin, gamma-actin, cytokeratin 8, cytokeratin 18, and c-myc are overexpressed in cultured hepatocytes, and they are also overexpressed during liver carcinogenesis and in transplantable tumors. Nuclear run-on assays showed no increase in the transcriptional activity of Pgp genes in transplantable liver tumors compared to normal liver. Studies of in vivo mRNA stability, however, revealed that all three Pgp mRNAs were relatively stable in transplantable liver tumors (t(1/2) > 12 h), in contrast to what was found in normal liver (t(1/2) < 2 h). In addition, mRNA for several other genes, including alpha-tubulin, c-myc, and cyclin D1, all appear to be stabilized in the tumors. These findings suggest that the overexpression of Pgp genes in rat liver tumors may be the result of a mechanism involving stabilization of a diverse group of mRNAs. PMID:9731740

  3. Lipopolysaccharide increases cell surface P-glycoprotein that exhibits diminished activity in intestinal epithelial cells.

    PubMed

    Mishra, Jayshree; Zhang, Qiuye; Rosson, Jessica L; Moran, John; Dopp, John M; Neudeck, Brien L

    2008-10-01

    Increasingly, it is recognized that commensal microflora regulate epithelial cell processes through the dynamic interaction of pathogen-associated molecular patterns and host pattern recognition receptors such as Toll-like receptor 4 (TLR4). We therefore investigated the effects of bacterial lipopolysaccharide (LPS) on intestinal P-glycoprotein (P-gp) expression and function. Human SW480 (P-gp+/TLR4+) and Caco-2 (P-gp+/TLR4-) cells were treated with medium control or LPS (100 ng/ml) for 24 h prior to study. P-gp function was assessed by measuring the intracellular concentration of rhodamine 123 (Rh123). To confirm P-gp-specific effects, breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 2 (MRP-2/ABCC2) were also analyzed. Treatment of SW480 cells with LPS led to diminished P-gp activity, which could be prevented with polymyxin B (control: 207+/-16 versus LPS: 402+/-22 versus LPS+polymyxin B: 238+/-26 pmoles Rh123/mg protein, p<0.05 control versus LPS). These effects could be blocked by using polymyxin B and were not seen in the P-gp+/TLR4--Caco-2 cell line (control: 771+/-28 versus LPS: 775+/-59 pmoles Rh123/mg protein). Total cellular levels of P-gp did not change in LPS-treated SW480 cells; however, a significant increase in cell surface P-gp was detected. No change in activity, total protein, or apically located MRP-2 was detected following LPS treatment. Sequence analysis confirmed wild-type status of SW480 cells. These data suggest that activation of TLR4 in intestinal epithelial cells leads to an increase in plasma membrane P-gp that demonstrates a diminished capacity to transport substrate. PMID:18687802

  4. Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes.

    PubMed

    Horger, Kim S; Liu, Haiyan; Rao, Divya K; Shukla, Suneet; Sept, David; Ambudkar, Suresh V; Mayer, Michael

    2015-02-01

    This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (Ps) of passive diffusion and rate constants of active transport (kT) by P-gp as a result of different experimental conditions. The Ps value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the kT value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in kT values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels. PMID:25450342

  5. Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes

    PubMed Central

    Horger, Kim S.; Liu, Haiyan; Rao, Divya K.; Shukla, Suneet; Sept, David; Ambudkar, Suresh V.; Mayer, Michael

    2015-01-01

    This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (Ps) of passive diffusion and rate constants of active transport (kT) by P-gp as a result of different experimental conditions. The Ps value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the kT value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in kT values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels. PMID:25450342

  6. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    PubMed

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids. PMID:26944019

  7. What kinds of substrates show P-glycoprotein-dependent intestinal absorption? Comparison of verapamil with vinblastine.

    PubMed

    Ogihara, Takuo; Kamiya, Masatsugu; Ozawa, Makoto; Fujita, Takuya; Yamamoto, Akira; Yamashita, Shinji; Ohnishi, Shuhei; Isomura, Yasuo

    2006-06-01

    The influence of P-glycoprotein (P-gp) on intestinal absorption of drugs was investigated by comparison of the uptakes of two P-gp substrates, verapamil and vinblastine, using intestinal segments of wild-type and mdr1a/1b gene-deficient (mdr1a/1b(-/-)) mice, and Caco-2 cells. When [(3)H]vinblastine was injected into intestinal segments of wild-type mice, vinblastine was absorbed from duodenum and ileum, but not from jejunum. This difference among intestinal regions could not be explained by segmental differences of mdr1a mRNA expression. In Caco-2 cells, it was found that vinblastine had a high value of efflux/influx ratio (an index of affinity for P-gp) of 12.1, and a low permeability of less than 1 x 10(-6) cm/sec. The corresponding values for verapamil were 4.9 and 10.6 x 10(-6) cm/sec, respectively. After oral administration of [(3)H]vinblastine to mice, the maximum concentration (C(max)) and the area under the plasma concentration time-curve from time 0 to 24 hr (AUC(0-24 hr)) for mdr1a/1b(-/-) mice were 1.5 times greater than those for wild-type mice, while these parameters were not significantly different between the two strains in the case of [(3)H]verapamil. Therefore, P-gp substrates may be classified into at least two types, i.e., verapamil-type, for which the intestinal absorption is unaffected by P-gp, and vinblastine-type, for which the intestinal absorption is influenced by P-gp. Vinblastine-type P-gp substrates, with low permeability and high affinity for P-gp, would be unfavorable candidates for oral drugs. PMID:16858128

  8. The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2015-01-01

    P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp. PMID:25987565

  9. Protein contacts and ligand binding in the inward-facing model of human P-glycoprotein.

    PubMed

    Pajeva, Ilza K; Hanl, Markus; Wiese, Michael

    2013-05-01

    The primary aim of this work was to analyze the contacts between residues in the nucleotide binding domains (NBDs) and at the interface between the transmembrane domains (TMDs) and the NBDs in the inward-open homology model of human P-glycoprotein (P-gp). The analysis revealed communication nets through hydrogen bonding in the NBD and at the NBD-TMD interface of each half involving residues from the adenosine triphosphate (ATP) motifs and the coupling helices of the intracellular loops. Similar networks have been identified in P-gp conformations generated by molecular dynamics simulation. Differences have been recorded in the networking between both halves of P-gp. Many of the residue contacts have also been observed in the X-ray crystal structures of other ATP binding cassette (ABC) transporters, which confirms their validity. Next, possible binding pockets involving residues of importance for the TMD-NBD communication were identified. By studying these pockets, binding sites were suggested for rhodamine 123 (R-site) and prazosin (regulatory site) at the NBD-TMD interface that agreed with the experimental data on their location. Additionally, one more R-site in the protein cavity was proposed, in accordance with the available biochemical data. Together with the previously suggested Hoechst 33342 site (H-site), all sites were interpreted with respect to their effects on the protein ATPase activity, in correspondence with the experimental observations. Several residues involved in key contacts in the P-gp NBDs were proposed for further targeted mutagenesis experiments. PMID:23564544

  10. Effect of the MDR1 C3435T variant and P-glycoprotein induction on dicloxacillin pharmacokinetics.

    PubMed

    Putnam, Wendy S; Woo, Jonathan M; Huang, Yong; Benet, Leslie Z

    2005-04-01

    This study investigated 2 hypotheses about genotype-phenotype relationships for the efflux transporter, P-glycoprotein: (1) the presence of a synonymous C3435T variant in exon 26 of the MDR1 gene correlates to higher plasma concentrations of a P-glycoprotein substrate, dicloxacillin, and (2) the effects of genotypic differences decrease under conditions of P-glycoprotein induction by rifampin. Eighteen healthy volunteers received two 1-g doses of dicloxacillin, one on the 1st study day and the other on the 11th day of rifampin dosing (600 mg daily). Dicloxacillin and its 5-hydroxymethyl metabolite were analyzed using liquid chromatography/tandem mass spectrometry. Mean dicloxacillin C(max) measurements were 30.5 +/- 13.5, 33.3 +/- 4.7, and 31.1 +/- 12.8 mug/mL in individuals with the CC, CT, and TT genotype at position 3435 in exon 26 of the MDR1 gene. Following rifampin dosing, the mean dicloxacillin C(max) across genotypes decreased from 31.4 +/- 10.8 to 22.9 +/- 7.0 microg/mL (P < .05), whereas the mean oral clearance increased from 235 +/- 82 to 297 +/- 71 mL/min (P < .001), and the mean absorption time increased from 0.71 +/- 0.55 to 1.34 +/- 0.77 h (P < .05). Rifampin treatment increased the formation clearance, C(max), and AUC of the 5-hydroxymethyl metabolite by 135%, 119%, and 59%, respectively. The C3435T variant had no effect on dicloxacillin pharmacokinetics. The data suggested that rifampin induced intestinal P-glycoprotein and increased dicloxacillin metabolism. PMID:15778422

  11. Brain sites mediating corticosteroid feedback inhibition of stimulated ACTH secretion

    SciTech Connect

    Jacobson, L.

    1989-01-01

    There is substantial evidence that the brain mediates stress-induced and circadian increases in ACTH secretion and that corticosteroid concentrations which normalize basal plasma ACTH are insufficient to normalize ACTH responses to circadian or stressful stimuli in adrenalectomized rats. To identify brain sites mediating corticosteroid inhibition of stimulated ACTH secretion, two approaches were used. The first compared brain ({sup 14}C)-2-deoxyglucose uptake in rats with differential ACTH responses to stress. Relative to sham-adrenalectomized (SHAM) rats, adrenalectomized rats replaced with low, constant corticosterone levels via a subcutaneous corticosterone pellet (B-PELLET) exhibited elevated and prolonged ACTH responses to a variety of stimuli. Adrenalectomized rate given a circadian corticosterone rhythm via corticosterone in their drinking water exhibited elevated ACTH levels immediately after stress, but unlike B-PELLET rats, terminated stress induced ACTH secretion normally relative to SHAMS. Therefore, the abnormal ACTH responses to stress in B-PELLET rats were due to the lack of both circadian variations and stress-induced increases in corticosterone. Hypoxia was selected as a standardized stimulus for correlating brain ({sup 14}C)-2-deoxyglucose uptake with ACTH secretion. In intact rats, increases in plasma ACTH and decreases in arterial PO{sub 2} correlated with the severity of hypoxia at arterial PCO{sub 2} below 60 mm Hg. Hypoxia PELLET vs. SHAM rats. However, in preliminary experiments, although hypoxia increased brain 2-deoxyglucose uptake in most brain regions, plasma ACTH correlated poorly with 2-deoxyglucose uptake at 12% and 10% O{sub 2}.

  12. Pore-exposed tyrosine residues of P-glycoprotein are important hydrogen-bonding partners for drugs.

    PubMed

    Dönmez Cakil, Yaprak; Khunweeraphong, Narakorn; Parveen, Zahida; Schmid, Diethart; Artaker, Matthias; Ecker, Gerhard F; Sitte, Harald H; Pusch, Oliver; Stockner, Thomas; Chiba, Peter

    2014-03-01

    The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein. PMID:24366667

  13. Decreased activity of hepatic P-glycoprotein in the isolated perfused liver of the adjuvant arthritis rat.

    PubMed

    Achira, M; Totsuka, R; Kume, T

    2002-11-01

    1. We investigated the hepatobiliary transport of doxorubicin in the isolated perfused liver prepared from the adjuvant arthritis rat, an animal model for rheumatoid arthritis, to examine the hepatic P-glycoprotein activity in the adjuvant arthritis rat. 2. Liver was isolated from the normal and the adjuvant arthritis rat and perfused for 60 min with recirculating buffer and the perfusate and bile samples were collected at timed interval. 3. The elimination of doxorubicin in the adjuvant arthritis rat tended to be reduced, but it was not significantly different from the normal rat. Biliary clearance (CL(bile)) in the normal rat was 1.93 +/- 0.48 ml min(-1), whereas, CL(bile) in the adjuvant arthritis rat was significantly decreased to 0.40 +/- 0.13 ml min(-1). 4. CL(bile) was markedly decreased to about 0.15 ml min(-1) in the presence of 100 microM verapamil in both types of rat. Methotrexate treatment had no effect on CL(bile) in both the normal and adjuvant arthritis rat (2.18 +/- 0.22 and 0.47 +/- 0.22 ml min(-1), respectively). 5. The results suggest that the hepatic P-glycoprotein activity was markedly decreased in the adjuvant arthritis rat and the effect of methotrexate on the hepatic P-glycoprotein activity did not corresponded to its anti-inflammatory effect. PMID:12487726

  14. Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis.

    PubMed

    Wang, Changhe; Wang, Yeshi; Hu, Meiqin; Chai, Zuying; Wu, Qihui; Huang, Rong; Han, Weiping; Zhang, Claire Xi; Zhou, Zhuan

    2016-01-01

    Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca(2+)-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural-functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis. PMID:26589353

  15. Neuroprotection mediated by inhibition of calpain during acute viral encephalitis

    PubMed Central

    Howe, Charles L.; LaFrance-Corey, Reghann G.; Mirchia, Kanish; Sauer, Brian M.; McGovern, Renee M.; Reid, Joel M.; Buenz, Eric J.

    2016-01-01

    Neurologic complications associated with viral encephalitis, including seizures and cognitive impairment, are a global health issue, especially in children. We previously showed that hippocampal injury during acute picornavirus infection in mice is associated with calpain activation and is the result of neuronal death triggered by brain-infiltrating inflammatory monocytes. We therefore hypothesized that treatment with a calpain inhibitor would protect neurons from immune-mediated bystander injury. C57BL/6J mice infected with the Daniel’s strain of Theiler’s murine encephalomyelitis virus were treated with the FDA-approved drug ritonavir using a dosing regimen that resulted in plasma concentrations within the therapeutic range for calpain inhibition. Ritonavir treatment significantly reduced calpain activity in the hippocampus, protected hippocampal neurons from death, preserved cognitive performance, and suppressed seizure escalation, even when therapy was initiated 36 hours after disease onset. Calpain inhibition by ritonavir may be a powerful tool for preserving neurons and cognitive function and preventing neural circuit dysregulation in humans with neuroinflammatory disorders. PMID:27345730

  16. Reversion of resistance to immunosuppressive agents in three patients with psoriatic arthritis by cyclosporine A: modulation of P-glycoprotein function.

    PubMed

    Diamanti, Andrea Picchianti; Rosado, Manuela; Germano, Valentina; Scarsella, Marco; Giorda, Ezio; Podestà, Edoardo; D'Amelio, Raffaele; Carsetti, Rita; Laganà, Bruno

    2011-01-01

    Secondary resistance may be a major problem in the management of autoimmune diseases. P-glycoprotein (P-gp) over-function has been described as a mechanism of drug resistance in autoimmune patients. P-gp function can in vitro be inhibited by cyclosporine A (CSA) and verapamil; moreover, P-gp reduction by CSA in systemic lupus erythematosus and rheumatoid arthritis has been demonstrated. Here, P-gp function before and after CSA administration in three psoriatic arthritis (PsA) patients, who developed a resistance to MTX/SSA, has been evaluated. P-gp function on patient cells was analyzed by measuring the changes in rhodamine-123 (Rh-123) fluorescence after verapamil incubation. CSA treatment resulted in good clinical outcome that was related with a significant P-gp function reduction at CD3+ and CD8+ levels. In addition to its immunosuppressive activity, CSA results may also be related to MTX/SSA effect restoration through P-gp inhibition. This is the first time that CSA has been demonstrated as being able to revert MTX/SSA resistance in PsA. PMID:21062675

  17. Chronic cyclosporin A nephrotoxicity, P-glycoprotein overexpression, and relationships with intrarenal angiotensin II deposits.

    PubMed Central

    del Moral, R. G.; Andujar, M.; Ramírez, C.; Gómez-Morales, M.; Masseroli, M.; Aguilar, M.; Olmo, A.; Arrebola, F.; Guillén, M.; García-Chicano, M. J.; Nogales, F. F.; O'Valle, F.

    1997-01-01

    P-glycoprotein (P-gp) expels hydrophobic substances from the cell, including chemotherapeutic agents and immunosuppressants such as cyclosporin A (CsA) and FK506. Exposure of cultured renal tubular cells to CsA induces P-gp overexpression in cell membranes. Angiotensin II has recently been implicated as the principal factor responsible for progression of interstitial fibrosis induced by CsA. To investigate the in vivo relationships between histological lesions, P-gp overexpression, and intrarenal angiotensin II deposits, we developed a model of chronic CsA toxicity in Sprague-Dawley rats treated with 25 mg/kg/day CsA for 28 and 56 days and fed either a standard maintenance diet or a low-salt diet. Immunohistochemical methods were used to study the expression of P-gp in renal tubular cells and the appearance of intrarenal angiotensin II deposits. Rats treated with CsA developed chronic nephrotoxicity lesions that were more evident in the group fed the low-salt diet. Treatment with CsA induced overexpression of P-gp in tubular cells of the kidney that increased with time. We found that immunohistochemical expression of P-gp was slightly more severe in rats fed a low-salt diet. Intrarenal deposits of angiotensin II were more evident in rats treated with CsA; these deposits also increased with time. This finding was also more relevant in rats given the low-salt diet. The up-regulation of P-gp was inversely related to the incidence of hyaline arteriopathy (r = -0.65; P < 0.05), periglomerular (r = -0.58; P < 0.05) and peritubular fibrosis (r = -0.63; P < 0.05), and intrarenal angiotensin H deposits in animals with severe signs of nephrotoxicity (r = -0.65; P < 0.05). These results support the hypothesis that the role of P-gp as a detoxicant in renal cells may be related to mechanisms that control the cytoplasmic removal of both toxic metabolites from CsA and those originating from the catabolism of signal transduction proteins (methylcysteine esters), which are produced

  18. Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion.

    PubMed

    Sato, Makoto; Yasugi, Tetsuo; Minami, Yoshiaki; Miura, Takashi; Nagayama, Masaharu

    2016-08-30

    Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or "proneural wave" accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation. PMID:27535937

  19. Regulation of P-glycoprotein expression in brain capillaries in Huntington's disease and its impact on brain availability of antipsychotic agents risperidone and paliperidone.

    PubMed

    Kao, Yu-Han; Chern, Yijuang; Yang, Hui-Ting; Chen, Hui-Mei; Lin, Chun-Jung

    2016-08-01

    Huntington's disease (HD) is a neurodegenerative disease marked by an expanded polyglutamine (polyQ) tract on the huntingtin (HTT) protein that may cause transcriptional dysfunction. This study aimed to investigate the regulation and function of P-glycoprotein, an important efflux transporter, in brain capillaries in HD. The results showed that, compared with the littermate controls, R6/2 HD transgenic mice with the human mutant HTT gene had higher levels of P-glycoprotein mRNA and protein and enhanced NF-κB activity in their brain capillaries. Higher P-glycoprotein expression was also observed in the brain capillaries of human HD patients. Consistent with this enhanced P-glycoprotein expression, brain extracellular levels and brain-to-plasma ratios of the antipsychotic agents risperidone and paliperidone were significantly lower in R6/2 mice than in their littermate controls. Exogenous expression of human mutant HTT protein with expanded polyQ (mHTT-109Q) in HEK293T cells enhanced the levels of P-glycoprotein transcripts and NF-κB activity compared with cells expressing normal HTT-25Q. Treatment with the IKK inhibitor, BMS-345541, decreased P-glycoprotein mRNA level in cells transfected with mHTT-109Q or normal HTT-25Q In conclusion, mutant HTT altered the expression of P-glycoprotein through the NF-κB pathway in brain capillaries in HD and markedly affected the availability of P-glycoprotein substrates in the brain. PMID:26661162

  20. Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185 yields Val-185 substitution in P-glycoprotein

    SciTech Connect

    Safa, A.R.; Stern, R.K.; Choi, Kyunghee; Agresti, M.; Tamai, Ikumi; Mehta, N.D.; Roninson, I.B. )

    1990-09-01

    Expression of P-glycoprotein, encoded by the human MDR1 gene, results in cross-resistance to many lipophilic cytotoxic drugs (multidrug resistance). P-glycoprotein is believed to function as an energy-dependent efflux pump that is responsible for decreased drug accumulation in multidrug-resistant cells. Previous work showed that preferential resistance to colchicine in a colchicine-selected multidrug-resistant cell line was caused by spontaneous mutations in the MDR1 gene that resulted in a Gly-185 {yields} Val-185 substitution in P-glycoprotein. The authors have now compared transfectant cell lines expressing the wild-type Gly-185 or the mutant Val-185 P-glycoprotein with regard to their levels of resistance to and accumulation and binding of different drugs. In cells expressing the mutant protein, increased resistance to colchicine and decreased resistance to vinblastine correlated with a decreased accumulation of colchicine and increased accumulation of vinblastine. Expression of the mutant P-glycoprotein also resulted in significantly increased resistance to epipodophyllotoxin and decreased resistance to vincristine and actinomycin D; smaller changes in resistance were observed for several other drugs. Unexpectedly, the mutant P-glycoprotein showed increased binding of photoactive analogs of vinblastine and verapamil and the photoactive compound azidopine and decreased binding of a photoactive colchicine analog. These results suggest that the Gly-185 {yields} Val-185 substitution affects not the initial drug-binding site of P-glycoprotein but another site, associated with the release of P-glycoprotein-bound drugs to the outside of the cell.

  1. Human Medial Frontal Cortex Mediates Unconscious Inhibition of Voluntary Action

    PubMed Central

    Sumner, Petroc; Nachev, Parashkev; Morris, Peter; Peters, Andrew M.; Jackson, Stephen R.; Kennard, Christopher; Husain, Masud

    2007-01-01

    Summary Within the medial frontal cortex, the supplementary eye field (SEF), supplementary motor area (SMA), and pre-SMA have been implicated in the control of voluntary action, especially during motor sequences or tasks involving rapid choices between competing response plans. However, the precise roles of these areas remain controversial. Here, we study two extremely rare patients with microlesions of the SEF and SMA to demonstrate that these areas are critically involved in unconscious and involuntary motor control. We employed masked-prime stimuli that evoked automatic inhibition in healthy people and control patients with lateral premotor or pre-SMA damage. In contrast, our SEF/SMA patients showed a complete reversal of the normal inhibitory effect—ocular or manual—corresponding to the functional subregion lesioned. These findings imply that the SEF and SMA mediate automatic effector-specific suppression of motor plans. This automatic mechanism may contribute to the participation of these areas in the voluntary control of action. PMID:17553420

  2. Molecular mechanism of statin-mediated LOX-1 inhibition.

    PubMed

    Biocca, Silvia; Iacovelli, Federico; Matarazzo, Sara; Vindigni, Giulia; Oteri, Francesco; Desideri, Alessandro; Falconi, Mattia

    2015-01-01

    Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins. PMID:25950192

  3. Glial GABA, synthesized by monoamine oxidase B, mediates tonic inhibition.

    PubMed

    Yoon, Bo-Eun; Woo, Junsung; Chun, Ye-Eun; Chun, Heejung; Jo, Seonmi; Bae, Jin Young; An, Heeyoung; Min, Joo Ok; Oh, Soo-Jin; Han, Kyung-Seok; Kim, Hye Yun; Kim, Taekeun; Kim, Young Soo; Bae, Yong Chul; Lee, C Justin

    2014-11-15

    GABA is the major inhibitory transmitter in the brain and is released not only from a subset of neurons but also from glia. Although neuronal GABA is well known to be synthesized by glutamic acid decarboxylase (GAD), the source of glial GABA is unknown. After estimating the concentration of GABA in Bergmann glia to be around 5-10 mM by immunogold electron microscopy, we demonstrate that GABA production in glia requires MAOB, a key enzyme in the putrescine degradation pathway. In cultured cerebellar glia, both Ca(2+)-induced and tonic GABA release are significantly reduced by both gene silencing of MAOB and the MAOB inhibitor selegiline. In the cerebellum and striatum of adult mice, general gene silencing, knock out of MAOB or selegiline treatment resulted in elimination of tonic GABA currents recorded from granule neurons and medium spiny neurons. Glial-specific rescue of MAOB resulted in complete rescue of tonic GABA currents. Our results identify MAOB as a key synthesizing enzyme of glial GABA, which is released via bestrophin 1 (Best1) channel to mediate tonic inhibition in the brain. PMID:25239459

  4. Molecular mechanism of statin-mediated LOX-1 inhibition

    PubMed Central

    Biocca, Silvia; Iacovelli, Federico; Matarazzo, Sara; Vindigni, Giulia; Oteri, Francesco; Desideri, Alessandro; Falconi, Mattia

    2015-01-01

    Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins. PMID:25950192

  5. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier

    PubMed Central

    Li, Yangfang; Wu, Qian; Li, Chen; Liu, Ling; Du, Kun; Shen, Jin; Wu, Yuqin; Zhao, Xiaofen; Zhao, Mei; Bao, Lingyun; Gao, Jin; Keep, Richard F.; Xiang, Jianming

    2016-01-01

    While the blood-brain barrier (BBB) protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC) transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP) but not those overexpressing human P-gp (MDCKII-MDR cells) had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated. PMID:27300692

  6. The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity.

    PubMed

    Szalóki, Gábor; Krasznai, Zoárd T; Tóth, Ágnes; Vízkeleti, Laura; Szöllősi, Attila G; Trencsényi, György; Lajtos, Imre; Juhász, István; Krasznai, Zoltán; Márián, Teréz; Balázs, Margit; Szabó, Gábor; Goda, Katalin

    2014-01-01

    P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC). PMID:25238617

  7. Multidrug resistance after retroviral transfer of the human MDR1 gene correlates with P-glycoprotein density in the plasma membrane and is not affected by cytotoxic selection.

    PubMed Central

    Choi, K; Frommel, T O; Stern, R K; Perez, C F; Kriegler, M; Tsuruo, T; Roninson, I B

    1991-01-01

    Multidrug resistance (MDR) in mammalian cells is associated with the expression of the MDR1 gene encoding P-glycoprotein (P-gp), an and active efflux pump for various lipophilic compounds. MDR transfectants can be isolated after MDR1 gene transfer and selection with cytotoxic drugs; low levels of drug resistance have also been observed in unselected NIH 3T3 mouse cells after retrovirus-mediated transfer of mouse mdr1 cDNA. MDR cell lines possess multiple phenotypic changes, suggesting that P-gp function could be complemented by some additional mechanisms associated with cytotoxic selection. To determine whether cytotoxic selection contributes to the MDR phenotype of MDR1-expressing cells, NIH 3T3 cells infected with a recombinant retrovirus carrying the human MDR1 gene were selected by two different procedures: (i) noncytotoxic selection for increased P-gp expression on the cell surface by multiple rounds of immunofluorescence labeling and flow sorting or (ii) one or more steps of selection with a cytotoxic drug. The levels of MDR in both types of infectants showed an excellent correlation with the P-gp density in the plasma membrane, expressed as immunoreactivity with a P-gp-specific antibody normalized by reactivity with an antibody against an unrelated antigen. Cytotoxic selection conferred no additional increase in resistance relative to P-gp density. These results indicate that P-gp density in the plasma membrane may be sufficient to determine the level of MDR. Images PMID:1678523

  8. Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix.

    PubMed

    Sperandio, S; Poksay, K; de Belle, I; Lafuente, M J; Liu, B; Nasir, J; Bredesen, D E

    2004-10-01

    Programmed cell death (pcd) may take the form of apoptotic or nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here, we report that paraptosis, an alternative, nonapoptotic cell death program that may be induced by the insulin-like growth factor I receptor (among other inducers), is mediated by mitogen-activated protein kinases (MAPKs) and inhibited by AIP-1/Alix. The inhibition by AIP-1/Alix is specific for paraptosis since apoptosis was not inhibited. Caspases were not activated in this paradigm, nor were caspase inhibitors effective in blocking cell death. However, insulin-like growth factor I receptor (IGFIR)-induced paraptosis was inhibited by MEK-2-specific inhibitors and by antisense oligonucleotides directed against c-jun N-terminal kinase-1 (JNK-1). These results suggest that IGFIR-induced paraptosis is mediated by MAPKs, and inhibited by AIP-1/Alix. PMID:15195070

  9. Doxycycline as an inhibitor of p-glycoprotein in the alpaca for the purpose of maintaining avermectins in the CNS during treatment for parelaphostrongylosis.

    PubMed

    Agbedanu, Prince N; Anderson, Kristi L; Brewer, Matthew T; Carlson, Steve A

    2015-09-15

    Meningeal worms (Parelaphostrongylus tenuis) are a common malady of alpacas, often refractory to conventional treatments. Ivermectin is a very effective anthelmintic used against a variety of parasites but this drug is not consistently effective against alpaca meningeal worms once the parasite has gained access to the CNS, even if used in a protracted treatment protocol. Ivermectin is not effective against clinical cases of P. tenuis, raising the possibility that the drug is not sustained at therapeutic concentrations in the central nervous system (CNS). A specific protein (designated as p-glycoprotein (PGP)) effluxes ivermectin from the brain at the blood-brain barrier, thus hampering the maintenance of therapeutic concentrations of the drug in the CNS. Minocycline is a synthetic tetracycline antibiotic with an excellent safety profile in all animals tested to date. Minocycline has three unique characteristics that could be useful for treating meningeal worms in conjunction with ivermectin. First, minocycline is an inhibitor of PGP at the blood-brain barrier and this inhibition could maintain effective concentrations of ivermectin in the brain and meninges. Second, minocycline protects neurons in vivo through a number of different mechanisms and this neuroprotection could alleviate the potential untoward neurologic effects of meningeal worms. Third, minocycline is a highly lipid-soluble drug, thus facilitating efficient brain penetration. We thus hypothesized that minocycline will maintain ivermectin, or a related avermectin approved in ruminants (abamectin, doramectin, or eprinomectin), in the alpaca CNS. To test this hypothesis, we cloned the gene encoding the alpaca PGP, expressed the alpaca PGP in a heterologous expression system involving MDCK cells, and measured the ability of minocycline to inhibit the efflux of avermectins from the MDCK cells; doxycycline was used as a putative negative control (based on studies in other species). Our in vitro studies

  10. Artemisinin induces doxorubicin resistance in human colon cancer cells via calcium-dependent activation of HIF-1α and P-glycoprotein overexpression

    PubMed Central

    Riganti, C; Doublier, S; Viarisio, D; Miraglia, E; Pescarmona, G; Ghigo, D; Bosia, A

    2009-01-01

    Background and purpose: Artemisinin is an antimalarial drug exerting pleiotropic effects, such as the inhibition of the transcription factor nuclear factor-kappa B and of the sarcoplasmic/endoplasmic reticulum Ca++-ATPase (SERCA) of P. falciparum. As the sesquiterpene lactone thapsigargin, a known inhibitor of mammalian SERCA, enhances the expression of P-glycoprotein (Pgp) by increasing the intracellular Ca++ ([Ca++]i) level, we investigated whether artemisinin and its structural homologue parthenolide could inhibit SERCA in human colon carcinoma HT29 cells and induce a resistance to doxorubicin. Experimental approach: HT29 cells were incubated with artemisinin or parthenolide and assessed for SERCA activity, [Ca++]i levels, Pgp expression, doxorubicin accumulation and toxicity, and translocation of the hypoxia-inducible factor, HIF-1α. Key results: Artemisinin and parthenolide, like the specific SERCA inhibitors thapsigargin and cyclopiazonic acid, reduced the activity of SERCA. They also increased intracellular calcium concentration ([Ca++]i) and Pgp expression and decreased doxorubicin accumulation and cytotoxicity. The intracellular Ca++ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, and the inhibitor of calmodulin-dependent kinase II (CaMKII) KN93 prevented these effects. CaMKII is known to promote the phosphorylation and the activation of HIF-1α, which may induce Pgp. In HT29 cells, artemisinin and parthenolide induced the phosphorylation of HIF-1α, which was inhibited by KN93. Conclusions and implications: Our results suggest that artemisinin and parthenolide may act as SERCA inhibitors and, like other SERCA inhibitors, induce resistance to doxorubicin in human colon cancer cells, via the CaMKII-dependent activation of HIF-1α and the induction of Pgp. PMID:19298255

  11. Osthole shows the potential to overcome P-glycoprotein‑mediated multidrug resistance in human myelogenous leukemia K562/ADM cells by inhibiting the PI3K/Akt signaling pathway.

    PubMed

    Wang, Hong; Jia, Xiu-Hong; Chen, Jie-Ru; Wang, Jian-Yong; Li, You-Jie

    2016-06-01

    P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) has been reported to play a pivotal role in tumor chemotherapy failure. Study after study has illustrated that the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with P-gp expression in many human malignancies. In the present study, osthole, an O-methylated coumarin, exhibited potent reversal capability of MDR in myelogenous leukemia K562/ADM cells. Simultaneously, the uptake and efflux of Rhodamine-123 (Rh-123) and the accumulation of doxorubicin assays combined with flow cytometric analysis suggested that osthole could increase intracellular drug accumulation. Furthermore, osthole decreased the expression of multidrug resistance gene 1 (MDR1) at both the mRNA and protein levels. Further experiments elucidated that osthole could suppress P-gp expression by inhibiting the PI3K/Akt signaling pathway which might be the main mechanism accounting for the reversal potential of osthole in the MDR in K562/ADM cells. In conclusion, osthole combats MDR and could be a promising candidate for the development of novel MDR reversal modulators. PMID:27109742

  12. Inhibitory Effects of Highly Oxygenated Lanostane Derivatives from the Fungus Ganoderma lucidum on P-Glycoprotein and α-Glucosidase.

    PubMed

    Zhao, Xi-Run; Huo, Xiao-Kui; Dong, Pei-Pei; Wang, Chao; Huang, Shan-Shan; Zhang, Bao-Jing; Zhang, Hou-Li; Deng, Sa; Liu, Ke-Xin; Ma, Xiao-Chi

    2015-08-28

    Twelve new highly oxygenated lanostane triterpenoids and nine known ganoderic acids were isolated from the fruiting body of Ganoderma lucidum. The new compounds were lanostane nortriterpenoids with 27 carbons (1-5 and 8), lanostane nor-triterpenoids with 25 carbons (6 and 7), and lanostane triterpenoids (9-12) based on multiple spectroscopic data analysis, including HRESIMS, 1D-NMR, 2D-NMR, and CD. Compounds 1-5 were identified as rare nor-lanostanoids that contain a 17β-pentatomic lactone ring. Compound 13, possessing a lactone ring, had been isolated previously. The P-glycoprotein (P-gp) inhibitory effects of compounds 1-21 were evaluated at a concentration of 20 μM using an adriamycin (ADM)-resistant human breast adenocarcinoma cell line (MCF-7/ADR). Compounds 1, 5, 18, and 20 and verapamil increased the accumulation of ADM in MCF-7/ADR cells approximately 3-fold when compared with the negative control. These data support the significant P-glycoprotein inhibitory activities of compounds 1, 5, 18, and 20. In silico docking analysis suggested these compounds had similar P-gp recognition mechanisms compared with those of verapamil (a classical inhibitor). Furthermore, in an in vitro bioassay, compounds 2, 4, 5, 6, and 18 showed moderate inhibitory effects against α-glucosidase compared with those of the positive control acarbose. PMID:26222905

  13. Interaction of anthelmintic drugs with P-glycoprotein in recombinant LLC-PK1-mdr1a cells.

    PubMed

    Dupuy, Jacques; Alvinerie, Michel; Ménez, Cecile; Lespine, Anne

    2010-08-01

    Given the widespread use of formulations combining anthelmintics which are possible P-glycoprotein interfering agents, the understanding of drug interactions with efflux ABC transporters is of concern for improving anthelmintic control. We determined the ability of 14 anthelmintics from different classes to interact with abcb1a (mdr1a, P-glycoprotein, Pgp) by following the intracellular accumulation of rhodamine 123 (Rho 123), a fluorescent Pgp substrate, in LLC-PK1 cells overexpressing Pgp. The cytotoxicity of the compounds that are able to interfere with Pgp activity was evaluated in cells overexpressing Pgp and compared with parental cells using the MTS viability assay. Among all the anthelmintics used, ivermectin (IVM), triclabendazole (TCZ), triclabendazole sulfoxide (TCZ-SO), closantel (CLOS) and rafoxanide (RAF) increased the intracellular Rho 123 in Pgp overexpressing cells, while triclabendazole sulfone, albendazole, mebendazole, oxfendazole, thiabendazole, nitroxynil, levamisole, praziquantel and clorsulon failed to have any effect. The concentration needed to reach the maximal Rho 123 accumulation (E(max)) was obtained with 10 microM for IVM, 80 microM for CLOS, 40 microM for TCZ and TCZ-SO, and 80 microM for RAF. We showed that for these five drugs parental cell line was more sensitive to drug toxicity compared with Pgp recombinant cell line. Such in vitro approach constitutes a powerful tool to predict Pgp-drug interactions when formulations combining several anthelmintics are administered and may contribute to the required optimization of efficacy of anthelmintics. PMID:20513441

  14. Synergistic effect of folate-mediated targeting and verapamil-mediated P-gp inhibition with paclitaxel -polymer micelles to overcome multi-drug resistance.

    PubMed

    Wang, Feihu; Zhang, Dianrui; Zhang, Qiang; Chen, Yuxuan; Zheng, Dandan; Hao, Leilei; Duan, Cunxian; Jia, Lejiao; Liu, Guangpu; Liu, Yue

    2011-12-01

    Multidrug resistance (MDR) in tumor cells is a significant obstacle for successful cancer chemotherapy. Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) is a key factor contributing to the development of tumor drug resistance. Verapamil (VRP), a P-gp inhibitor, has been reported to be able to reverse completely the resistance caused by P-gp. For optimal synergy, the drug and inhibitor combination may need to be temporally colocalized in the tumor cells. Herein, we investigated the effectiveness of simultaneous and targeted delivery of anticancer drug, paclitaxel (PTX), along with VRP, using DOMC-FA micelles to overcome tumor drug resistance. The floate-functionalized dual agent loaded micelles resulted in the similar cytotoxicity to PTX-loaded micelles/free VRP combination and co-administration of two single-agent loaded micelles, which was higher than that of PTX-loaded micelles. Enhanced therapeutic efficacy of dual agent micelles could be ascribe to increased accumulation of PTX in drug-resistant tumor cells. We suggest that the synergistic effect of folate receptor-mediated internalization and VRP-mediated overcoming MDR could be beneficial in treatment of MDR solid tumors by targeting delivery of micellar PTX into tumor cells. As a result, the difunctional micelle systems is a very promising approach to overcome tumor drug resistance. PMID:21903258

  15. Raman, SERS, and induced circular dichroism techniques as a probe of pharmaceuticals in their interactions with the human serum albumin and p-glycoprotein

    NASA Astrophysics Data System (ADS)

    Fleury, Fabrice; Ianoul, Anatoli I.; Baggetto, Loris; Jardillier, Jean-Claude; Alix, Alain J.; Nabiev, Igor R.

    1999-04-01

    Camptothecin (CPT) derivatives are the well known inhibitors of the human DNA topoisomerase (topo) I. Two of them, irinotecan and topotecan, are just in the clinics; 9-amino- CPT is on the stage II of clinical trials, and the active search for new derivatives is now in progress. Stability of the CPT derivatives on their way to the target and resistance of cancer cells to these drugs present the crucial problem of the chemotherapy. Human serum albumin (HSA) is the mediator of transport and metabolism of numerous pharmaceuticals in the blood and P-glycoprotein (P- gp) plays a crucial role of the mediator of the multidrug resistance (MDR) of the cancer cells. This paper present the result of analysis of molecular interactions of some drugs of CPT family with the HSA and P-gp. Induced circular dichroism (CD) and Raman techniques have been applied for monitoring molecular interaction of drugs with HSA as well as to identify the conformational transition of the protein induced by the drug binding. Drug molecular determinants responsible for interaction have been identified and their binding sites within the HSA have been localized. New cancer cells lines exhibiting an extremely high level of MDR resistance have been established and were shown to contain the P-gp overproduced in the quantities of 35 percent from the all membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental sensitive cells may be used as a model system for spectroscopic analysis of the specific pharmaceuticals/P-gp interactions.

  16. Nonlinear accumulation in the brain of the new taxoid TXD258 following saturation of P-glycoprotein at the blood–brain barrier in mice and rats

    PubMed Central

    Cisternino, Salvatore; Bourasset, Fanchon; Archimbaud, Yves; Sémiond, Dorothée; Sanderink, Gérard; Scherrmann, Jean-Michel

    2003-01-01

    TXD258, a new taxoid antitumor agent, is a poor substrate for the P-glycoprotein (P-gp) in Caco-2 cells. In this study, we investigated the amount of drug accumulating in the brains of rats and mice under a variety of conditions (dose and infusion time, species and plasma concentration) using conventional in vivo pharmacokinetic techniques and in situ brain perfusion. Mice were infused with radiolabeled TXD258 at 15, 30, 45 and 90 mg m−2 for 45 s or 1 h and rats were infused with 15 and 60 mg m−2 over 2.3 min. The radioactivity in the plasma and brains was measured. The brain concentrations of TXD258 in mice and rats were maximal from 2 min to 1 h postinfusion and radioactivity was still detectable at 168 h. While the plasma concentration of TXD258 increased linearly in mice with the infused dose, the brain content increased more than proportionally with the dose between 15 and 90 mg m−2. This nonlinear uptake of TXD258 also occurred in the plasma and brain of the rat. These findings suggest that the protein-mediated efflux across the blood–brain barrier (BBB) becomes saturated. In situ brain perfusion studies confirmed that TXD258 is a P-gp substrate at the BBB of mice and rats. The P-gp of both species was saturated at the half-inhibitory concentration (∼13 μM) produced by i.v. infusion. Thus, the observed nonlinear accumulation of TXD258 in the brain seems to occur by saturation of the P-gp at the rodent BBB. This saturation could have several advantages, such as overcoming a P-gp-mediated efflux, but the nonlinear pharmacokinetics could increase the risk of toxicity. PMID:12711638

  17. P-glycoprotein differentially affects escitalopram, levomilnacipran, vilazodone and vortioxetine transport at the mouse blood-brain barrier in vivo.

    PubMed

    Bundgaard, Christoffer; Eneberg, Elin; Sánchez, Connie

    2016-04-01

    P-glycoprotein (P-gp)-mediated brain efflux of xenobiotics is a well-known process, which may result in suboptimal target engagement and consequently reduced efficacy of drugs exerting their therapeutic effects in the central nervous system. In the present study the role of P-gp in transport across the blood-brain barrier (BBB) was investigated with a series of newer antidepressants (levomilnacipran, vilazodone and vortioxetine) and a control substrate (escitalopram) using P-gp knock-out (KO) and P-gp competent wild-type (WT) mice. Brain and plasma exposure time-courses were measured after an acute subcutaneous dose and at steady-state obtained after subcutaneous drug infusion by osmotic minipumps. Following acute dosing, the brain-to-plasma KO/WT exposure enhancement ratios ((AUCbrain ko/AUCplasma ko)/(AUCbrain WT/AUCplasma WT)) were 5.8 (levomilnacipran), 5.4 (vilazodone), 3.1 (escitalopram) and 0.9 (vortioxetine), respectively. At steady-state, assessment of Kp,uu (unbound brain concentrations/unbound plasma concentrations) revealed a restriction in the brain distribution in WT mice for all compounds except vortioxetine. Levomilnacipran exhibited the most pronounced efflux with a Kp,uu-value of 0.038 in WT mice which was increased to 0.37 in KO mice. Based on both the acute and steady-state distribution data, the results suggest that levomilnacipran, vilazodone and escitalopram are susceptible to P-gp mediated efflux at the BBB in vivo in mice, whereas vortioxetine was practically devoid of being affected by P-gp in vivo. The functional impact of the drug transport-controlling role of P-gp at the BBB was demonstrated by in vivo cortical serotonin transporter occupancy of vilazodone, which exhibited a 20-fold higher plasma EC50 in WT mice compared to KOs. PMID:26700248

  18. Efficient Trafficking of MDR1/P-Glycoprotein to Apical Canalicular Plasma Membranes in HepG2 Cells Requires PKA-RIIα Anchoring and Glucosylceramide

    PubMed Central

    Wojtal, Kacper A.; de Vries, Erik; Hoekstra, Dick

    2006-01-01

    In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIα from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5′NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5′NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIα displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIα anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1. PMID:16723498

  19. Digoxin and ouabain induce P-glycoprotein by activating calmodulin kinase II and hypoxia-inducible factor-1alpha in human colon cancer cells

    SciTech Connect

    Riganti, Chiara

    2009-11-01

    Digoxin and ouabain are cardioactive glycosides, which inhibit the Na{sup +}/K{sup +}-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca{sup ++}]{sub i}). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca{sup ++}]{sub i} and this event was dependent on the calcium influx via the Na{sup +}/Ca{sup ++} exchanger. The increased [Ca{sup ++}]{sub i} enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1alpha. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na{sup +}/Ca{sup ++} exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.

  20. Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

    PubMed Central

    Šereš, Mário; Cholujová, Dana; Bubenčíkova, Tatiana; Breier, Albert; Sulová, Zdenka

    2011-01-01

    P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump. PMID:22174631

  1. Expression and significance of hypoxia-inducible factor-1α and MDR1/P-glycoprotein in laryngeal carcinoma tissue and hypoxic Hep-2 cells

    PubMed Central

    XIE, JIN; LI, DA-WEI; CHEN, XIN-WEI; WANG, FEI; DONG, PIN

    2013-01-01

    The present study aimed to evaluate the expression of hypoxia-inducible factor-1α (HIF-1α) and MDR1/P-glycoprotein (P-gp) in human laryngeal squamous cell carcinoma (LSCC) tissues, and also to investigate the regulation of MDR1 gene expression by HIF-1α in Hep-2 cells under hypoxic conditions. The expression of HIF-1α and MDR1/P-gp in human LSCC tissues was examined using immunohistochemistry. The HIF-1α and MDR1 gene expression in the Hep-2 cells was detected using real-time quantitative reverse transcription (QRT)-PCR and western blot analysis under normoxic and hypoxic conditions. In hypoxia, HIF-1α expression was inhibited by RNA interference. HIF-1α and MDR1/P-gp expression was high in the LSCC tissues and was associated with the clinical stage and lymph node metastasis (P<0.05). HIF-1α expression was positively correlated with MDR1/P-gp expression (P<0.01). In the Hep-2 cells, HIF-1α and MDR1/P-gp expression significantly increased in response to hypoxia. The inhibition of HIF-1α expression synergistically downregulated the expression of the MDR1 gene in hypoxic Hep-2 cells. HIF-1α expression is positively correlated with MDR1/P-gp expression in LSCC, and the two proteins may be able to serve as potential biomarkers for predicting the malignant progression and metastasis of LSCC. HIF-1α may be critical for the upregulation of MDR1 gene expression induced by hypoxia in Hep-2 cells. PMID:23946810

  2. Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor.

    PubMed

    Savage, J; Meaney, M; Brennan, G P; Hoey, E; Trudgett, A; Fairweather, I

    2014-07-01

    A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance. PMID:24889697

  3. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    PubMed Central

    Prados, Jose; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Methods Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Results Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. Conclusions These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma

  4. A novel phenylcyclohex-1-enecarbothioamide derivative inhibits CXCL8-mediated chemotaxis through selective regulation of CXCR2-mediated signalling

    PubMed Central

    Ha, Helen; Bensman, Tim; Ho, Henry; Beringer, Paul M; Neamati, Nouri

    2014-01-01

    Background and Purpose Since the CXC chemokine receptor CXCR2 and its cognate ligand CXCL8 (IL-8) critically regulate neutrophil trafficking during inflammation, they have been implicated in a number of inflammatory lung diseases. Several CXCR2 antagonists have been described and the blockade of CXCR2 has shown promise in pre-clinical disease models and early clinical trials. However, given its potential, there are fewer distinct classes of antagonists of CXCR2 than of other clinically relevant molecular targets. Thus, we sought to identify additional classes of compounds that alter CXCR2 function. Experimental Approach We used the CXCR2 Tango™ assay to screen an in-house library of highly diverse chemical compounds. CX4338 [2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-enecarbothioamide] was identified from our screen and additional studies to characterize the compound were performed. Receptor internalization and second-messenger assays were used to assess the effects of CX4338 on CXCR2-mediated signalling. Wound healing, transwell cell migration and LPS-induced lung inflammation in mice were used to determine the in vitro and in vivo effects of CX4338. Key Results CX4338 selectively inhibited CXCR2-mediated recruitment of β-arrestin-2 and receptor internalization, while enhancing CXCR2-mediated MAPK activation. Additionally, CX4338 inhibited CXCL8-induced chemotaxis in CXCR2-overexpressing cells and human neutrophils. In vivo, CX4338 significantly reduced neutrophils in bronchoalveolar lavage induced by LPS in mice. Conclusions and Implications A novel compound CX4338 inhibited CXCR2-mediated cell migration with a mechanism of action not previously reported. Also, selective inhibition of CXCR2-mediated β-arrestin-2 activation is sufficient to inhibit CXCL8-mediated chemotaxis. PMID:24354854

  5. Capsaicin pretreatment increased the bioavailability of cyclosporin in rats: involvement of P-glycoprotein and CYP 3A inhibition.

    PubMed

    Zhai, Xue-jia; Shi, Fang; Chen, Fen; Lu, Yong-ning

    2013-12-01

    Capsaicin (CAP), the main ingredient responsible for the hot pungent taste of chilli peppers. This study investigated the effect of CAP on the pharmacokinetics of Cyclosporin A (CyA) in rats and the mechanism of this food-drug interaction. The results indicated that after 7 days of low or middle dose of CAP (0.3 or 1.0 mg/kg), the blood concentration of CyA was not significantly changed compared with that of vehicle-treated rats, whereas the blood concentration of CyA in high dose group (3.0 mg/kg) was significantly increased. The total clearance (CL/F) of CyA was decreased, and the bioavailability was significantly increased to about 1.44-fold of that in vehicle-treated rats after 7 days of high dose CAP treatment. At this time, the P-gp and CYP3A1/2 in the liver and intestine were decreased at both the mRNA and protein levels. These results demonstrated that chronic ingestion of high doses of CAP will increase the bioavailability of CyA to a significant extent in rats and the food-drug interaction between CAP and CyA appears to be due to modulation of P-gp and CYP3A gene expression by CAP, with differential dose-dependence. PMID:24013073

  6. Novel dihydro-beta-agarofuran sesquiterpenes as potent modulators of human P-glycoprotein dependent multidrug resistance.

    PubMed

    Torres-Romero, David; Muñoz-Martínez, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Gamarro, Francisco; Bazzocchi, Isabel L

    2009-12-21

    P-Glycoprotein (Pgp) overexpression is one factor contributing to multidrug resistance (MDR) in cancer cells and represents one drawback in the treatment of cancer. In an attempt to find more specific and less toxic anticancer MDR-reversal agents, we report herein the isolation, structure elucidation and biological activity of nine new (, and ) and seven known (, and ) dihydro-beta-agarofuran sesquiterpenes from the leaves of Celastrus vulcanicola. Their stereostructures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies and biogenetic means. All the compounds were assayed on human MDR1-transfected NIH-3T3 cells, in order to determine their ability to reverse the MDR phenotype due to Pgp overexpression. Six compounds from these series (, , , , and ) showed an effectiveness that was similar to (or higher than) the classical Pgp reversal agent verapamil for the reversal of resistance to daunomycin and vinblastine. The structure-activity relationships are discussed. PMID:20024113

  7. Differential expression of sphingolipids in P-glycoprotein or multidrug resistance-related protein 1 expressing human neuroblastoma cell lines.

    PubMed

    Dijkhuis, Anne-Jan; Douwes, Jenny; Kamps, Willem; Sietsma, Hannie; Kok, Jan Willem

    2003-07-31

    The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established. SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in SK-N-AS cells. These two cell lines exhibited higher sphingolipid levels, compared to SK-N-DZ, which had the lowest activity of either ATP-binding cassette transporter protein. SK-N-DZ cells also differed in ganglioside composition with predominant expression of b-series gangliosides. In conclusion, these three neuroblastoma cell lines offer a good model system to study sphingolipid metabolism in relation to ATP-binding cassette transporter protein function. PMID:12885402

  8. Drugs Modulate Interactions between the First Nucleotide-Binding Domain and the Fourth Cytoplasmic Loop of Human P-Glycoprotein.

    PubMed

    Loo, Tip W; Clarke, David M

    2016-05-24

    Drug substrates stimulate ATPase activity of the P-glycoprotein (P-gp) ATP-binding cassette drug pump by an unknown mechanism. Cross-linking analysis was performed to test if drug substrates stimulate P-gp ATPase activity by altering cross-talk at the first transmission interface linking the drug-binding [intracellular loop 4 (S909C)] and first nucleotide-binding domains [NBD1 (V472C or L443C)]. In the absence of lipid (inactive P-gp), only V472C/S909C showed cross-linking. Drugs blocked V472C/S909C cross-linking. In the presence of lipids (active P-gp), drug substrates promoted only L443C/S909C cross-linking. This suggests that drug substrates stimulate ATPase activity through a conformational change that shifts Ser909 away from Val472 and toward Leu443. PMID:27159830

  9. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice

    PubMed Central

    Wang, Xueqian; Cabrera, Robert M.; Li, Yue; Miller, David S.; Finnell, Richard H.

    2013-01-01

    Folate deficiency has been associated with many adverse clinical manifestations. The blood-brain barrier (BBB), formed by brain capillary endothelial cells, protects the brain from exposure to neurotoxicants. The function of BBB is modulated by multiple ABC transporters, particularly P-glycoprotein. A proton-coupled folate transporter (PCFT)-deficient mouse has been previously described as a model for systemic folate deficiency. Herein, we demonstrate that exposing mouse brain capillaries to the antiepileptic drug, valproic acid (VPA; 5 μM), significantly increased P-glycoprotein transport function in the wild-type animals. A ligand to the aryl hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produced a similar induction of P-glycoprotein, which tightened the BBB, thereby increasing the neuroprotection. However, VPA- or TCDD-induced P-glycoprotein transport was blocked in the PCFT-nullizygous mice, indicating that multiple neuroprotective mechanisms are compromised under folate-deficient conditions. Brain capillaries from S-folinic acid (SFA; 40 mg/kg)-treated PCFT-nullizygous mice exhibited increased P-glycoprotein transport following VPA exposure. This suggests that SFA supplementation restored the normal BBB function. In addition, we show that tight-junction proteins are disintegrated in the PCFT mutant mice. Taken together, these findings strongly suggest that folate deficiency disrupts the BBB function by targeting the transporter and tight junctions, which may contribute to the development of neurological disorders.—Wang, X., Cabrera, R. M., Li, Y., Miller, D. S., Finnell, R. H. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice. PMID:23212123

  10. ¹⁸FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein.

    PubMed

    Krasznai, Zoárd T; Trencsényi, György; Krasznai, Zoltán; Mikecz, Pál; Nizsalóczki, Enikő; Szalóki, Gábor; Szabó, Judit P; Balkay, László; Márián, Teréz; Goda, Katalin

    2014-11-20

    2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp. PMID:25149126

  11. Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense

    SciTech Connect

    Leslie, Elaine M.; Deeley, Roger G.; Cole, Susan P.C. . E-mail: coles@post.queensu.ca

    2005-05-01

    In tumor cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation which is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter proteins. ABC proteins that confer drug resistance include (but are not limited to) P-glycoprotein (gene symbol ABCB1), the multidrug resistance protein 1 (MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), and the breast cancer resistance protein (BCRP, gene symbol ABCG2). In addition to their role in drug resistance, there is substantial evidence that these efflux pumps have overlapping functions in tissue defense. Collectively, these proteins are capable of transporting a vast and chemically diverse array of toxicants including bulky lipophilic cationic, anionic, and neutrally charged drugs and toxins as well as conjugated organic anions that encompass dietary and environmental carcinogens, pesticides, metals, metalloids, and lipid peroxidation products. P-glycoprotein, MRP1, MRP2, and BCRP/ABCG2 are expressed in tissues important for absorption (e.g., lung and gut) and metabolism and elimination (liver and kidney). In addition, these transporters have an important role in maintaining the barrier function of sanctuary site tissues (e.g., blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier or placenta). Thus, these ABC transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, excretion, and toxicity of xenobiotics. In this review, the role of these four ABC transporter proteins in protecting tissues from a variety of toxicants is discussed. Species variations in substrate specificity and tissue distribution of these transporters are also addressed since these properties have implications for in vivo models of toxicity used for drug discovery and development.

  12. Comparison of P-glycoprotein expression in cell lines and xenogragraft sections using I-125 MRK-16 monoclonal antibody (MAB)

    SciTech Connect

    Mehta, B.M.; Kostakoglu, L.; Levchenko, A.

    1994-05-01

    P-glycoprotein (Pgp) is known to be associated with multidrug resistance (MDR). Quantitation of P-glycoprotein expression may permit appropriate therapy depending on Pgp expression in tumors. The present study was undertaken to evaluate the utility of quantitative autoradiography (QAR) in the quantification of MDR using MRK-16, a murine IgG mAb reactive against Pgp. Balb/c mice were xenografted with colchicine resistant BE(2)C/CHC cells. Animals with established tumors were sacrificed, and 8 {mu}m tumor sections were prepared. Mab MRK-16 was labeled with I-125 (150 {mu}Ci/0.625 nmole) by the iodogen method and subsequently purified by size exclusion chromatography. Consecutive tumor sections were incubated overnight at 4{degrees}C with serial dilutions of I-125 MRK-16. Similarly cell suspensions containing 1 X 10{sup 7} cells per ml were also incubated with serial dilutions. QAR analysis of tissue sections of BE(2)C/CHC tumors growing as xenografts in nude mice, determined the binding affinity (K{sub a}) for MRK-16 to be 1 x 10{sup 9} L/M and the number of binding sites (B{sub max}) to be 137, 700 per cell (222 picomols/g); it compared very well with the K{sub a} value of 5 x 10{sup 8} L/M and the B{sub max} value of 130,000 per cell (217 picomols/g) obtained from binding analysis with cell suspensions.

  13. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation.

    PubMed

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li2CO3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li2CO3 did not affect PI3K-mediated PI(3,4,5)P3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li2CO3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li2CO3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li2CO3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity. PMID:24950409

  14. Lactobacillus acidophilus stimulates intestinal P-glycoprotein expression via a c-Fos/c-Jun-dependent mechanism in intestinal epithelial cells.

    PubMed

    Priyamvada, Shubha; Anbazhagan, Arivarasu N; Kumar, Anoop; Soni, Vikas; Alrefai, Waddah A; Gill, Ravinder K; Dudeja, Pradeep K; Saksena, Seema

    2016-04-15

    Our previous studies showed that Lactobacillus acidophilus (LA) culture supernatant (CS) increased P-glycoprotein [Pgp/multidrug resistance 1 (MDR1)] function, expression, and promoter activity in Caco-2 cells. The current studies were designed to elucidate the molecular mechanisms mediating the stimulatory effects of LA CS on Pgp promoter activity. Deletion analysis indicated that the LA CS response element(s) is located in the -172/+428-bp region, and sequence analysis of this region revealed three potential binding sites for c-Fos or c-Jun: proximal activating protein (AP) 1a (-119/-98 bp), distal AP1b (-99/-78 bp), and AP1c (+175/+196 bp). LA CS (24 h) showed an approximately twofold increase in the protein expression of c-Fos and c-Jun in Caco-2 cells. Electrophoretic mobility shift assay showed that LA CS markedly increased the binding of Caco-2 nuclear proteins to AP1a and AP1b, but not AP1c. The DNA-protein complex was completely eliminated by c-Fos antibody, while c-Jun antibody partially eliminated the complex. Chromatin immunoprecipitation analysis also showed that LA CS enhanced the association of c-Fos and c-Jun (by ∼4- and 1.5-fold, respectively) with endogenous Pgp promoter in Caco-2 cells (p-172/+1). Interestingly, overexpression of c-Fos or c-Jun activated Pgp promoter by nearly twofold each. This increase was further enhanced (∼14-fold) when c-Fos and c-Jun were simultaneously overexpressed, suggesting that the presence of one of these transcription factors potentiates the effect of the other. These studies, for the first time, provide evidence for the involvement of c-Fos/c-Jun in stimulation of Pgp gene expression by LA CS in the human intestine. PMID:26867563

  15. Acetaminophen Modulates P-Glycoprotein Functional Expression at the Blood-Brain Barrier by a Constitutive Androstane Receptor–Dependent Mechanism

    PubMed Central

    Thompson, Brandon J.; Sanchez-Covarrubias, Lucy; Zhang, Yifeng; Laracuente, Mei-Li; Vanderah, Todd W.; Ronaldson, Patrick T.; Davis, Thomas P.

    2013-01-01

    Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4–1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp–mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy. PMID:24019224

  16. Targeting T Cell Bioenergetics by Modulating P-Glycoprotein Selectively Depletes Alloreactive T Cells To Prevent Graft-versus-Host Disease.

    PubMed

    McIver, Zachariah A; Grayson, Jason M; Coe, Benjamin N; Hill, Jacqueline E; Schamerhorn, Gregory A; Ohulchanskyy, Tymish Y; Linder, Michelle K; Davies, Kellie S; Weiner, Roy S; Detty, Michael R

    2016-09-01

    T lymphocytes play a central role in many human immunologic disorders, including autoimmune and alloimmune diseases. In hematopoietic stem cell transplantation, acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Selectively depleting GVHD-causing cells prior to transplant may prevent GVHD. In this study, we evaluated 24 chalcogenorhodamine photosensitizers for their ability to selectively deplete reactive T lymphocytes and identified the photosensitizer 2-Se-Cl, which accumulates in stimulated T cells in proportion to oxidative phosphorylation. The photosensitizer is also a potent stimulator of P-glycoprotein (P-gp). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria and protects resting lymphocytes that are essential for antipathogen and antitumor responses. To evaluate the selective depletion of alloimmune responses, donor C57BL/6 splenocytes were cocultured for 5 d with irradiated BALB/c splenocytes and then photodepleted (PD). PD-treated splenocytes were infused into lethally irradiated BALB/c (same-party) or C3H/HeJ (third-party) mice. Same-party mice that received PD-treated splenocytes at the time of transplant lived 100 d without evidence of GVHD. In contrast, all mice that received untreated primed splenocytes and third-party mice that received PD-treated splenocytes died of lethal GVHD. To evaluate the preservation of antiviral immune responses, acute lymphocytic choriomeningitis virus infection was used. After photodepletion, expansion of Ag-specific naive CD8(+) T cells and viral clearance remained fully intact. The high selectivity of this novel photosensitizer may have broad applications and provide alternative treatment options for patients with T lymphocyte-mediated diseases. PMID:27456485

  17. Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1

    PubMed Central

    Hawley, Teresa S.; Riz, Irene; Yang, Wenjing; Wakabayashi, Yoshiyuki; DePalma, Louis; Chang, Young-Tae; Peng, Weiqun; Zhu, Jun; Hawley, Robert G.

    2013-01-01

    Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy-resistant cancer cells is responsible for propagation of the tumor. Herein we report that efflux of the pluripotent stem cell dye CDy1 identifies a subpopulation in MM cell lines characterized by increased expression of P-glycoprotein, a member of the ABC (ATP-binding cassette) superfamily of transporters encoded by ABCB1. We also demonstrate that ABCB1-overexpressing MM cells are resistant to the second-generation proteasome inhibitor carfilzomib that recently received accelerated approval for the treatment of therapy-refractive MM by the U.S. Food and Drug Administration. Moreover, increased resistance to carfilzomib in sensitive MM cells following drug selection was associated with upregulation of ABCB1 cell-surface expression which correlated with increased transporter activity as measured by CDy1 efflux. We further show that chemosensitization of MM cells to carfilzomib could be achieved in vitro by cotreatment with vismodegib, a hedgehog pathway antagonist which is currently in MM clinical trials. CDy1 efflux may therefore be a useful assay to determine whether high expression of ABCB1 is predictive of poor clinical responses in MM patients treated with carfilzomib. Our data also suggest that inclusion of vismodegib might be a potential strategy to reverse ABCB1-mediated drug resistance should it occur. PMID:23475625

  18. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    SciTech Connect

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li{sub 2}CO{sub 3} significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li{sub 2}CO{sub 3} did not affect PI3K-mediated PI(3,4,5)P{sub 3} production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li{sub 2}CO{sub 3} on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li{sub 2}CO{sub 3} significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li{sub 2}CO{sub 3} significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity.

  19. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    PubMed Central

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  20. Apoptosis induction and modulation of P-glycoprotein mediated multidrug resistance by new macrocyclic lathyrane-type diterpenoids.

    PubMed

    Duarte, Noélia; Varga, Andras; Cherepnev, Georg; Radics, Rita; Molnár, Joseph; Ferreira, Maria-José U

    2007-01-01

    The macrocyclic lathyrane diterpenes, latilagascenes D-F (1-3) and jolkinol B (4), were isolated from the methanol extract of Euphorbia lagascae, and evaluated for multidrug resistance reversing activity on mouse lymphoma cells. All compounds displayed very strong activity compared with that of the positive control, verapamil. The structure-activity relationship is discussed. The evaluation of compounds 1 and 4, and of latigascenes A-C (5-7), isolated from the same species, as apoptosis-inducers was also carried out. Compound 1 was the most active. Furthermore, in the model of combination chemotherapy, the interaction between the doxorubicine and latilagascene B (6) was studied in vitro, on human MDR1 gene transfected mouse lymphoma cells, showing that the type of interaction was synergistic. Latilagascenes D-F (1-3) are new compounds whose structures were established on the basis of spectroscopic methods, including 2D NMR experiments (COSY, HMQC, HMBC and NOESY). PMID:17035027

  1. Combating P-glycoprotein-mediated multidrug resistance with 10-O-phenyl dihydroartemisinin ethers in MCF-7 cells.

    PubMed

    Zhong, Hang; Zhao, Xuan; Zuo, Zhizhong; Sun, Jingwei; Yao, Yao; Wang, Tao; Liu, Dan; Zhao, Linxiang

    2016-01-27

    A series of 10-β-phenyl ethers of dihydroartemisinin (DHA) with piperazine substitutions were synthesized with the goal of overcoming multidrug resistance in cancer therapy. These novel compounds exerted significant antiproliferative activities in breast cancer MCF-7 and MCF-7/Adr cell lines at the submicromolar level and were shown to be approximately 100- to 300-times more potent than the lead compound DHA. Remarkably, the P-gp-overexpressed MCF-7/Adr cell line showed collateral sensitivity towards these derivatives. Furthermore, compounds 3d and 5c, with the highest selectivity for MCF-7/Adr towards MCF-7 cells, were free from P-gp efflux in a MDCK-MDR1 assay. Flow cytometry and western blot assays suggested that the antiproliferative effects of 5c were associated with cell cycle arrest at G1 phase through the downregulation of Cyclin D1 and Cyclin B1. PMID:26741854

  2. Pluronic P85-coated poly(butylcyanoacrylate) nanoparticles overcome phenytoin resistance in P-glycoprotein overexpressing rats with lithium-pilocarpine-induced chronic temporal lobe epilepsy.

    PubMed

    Fang, Ziyan; Chen, Shuda; Qin, Jiaming; Chen, Bao; Ni, Guanzhong; Chen, Ziyi; Zhou, Jueqian; Li, Ze; Ning, Yuping; Wu, Chuanbin; Zhou, Liemin

    2016-08-01

    P-glycoprotein (Pgp) overexpression in the blood brain barrier (BBB) is hypothesized to lower brain drug concentrations and thus inhibit anticonvulsant effects in drug-resistant epilepsy. Recently, the poly(butylcyanoacrylate) (PBCA) nanoparticle system was shown to overcome the obstacle of the BBB to deliver drugs into the brain. To determine whether pluronic P85-coated phenytoin poly(butylcyanoacrylate) nanoparticles (P85-PHT-PBCA-NPs) target PHT to the brain, PHT-resistant rats overexpressing Pgp in the BBB were screened by response to PHT treatment after chronic temporal lobe epilepsy induced by lithium-pilocarpine, followed by direct verification of PHT transport via measurement of brain PHT concentrations using microdialysis. Thereafter, the PHT-resistant rats were divided into three groups, which were treated with PHT, PHT + tariquidar (TQD), or P85-PHT-PBCA-NPs. PHT + TQD and P85-PHT-PBCA-NPs showed anticonvulsant activity in the PHT-resistant rats and increased the ratio of the area under the curve of the PHT concentrations in the brain/plasma in comparison with that observed in animals subjected to PHT treatment. However, the ratios of the PHT concentrations in the liver/plasma and kidney/plasma following P85-PHT-PBCA-NPs treatment were much lower than those measured following PHT + TQD treatment. Thus, Pgp overexpression decreases therapeutic drug concentrations in the brains of subjects with drug-resistant epilepsy and P85-PHT-PBCA-NPs could increase these drug concentrations. PMID:27162079

  3. Evaluation of the role of P-glycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells.

    PubMed

    Maines, Lynn W; Antonetti, David A; Wolpert, Ellen B; Smith, Charles D

    2005-10-01

    Expression of the drug transport proteins, including P-glycoprotein (Pgp), in the brain vascular endothelium represents a challenge for the effective delivery of drugs for the treatment of several central nervous system (CNS) disorders including depression, schizophrenia and epilepsy. It has been hypothesized that Pgp plays a major role in drug efflux at the blood-brain barrier, and may be an underlying factor in the variable responses of patients to CNS drugs. However, the role of Pgp in the transport of many CNS drugs has not been directly demonstrated. To explore the role of Pgp in drug transport across an endothelial cell barrier derived from the central nervous system, the expression and activity of Pgp in bovine retinal endothelial cells (BRECs) and the effects of representative CNS drugs on Pgp activity were examined. Significant Pgp expression in BRECs was demonstrated by western analyses, and expression was increased by treatment of the cells with hydrocortisone. Intracellular accumulation of the well-characterized Pgp-substrate Taxol was markedly increased by the non-selective transporter inhibitor verapamil and the Pgp-selective antagonist PGP-4008, demonstrating that Pgp is active in these endothelial cells. In contrast, neither verapamil nor PGP-4008 affected the intracellular accumulation of [3H]paroxetine, [14C]phenytoin, [3H]clozapine or [14C]carbamazapine, indicating that these drugs are not substrates for Pgp. Paroxetine, clozapine and phenytoin were shown to be Pgp inhibitors, while carbamazapine did not inhibit Pgp at any concentration tested. These results indicate that Pgp is not likely to modulate patient responses to these drugs. PMID:15961125

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

    PubMed Central

    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 < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/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 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

  5. The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction

    PubMed Central

    Martin, Catherine; Berridge, Georgina; Higgins, Christopher F; Callaghan, Richard

    1997-01-01

    The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter. The steady-state accumulation of [3H]-vinblastine in P-gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously. Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non-competitive interaction, most likely at distinct sites. The specific [3H]-vinblastine binding to P-gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3±4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric effect on the vinca alkaloid acceptor site. These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model. PMID:9375975

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

    PubMed

    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 < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/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 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

  7. Macrocyclic lactones differ in interaction with recombinant P-glycoprotein 9 of the parasitic nematode Cylicocylus elongatus and ketoconazole in a yeast growth assay.

    PubMed

    Kaschny, Maximiliane; Demeler, Janina; Janssen, I Jana I; Kuzmina, Tetiana A; Besognet, Bruno; Kanellos, Theo; Kerboeuf, Dominique; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

    2015-04-01

    Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the

  8. The bisbenzylisoquinoline alkaloids, tetrandine and fangchinoline, enhance the cytotoxicity of multidrug resistance-related drugs via modulation of P-glycoprotein.

    PubMed

    Choi, S U; Park, S H; Kim, K H; Choi, E J; Kim, S; Park, W K; Zhang, Y H; Kim, H S; Jung, N P; Lee, C O

    1998-03-01

    The occurrence of resistance to chemotherapeutic drugs is a major problem for successful cancer treatment and reducing drug accumulation by P-glycoprotein (P-gp) is one of the major mechanisms of multidrug resistance (MDR). The present study was performed to evaluate the MDR-reversal abilities of two bisbenzylisoquinoline alkaloids, tetrandine (TET) and fangchinoline (FAN), compared with verapamil (VER), a well-known P-gp modulator. TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) reduced the paclitaxel (TAX) concentration required to achieve 50% inhibition of cell growth (EC50) to HCT15 (P-gp-positive) cells about 3100-, 1900- and 410-fold, and these compounds also reduced the EC50 value of actinomycin D (AMD) about 36.0-, 45.9- and 18.2-fold in the cells, respectively. Meanwhile, TET, FAN and VER had no effect on the cytotoxicity of the drugs to SK-OV-3 (P-gp-negative) cells. On the other hand, TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) similarly enhanced the accumulation rates of rhodamine 123, a well known P-gp substrate, in HCT15 cells (200-250%). After efflux for 2 h with fresh medium, TET and FAN also enhanced the residual rate of rhodamine 123 about 5.0- and 2.6-fold in comparison with control, respectively. TET, FAN and VER could not affect the accumulation and residual rate of rhodamine 123 in SK-OV-3 cells. From the result, we conclude that TET and FAN enhanced the cytotoxicity of MDR-related drugs via modulation of P-gp. PMID:9625436

  9. Macrocyclic Lactones Differ in Interaction with Recombinant P-Glycoprotein 9 of the Parasitic Nematode Cylicocylus elongatus and Ketoconazole in a Yeast Growth Assay

    PubMed Central

    Kaschny, Maximiliane; Demeler, Janina; Janssen, I. Jana I.; Kuzmina, Tetiana A.; Besognet, Bruno; Kanellos, Theo; Kerboeuf, Dominique; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

    2015-01-01

    Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the

  10. Alpha-2 adrenergic receptor-mediated inhibition of thermogenesis

    PubMed Central

    Madden, Christopher J.; Tupone, Domenico; Cano, Georgina; Morrison, Shaun F.

    2013-01-01

    Alpha2-adrenergic receptor (α2-AR) agonists have been use as anti-hypertensive agents, in the management of drug withdrawal, and as sedative analgesics. Since α2-AR agonists also influence the regulation of body temperature, we explored their potential as antipyretic agents. This study delineates the central neural substrate for the inhibition of rat brown adipose tissue (BAT) and shivering thermogenesis by α2-AR agonists. Nanoinjection of the α2-AR agonist, clonidine (1.2 nmol), into the rostral raphe pallidus (rRPa) inhibited BAT sympathetic nerve activity (SNA) and BAT thermogenesis. Subsequent nanoinjection of the α2-AR antagonist, idazoxan (6nmol) into the rRPa reversed the clonidine-evoked inhibition of BAT SNA and BAT thermogenesis. Systemic administration of the α2-AR agonists, dexmedetomidine (25ug/kg, iv) or clonidine (100ug/kg, iv) inhibited shivering EMGs, BAT SNA and BAT thermogenesis effects that were reversed by nanoinjection of idazoxan (6nmol) into the rRPa. Dexmedetomidine (100µg/kg, ip) prevented and reversed lipopolysaccharide (10µg/kg ip)-evoked thermogenesis in free-behaving rats. Cholera toxin subunit b retrograde tracing from rRPa and pseudorabies virus transynaptic retrograde tracing from BAT combined with immunohistochemistry for catecholaminergic biosynthetic enzymes revealed the ventrolateral medulla as the source of catecholaminergic input to the rRPa and demonstrated that these catecholaminergic neurons are synaptically connected to BAT. Photostimulation of VLM neurons expressing of the PRSx8-ChR2-mCherry lentiviral vector inhibited BAT SNA via activation of α2-ARs in the rRPa. These results indicate a potent inhibition of BAT and shivering thermogenesis by α2-AR activation in the rRPa, and suggest a therapeutic potential of α2-AR agonists for reducing potentially-lethal elevations in body temperature during excessive fever. PMID:23365239

  11. Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

    PubMed Central

    Gannon, Michael K.; Holt, Jason J.; Bennett, Stephanie M.; Wetzel, Bryan R.; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.; Sawada, Geri A.; Higgins, J. William; Tombline, Gregory; Raub, Thomas J.; Detty, Michael R.

    2012-01-01

    We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells. PMID:19402665

  12. Differential growth inhibition of isoquinolinesulfonamides H-8 and H-7 towards multidrug-resistant P388 murine leukaemia cells.

    PubMed Central

    Ido, M.; Nagao, Y.; Higashigawa, M.; Shibata, T.; Taniguchi, K.; Hamazaki, M.; Sakurai, M.

    1991-01-01

    The effects of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on the growth of P388 and its multidrug-resistant (MDR) variants were examined with the objective of assessing the possible changes in cyclic nucleotide-dependent protein kinases and protein kinase C-mediated pathways associated with MDR. H-8, an inhibitor of cyclic nucleotide-dependent protein kinases, inhibited the growth of the parental P388 murine leukaemic cells, but not that of MDR variants up to 200 microM. However the growth of both drug-sensitive and resistant cell lines were uniformly inhibited by H-7. Both the cytotoxic and cytokinetic results revealed that the growth-inhibition by H-8 of P388 cells is mainly due to a blockade of cell-cycle progression rather than due to a killing of cells. The degree of resistance to H-8 was directly proportional to their extent of resistance to vincristine, adriamycin, and 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene)-beta-D-gluco pyr anoside (VP-16) and to that of the expression of P-glycoprotein. These findings raised the possibility that P-glycoprotein might play a role in the cross-resistance to H-8. To test the hypothesis, we examined the effect of H-8 on the binding of 3H-vincristine to membrane fraction isolated from P388/VCR-600 cells and on the enhancement of cytotoxicity to anticancer drugs in MDR cells. H-8 did not have any influences on these reactions. Thus, the cross-resistance to H-8 may be mediated through a mechanism different from an overexpression of P-glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 PMID:1684908

  13. The mechanism of OTUB1-mediated inhibition of ubiquitination

    SciTech Connect

    Wiener, Reuven; Zhang, Xiangbin; Wang, Tao; Wolberger, Cynthia

    2013-04-08

    Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13-Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13-Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13-Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13-Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13-Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13-Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1

  14. Evidence for the involvement of P-glycoprotein on the extrusion of taken up L-DOPA in cyclosporine A treated LLC-PK1 cells

    PubMed Central

    Soares-da-Silva, P; Serrão, M P; Vieira-Coelho, M A; Pestana, M

    1998-01-01

    .05).Detection of P-glycoprotein activity was carried out by measuring verapamil- or UIC2-sensitive rhodamine 123 accumulation. Both UIC2 (3 μg ml−1) and verapamil (25 μM) significantly increased the accumulation of rhodamine 123 in LLC-PK1 cells. A 30 min exposure to CsA was found not to affect the accumulation of rhodamine 123 in the presence of verapamil (25 μM), whereas a 14 h exposure to CsA was found to reduce the accumulation of rhodamine 123.In conclusion, the increase and the reduction in the formation of dopamine after short- and long-term exposure to CsA, respectively, correlate with the effects of the immunosuppressant on the apical cell extrusion of taken up L-DOPA, suggesting the involvement of P-glycoprotein. The effects of CsA on the fractional outflow of newly-formed dopamine appear to be mediated by a different mechanism. PMID:9484849

  15. Cetuximab enhanced the efficacy of chemotherapeutic agent in ABCB1/P-glycoprotein-overexpressing cancer cells

    PubMed Central

    Liu, Tao; Huang, Yue; Zhao, Jianming; Wang, Xiaokun; Yang, Ke; Ma, Shaolin; Huang, Liyan; Wah To, Kenneth Kin; Gu, Yong; Fu, Liwu

    2015-01-01

    The overexpression of ATP-binding cassette (ABC) transporters is closely associated with the development of multidrug resistance (MDR) in certain types of cancer, which represents a formidable obstacle to the successful cancer chemotherapy. Here, we investigated that cetuximab, an EGFR monoclonal antibody, reversed the chemoresistance mediated by ABCB1, ABCG2 or ABCC1. Our results showed that cetuximab significantly enhanced the cytotoxicity of ABCB1 substrate agent in ABCB1-overexpressing MDR cells but had no effect in their parental drug sensitive cells and ABCC1, ABCG2 overexpressing cells. Furthermore, cetuximab markedly increased intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABCB1-overexpressing MDR cancer cells in a concentration-dependent manner. Cetuximab stimulated the ATPase activity but did not alter the expression level of ABCB1 or block phosphorylation of AKT and ERK. Interestingly, cetuximab decreased the cell membrane fluidity which was known to decrease the function of ABCB1. Our findings advocate further clinical investigation of combination chemotherapy of cetuximab and conventional chemotherapeutic drugs in ABCB1 overexpressing cancer patients. PMID:26506420

  16. Natural lignans from Arctium lappa modulate P-glycoprotein efflux function in multidrug resistant cancer cells.

    PubMed

    Su, Shan; Cheng, Xinlai; Wink, Michael

    2015-02-15

    Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites which have a wide range of bioactivities. Yet, their ability to reverse multidrug resistance (MDR) in cancer cells has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The MDR reversal potential of the isolated lignans and the underlying mechanism of action were studied using two MDR cancer cell lines, CaCo2 and CEM/ADR 5000 which overexpress P-gp and other ABC transporters. In two-drug combinations of lignans with the cytotoxic doxorubicin, all lignans exhibited synergistic effects in CaCo2 cells and matairesinol, arctiin, lappaol C and lappaol F display synergistic activity in CEM/ADR 5000 cells. Additionally, in three-drug combinations of lignans with the saponin digitonin and doxorubicin MDR reversal activity was even stronger enhanced. The lignans can increase the retention of the P-gp substrate rhodamine 123 in CEM/ADR 5000 cells, indicating that lignans can inhibit the activity of P-gp. Our study provides a first insight into the potential chemosensitizing activity of a series of natural lignans, which might be candidates for developing novel adjuvant anticancer agents. PMID:25765837

  17. Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

    PubMed Central

    Besong-Ndika, Jane; Ivanov, Konstantin I.; Hafrèn, Anders; Michon, Thierry

    2015-01-01

    ABSTRACT Potato virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection. IMPORTANCE The main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the

  18. Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

    PubMed Central

    Bunda, Severa; Burrell, Kelly; Heir, Pardeep; Zeng, Lifan; Alamsahebpour, Amir; Kano, Yoshihito; Raught, Brian; Zhang, Zhong-Yin; Zadeh, Gelareh; Ohh, Michael

    2015-01-01

    Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously ‘undruggable' oncogenic or hyperactive Ras. PMID:26617336

  19. Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity

    PubMed Central

    Menzies, F M; Garcia-Arencibia, M; Imarisio, S; O'Sullivan, N C; Ricketts, T; Kent, B A; Rao, M V; Lam, W; Green-Thompson, Z W; Nixon, R A; Saksida, L M; Bussey, T J; O'Kane, C J; Rubinsztein, D C

    2015-01-01

    Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease. PMID:25257175

  20. Nitrite-Mediated Antagonism of Cyanide Inhibition of Cytochrome c Oxidase in Dopamine Neurons

    PubMed Central

    Leavesley, Heather B.; Li, Li; Mukhopadhyay, Soma; Borowitz, Joseph L.; Isom, Gary E.

    2010-01-01

    Cyanide inhibits aerobic metabolism by binding to the binuclear heme center of cytochrome c oxidase (CcOX). Amyl nitrite and sodium nitrite (NaNO2) antagonize cyanide toxicity in part by oxidizing hemoglobin to methemoglobin (mHb), which then scavenges cyanide. mHb generation is thought to be a primary mechanism by which the NO2− ion antagonizes cyanide. On the other hand, NO2− can undergo biotransformation to generate nitric oxide (NO), which may then directly antagonize cyanide inhibition of CcOX. In this study, nitrite-mediated antagonism of cyanide inhibition of oxidative phosphorylation was examined in rat dopaminergic N27 cells. NaNO2 produced a time- and concentration-dependent increase in whole-cell and mitochondrial levels of NO. The NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxy 3-oxide (PTIO) reversed this increase in cellular and mitochondrial NO. NO generated from NaNO2 decreased cellular oxygen consumption and inhibited CcOX activity. PTIO reversed the NO-mediated inhibition, thus providing strong evidence that NO mediates the action of NaNO2. Under similar conditions, KCN (20μM) inhibited cellular state-3 oxygen consumption and CcOX activity. Pretreatment with NaNO2 reversed KCN-mediated inhibition of both oxygen consumption and CcOX activity. The NaNO2 antagonism of cyanide was blocked by pretreatment with the NO scavenger PTIO. It was concluded that NaNO2 antagonizes cyanide inhibition of CcOX by generating of NO, which then interacts directly with the binding of KCN × CcOX to reverse the toxicity. In vivo antagonism of cyanide by NO2− appears to be due to both generation of mHb and direct displacement of cyanide from CcOX by NO. PMID:20335280

  1. Studies on the mechanism of acetaldehyde-mediated inhibition of rat liver transaminases.

    PubMed

    Solomon, L R

    1987-09-30

    Incubation of mitochondria-depleted rat liver homogenates with 5 mmol/l acetaldehyde at 37 degrees C for 1 h inhibited both aspartate and alanine aminotransferases by 30%. Inhibition was prevented by decreasing temperature to 4 degrees C or by preincubating homogenates with cyanate but was unaffected by cyanamide and methylpyrazole which block acetaldehyde oxidation and reduction respectively. Cyanate-sensitive acetaldehyde-mediated inhibition of purified porcine heart transaminases was also demonstrated in the presence of rat liver homogenate but not in Tris/sucrose medium. Moreover, porcine transaminases were inhibited by trichloroacetic acid extracts of rat liver homogenates previously incubated with acetaldehyde but not by extracts of homogenates incubated with both acetaldehyde and cyanate. These findings suggest that acetaldehyde-mediated transaminase inhibition requires further non-oxidative metabolism of acetaldehyde. Since transaminase activities were not restored by addition of pyridoxal 5'-phosphate to the assay systems, acetaldehyde-induced transaminase inhibition does not appear to be mediated by displacement or depletion of this B6 coenzyme. PMID:3677417

  2. Identification of an elaborate complex mediating postsynaptic inhibition.

    PubMed

    Uezu, Akiyoshi; Kanak, Daniel J; Bradshaw, Tyler W A; Soderblom, Erik J; Catavero, Christina M; Burette, Alain C; Weinberg, Richard J; Soderling, Scott H

    2016-09-01

    Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remain poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. Clustered regularly interspaced short palindromic repeats (CRISPR) depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced the frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders. PMID:27609886

  3. Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

    PubMed Central

    Dijk, A. P. M. van; Tak, C. J. A. M.; Wilson, J. H. P.; Zijlstra, F. J.

    1996-01-01

    In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF-∝, IL-6, PGE2 and LTB4 by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF-∝, IL-6 and PGE2 production but had no effect on LTB4 production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the Kd or receptor number. PMID:18475705

  4. Influence of Panax ginseng on Cytochrome P450 (CYP)3A and P-glycoprotein (Pgp) Activity in Healthy Subjects

    PubMed Central

    Malati, Christine Y.; Robertson, Sarah M.; Hunt, Jennifer D.; Chairez, Cheryl; Alfaro, Raul M.; Kovacs, Joseph A.; Penzak, Scott R.

    2012-01-01

    A number of herbal preparations have been shown to interact with prescription medications secondary to modulation of cytochrome P450 (CYP) and/or P-glycoprotein (P-gp). The purpose of this study was to determine the influence of Panax ginseng on CYP3A and P-gp function using the probe substrates midazolam and fexofenadine, respectively. Twelve healthy subjects (8 males) completed this open label, single sequence pharmacokinetic study. Healthy volunteers received single oral doses of midazolam 8 mg and fexofenadine 120 mg, before and after 28 days of P. ginseng 500 mg twice daily. Midazolam and fexofenadine pharmacokinetic parameter values were calculated and compared pre-and post P. ginseng administration. Geometric mean ratios (post-ginseng/pre-ginseng) for midazolam area under the concentration vs. time curve from zero to infinity (AUC0-∞), half life (T1/2), and maximum concentration (Cmax) were significantly reduced at 0.66 (0.55 – 0.78), 0.71 (0.53 – 0.90), and 0.74 (0.56 – 0.93), respectively. Conversely, fexofenadine pharmacokinetics were unaltered by P. ginseng administration. Based on these results, Panax ginseng appeared to induce CYP3A activity in the liver and possibly the gastrointestinal tract. Patients taking Panax ginseng in combination with CYP3A substrates with narrow therapeutic ranges should be monitored closely for adequate therapeutic response to the substrate medication. PMID:21646440

  5. Contribution of radixin to P-glycoprotein expression and transport activity in mouse small intestine in vivo.

    PubMed

    Yano, Kentaro; Tomono, Takumi; Sakai, Riyo; Kano, Takashi; Morimoto, Kaori; Kato, Yukio; Ogihara, Takuo

    2013-08-01

    The ERM proteins, ezrin, radixin, and moesin, are membrane-cytoskeleton cross-linkers with multiple physiological functions. We previously showed that radixin is involved in posttranslational regulation of P-glycoprotein (P-gp) in human hepatoblastoma HepG2 cells. Here, we investigated the physiological role of radixin in regulating P-gp expression and activity in the small intestine by comparing wild-type- and radixin knockout (Rdx) mice. In intestinal tissue homogenates, P-gp protein levels increased markedly from the upper part to the lower part of the small intestine in both wild-type- and Rdx(-/-) mice. In the membrane fractions, a similar pattern was seen in wild-type mice. However, the membrane expression of P-gp protein remained at the same level from the upper to the lower part of the small intestine in Rdx(-/-) mice. When rhodamine123 (Rho123), a substrate of P-gp, was orally administered to Rdx(-/-) and wild-type mice, the absorption phase of Rho123 was greater in Rdx(-/-) than in wild-type mice, whereas the elimination phase in Rdx(-/-) mice was not different from that of wild-type mice. Our results indicate that radixin plays an important role in regulating P-gp localization and P-gp functional activity at the intestinal membrane. PMID:23754525

  6. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors

    PubMed Central

    Noack, Andreas; Noack, Sandra; Buettner, Manuela; Naim, Hassan Y.; Löscher, Wolfgang

    2016-01-01

    The blood–brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity. PMID:27375084

  7. Temozolomide down-regulates P-glycoprotein in human blood-brain barrier cells by disrupting Wnt3 signaling.

    PubMed

    Riganti, Chiara; Salaroglio, Iris C; Pinzòn-Daza, Martha L; Caldera, Valentina; Campia, Ivana; Kopecka, Joanna; Mellai, Marta; Annovazzi, Laura; Couraud, Pierre-Olivier; Bosia, Amalia; Ghigo, Dario; Schiffer, Davide

    2014-02-01

    Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/β-catenin signaling, and reduces the binding of β-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma. PMID:23771630

  8. Poor oral bioavailability of a promising anticancer agent andrographolide is due to extensive metabolism and efflux by P-glycoprotein.

    PubMed

    Ye, Ling; Wang, Tao; Tang, Lan; Liu, Wei; Yang, Zhen; Zhou, Juan; Zheng, Zhijie; Cai, Zheng; Hu, Ming; Liu, Zhongqiu

    2011-11-01

    Andrographolide (AP), isolated from Andrographis paniculata (Burm. F.) Nees, is an anticancer agent with significant clinical potential. This study determined its oral bioavailability and how intestinal disposition affects its bioavailability. Pharmacokinetics was evaluated in rats. Intestinal disposition was determined using a single-pass rat intestinal perfusion model and the cultured Caco-2 cells and Madin-Darby canine kidney II cells over expressing human P-gp (MDR1-MDCKII). Absolute bioavailability of AP was 2.67%. In the duodenum and jejunum, AP was rapidly metabolized to a sulfonate, identified as 14-deoxy-12-sulfo- andrographolide. AP was also rapidly metabolized by liver S9 fraction and in blank perfusates collected from duodenum and jejunum. The apparent permeability (P(app) ) of AP from basolateral (B) to apical (A) (4.94 × 10 cm/s) in the Caco-2 model was four times higher than the P(app) from A to B (1.14 × 10(-5) cm/s). Moreover, AP was significantly more permeable in the B to A direction than the opposite direction in MDR1-MDCKII cells. In the perfusion model, the effective permeability (P*(eff) ) for AP was highest in the duodenum, followed by jejunum, and then ileum and colon. In the ileum and colon, the P*(eff) for AP was significantly increased by verapamil, a P-glycoprotein (P-gp) inhibitor. AP has poor oral bioavailability because of its rapid biotransformation and efflux by P-gp. PMID:21721007

  9. Establishment and characterization of an MDCK cell line stably-transfected with chicken Abcb1 encoding P-glycoprotein.

    PubMed

    Sun, Yong; Guo, Tingting; Guo, Dawei; Guo, Li; Chen, Li; Zhang, Yu; Wang, Liping

    2016-06-01

    Chicken P-glycoprotein (chP-gp), encoded by Abcb1, determines the bioavailability because of its effect on pharmacokinetics of various drugs. However, comprehensive studies on chP-gp are still limited. In this study, the chicken full-length cDNA was first successfully cloned and then stably expressed in MDCK cell line. The open reading frame of chicken Abcb1 consists of 3864 nucleotides, encoding for a 1287-amino acid protein. Sequence alignments analysis showed that chicken P-gp had high identities with the homologues of turkey (95%), human (72%), pig (72%), rat (71%) and cattle (68%). The efflux ratio of rhodamine123 (Rho123, a human P-gp substrate) in chAbcb1 transfected MDCK cells was significantly higher than that in the wild type MDCK cell (6.24 vs 1.64, P<0.05), suggesting a good transporting function of chicken P-gp overexpressed in the transfected cell. Importantly, MDCK-chAbcb1 cells, unlike Caco-2 cells, exhibited biphasic saturation kinetics in transporting Rho123. In conclusion, an MDCK cell line stably expressing chAbcb1 was successfully established, which could provide a new cell model to screen its substrates and inhibitors and study the drug-drug interaction medicated via chicken P-gp. PMID:27234533

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

    PubMed Central

    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

  11. P-glycoprotein interactions of novel psychoactive substances - stimulation of ATP consumption and transport across Caco-2 monolayers.

    PubMed

    Meyer, Markus R; Wagmann, Lea; Schneider-Daum, Nicole; Loretz, Brigitta; de Souza Carvalho, Cristiane; Lehr, Claus-Michael; Maurer, Hans H

    2015-04-01

    In contrast to drugs for therapeutic use, there are only few data available concerning interactions between P-glycoprotein (P-gp) and drugs of abuse (DOA). In this work, interactions between structurally diverse DOA and P-gp were investigated using different strategies. First, the effect on the P-gp ATPase activity was studied by monitoring of ATP consumption after addition to recombinant, human P-gp. Second, DOA showing an increased ATP consumption were further characterized regarding their transport across filter grown Caco-2- monolayers. Analyses were performed by luminescence and liquid chromatography-mass spectrometry, respectively. Among the nine DOA initially screened, benzedrone, diclofensine, glaucine, JWH-200, MDBC, WIN-55,212-2 showed an increase of ATP consumption in the ATPase stimulation assay. In Caco-2 transport studies, Glaucine, JWH-200, mitragynine, WIN-55,212-2 could moreover be identified as non-transported substrates, but inhibitors of P-gp activity. Thus, drug-drug or drug-food interactions should be very likely for these compounds. PMID:25637762

  12. Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models.

    PubMed

    Zarrabi, Nawid; Ernst, Stefan; Verhalen, Brandy; Wilkens, Stephan; Börsch, Michael

    2014-03-15

    Single-molecule Förster resonance energy (smFRET) transfer has become a powerful tool for observing conformational dynamics of biological macromolecules. Analyzing smFRET time trajectories allows to identify the state transitions occuring on reaction pathways of molecular machines. Previously, we have developed a smFRET approach to monitor movements of the two nucleotide binding domains (NBDs) of P-glycoprotein (Pgp) during ATP hydrolysis driven drug transport in solution. One limitation of this initial work was that single-molecule photon bursts were analyzed by visual inspection with manual assignment of individual FRET levels. Here a fully automated analysis of Pgp smFRET data using hidden Markov models (HMM) for transitions up to 9 conformational states is applied. We propose new estimators for HMMs to integrate the information of fluctuating intensities in confocal smFRET measurements of freely diffusing lipid bilayer bound membrane proteins in solution. HMM analysis strongly supports that under conditions of steady state turnover, conformational states with short NBD distances and short dwell times are more populated compared to conditions without nucleotide or transport substrate present. PMID:23891547

  13. Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

    PubMed Central

    Kuo, M T; Vyas, R C; Jiang, L X; Hittelman, W N

    1994-01-01

    Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification. Images PMID:7913517

  14. Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

    PubMed

    Kuo, M T; Vyas, R C; Jiang, L X; Hittelman, W N

    1994-08-01

    Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification. PMID:7913517

  15. Molecular cloning and characterization of a P-glycoprotein from the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

    PubMed

    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

  16. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors.

    PubMed

    Noack, Andreas; Noack, Sandra; Buettner, Manuela; Naim, Hassan Y; Löscher, Wolfgang

    2016-01-01

    The blood-brain barrier (BBB) controls the entry of compounds into the brain, thereby regulating brain homeostasis. Efflux transporters such as P-glycoprotein (Pgp) significantly contribute to BBB function. Multiple signaling pathways modulate the expression and activity of Pgp in response to xenobiotics and disease. A non-genetic way of intercellular transfer of Pgp occurs in cancer cells, but whether this also occurs in non-cancer cells such as endothelial cells that form the BBB is not known. A human brain endothelial cell line (hCMEC/D3) was used to study whether cell-to-cell Pgp transfer occurs during co-culturing with Pgp-EGFP expressing hCMEC/D3 cells. The Pgp-EGFP fusion protein was transferred from donor to recipient cells by cell-to-cell contact and Pgp-EGFP enriched vesicles, which were exocytosed by donor cells and endocytosed by adherent recipient cells. Flow cytometry experiments with the Pgp substrate eFLUXX-ID Gold demonstrated that the transferred Pgp is functional in the recipient cells. Exposure of the donor cells with inhibitors of histone deacetylases (HDACs) resulted in an enhanced intercellular Pgp transfer. Non-genetic transfer of a resistance phenotype and its regulation by HDACs is a novel mechanism of altering BBB functionality. This mechanism may have important implications for understanding drug-induced alterations in Pgp expression and activity. PMID:27375084

  17. Induction of P-glycoprotein expression and activity by Aconitum alkaloids: Implication for clinical drug-drug interactions.

    PubMed

    Wu, Jinjun; Lin, Na; Li, Fangyuan; Zhang, Guiyu; He, Shugui; Zhu, Yuanfeng; Ou, Rilan; Li, Na; Liu, Shuqiang; Feng, Lizhi; Liu, Liang; Liu, Zhongqiu; Lu, Linlin

    2016-01-01

    The Aconitum species, which mainly contain bioactive Aconitum alkaloids, are frequently administered concomitantly with other herbal medicines or chemical drugs in clinics. The potential risk of drug-drug interactions (DDIs) arising from co-administration of Aconitum alkaloids and other drugs against specific targets such as P-glycoprotein (P-gp) must be evaluated. This study focused on the effects of three representative Aconitum alkaloids: aconitine (AC), benzoylaconine (BAC), and aconine, on the expression and activity of P-gp. We observed that Aconitum alkaloids increased P-gp expression in LS174T and Caco-2 cells in the order AC > BAC > aconine. Nuclear receptors were involved in the induction of P-gp. AC and BAC increased the P-gp transport activity. Strikingly, intracellular ATP levels and mitochondrial mass also increased. Furthermore, exposure to AC decreased the toxicity of vincristine and doxorubicin towards the cells. In vivo, AC significantly up-regulated the P-gp protein levels in the jejunum, ileum, and colon of FVB mice, and protected them against acute AC toxicity. Taken together, the findings of our in vitro and in vivo experiments indicate that AC can induce P-gp expression, and that co-administration of AC with P-gp substrate drugs may cause DDIs. Our findings have important implications for Aconitum therapy in clinics. PMID:27139035

  18. Expression of P-glycoprotein in excised human nasal mucosa and optimized models of RPMI 2650 cells.

    PubMed

    Dolberg, Anne M; Reichl, Stephan

    2016-07-11

    To assess the transmucosal drug transport in the development of medications for intranasal administration, cellular in vitro models are preferred over the use of animal tissues due to inter-species variations and ethical concerns. With regard to the distribution of active agents and multidrug resistance, the ABC transporter P-glycoprotein plays a major role in several mammalian tissues. The present study compares the expression of this efflux pump in optimized in vitro models based on the human RPMI 2650 cell line with specimens of human turbinate mucosa. The presence of the ABCB1 gene was investigated at the mRNA and protein levels using RT-PCR and Western blot analysis in differently cultured RPMI 2650 cells and excised human nasal epithelium. Furthermore, the localization and activity of P-gp was examined by immunohistochemical staining and functionality assays using different substrates in both in vitro and ex vivo models. Both mRNA and protein expression of P-gp was found in all studied models. Furthermore, transporter functionality was detected in both RPMI 2650 cell culture models and excised human mucosa. The results demonstrated a highly promising comparability between RPMI 2650 models and explants of human nasal tissue concerning the influence of MDR1 on drug disposition. The RPMI 2650 cell line might become a useful tool in preclinical trials to improve reproducibility and achieve greater applicability to humans of experimental data regarding passive diffusion and active efflux of drug candidates. PMID:27155589

  19. 3D-QSAR modelling dataset of bioflavonoids for predicting the potential modulatory effect on P-glycoprotein activity.

    PubMed

    Wongrattanakamon, Pathomwat; Lee, Vannajan Sanghiran; Nimmanpipug, Piyarat; Jiranusornkul, Supat

    2016-12-01

    The data is obtained from exploring the modulatory activities of bioflavonoids on P-glycoprotein function by ligand-based approaches. Multivariate Linear-QSAR models for predicting the induced/inhibitory activities of the flavonoids were created. Molecular descriptors were initially used as independent variables and a dependent variable was expressed as pFAR. The variables were then used in MLR analysis by stepwise regression calculation to build the linear QSAR data. The entire dataset consisted of 23 bioflavonoids was used as a training set. Regarding the obtained MLR QSAR model, R of 0.963, R (2)=0.927, [Formula: see text], SEE=0.197, F=33.849 and q (2)=0.927 were achieved. The true predictabilities of QSAR model were justified by evaluation with the external dataset (Table 4). The pFARs of representative flavonoids were predicted by MLR QSAR modelling. The data showed that internal and external validations may generate the same conclusion. PMID:27626051

  20. P-glycoprotein in adriamycin-resistant cells functions as an efflux pump for benzopyrene, a chemical carcinogen

    SciTech Connect

    Chao Yeh, G.; Poore, C.M.; Lopaczynska, J.; Phang, J.M. )

    1991-03-15

    The physiological function of multidrug resistant gene (MDR 1) coded P-glycoprotein 170 (P-gp) in normal tissues remains unknown. The authors propose that P-gp functions as an efflux pump in normal tissues for benzopyrene and other xenobiotic substances. To examine their hypothesis the authors used a series of MDR human breast cancer MCF-7 cells with increasing degrees of drug resistance, expression of MDR and levels of P-gp. First, they found the IC{sub 50} for benzopyrene is linearly correlated with the levels of P-gp at different stages of adriamycin resistant MCF-7 cells. Using P-gp ({sup 3}H)azidopine labeling as a measurement of P-gp they found benzopyrene competes for labeling of P-gp. Finally, they directly measured cellular efflux of benzopyrene with adherent cell laser cytometry and found that resistant cells expressing high levels of P-gp showed rapid efflux of benzopyrene. By contrast, drug sensitive wild type cells with undetectable P-gp showed negligible efflux. They conclude that P-gp can function as an efflux pump for benzopyrene and suggest that P-gp may be a cellular mechanism for resistance to carcinogens.

  1. Induction of P-glycoprotein expression and activity by Aconitum alkaloids: Implication for clinical drug–drug interactions

    PubMed Central

    Wu, Jinjun; Lin, Na; Li, Fangyuan; Zhang, Guiyu; He, Shugui; Zhu, Yuanfeng; Ou, Rilan; Li, Na; Liu, Shuqiang; Feng, Lizhi; Liu, Liang; Liu, Zhongqiu; Lu, Linlin

    2016-01-01

    The Aconitum species, which mainly contain bioactive Aconitum alkaloids, are frequently administered concomitantly with other herbal medicines or chemical drugs in clinics. The potential risk of drug–drug interactions (DDIs) arising from co-administration of Aconitum alkaloids and other drugs against specific targets such as P-glycoprotein (P-gp) must be evaluated. This study focused on the effects of three representative Aconitum alkaloids: aconitine (AC), benzoylaconine (BAC), and aconine, on the expression and activity of P-gp. We observed that Aconitum alkaloids increased P-gp expression in LS174T and Caco-2 cells in the order AC > BAC > aconine. Nuclear receptors were involved in the induction of P-gp. AC and BAC increased the P-gp transport activity. Strikingly, intracellular ATP levels and mitochondrial mass also increased. Furthermore, exposure to AC decreased the toxicity of vincristine and doxorubicin towards the cells. In vivo, AC significantly up-regulated the P-gp protein levels in the jejunum, ileum, and colon of FVB mice, and protected them against acute AC toxicity. Taken together, the findings of our in vitro and in vivo experiments indicate that AC can induce P-gp expression, and that co-administration of AC with P-gp substrate drugs may cause DDIs. Our findings have important implications for Aconitum therapy in clinics. PMID:27139035

  2. Rhubarb decreased the systemic exposure of cyclosporine, a probe substrate of P-glycoprotein and CYP 3A.

    PubMed

    Yu, Chung-Ping; Lin, Hui-Ju; Lin, Shiuan-Pey; Shia, Chi-Sheng; Chang, Pei-Hua; Hou, Yu-Chi; Hsieh, Yo-Wen

    2016-08-01

    1. Rhubarb, rhizome of Rheum palmatum L. (RP), is an important herb in clinical Chinese medicine. 2. Cyclosporine (CSP) is an immunosuppressant with narrow therapeutic window. The oral bioavailability of CSP was associated with P-glycoprotein (P-gp) and CYP 3A4. CSP was used as a probe substrate to investigate the in vivo modulation effects of RP on P-gp and CYP 3A. 3. Rats were orally administered 2.5 mg/kg of CSP with and without 0.25 and 1.0 g/kg of RP. The blood CSP concentration was determined by a specific monoclonal fluorescence polarization immunoassay. 4. Both dosages of RP significantly decreased the Cmax and AUC0-t of CSP in rats. Mechanism studies indicated that RP activated the functions of P-gp and CYP 3A. 5. RP ingestion reduced the systemic exposure of CSP through activating P-gp and CYP 3A. PMID:26634287

  3. BET bromodomain inhibition releases the Mediator complex from select cis-regulatory elements

    PubMed Central

    Bhagwat, Anand S.; Roe, Jae-Seok; Mok, Beverly A.; Hohmann, Anja F.; Shi, Junwei; Vakoc, Christopher R.

    2016-01-01

    The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. An shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance. PMID:27068464

  4. Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase Cγ-mediated Ca2+ mobilization

    PubMed Central

    Smith, Norah L.; Abi Abdallah, Delbert S.; Butcher, Barbara A.; Denkers, Eric Y.; Baird, Barbara; Holowka, David

    2013-01-01

    Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca2+-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca2+ responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca2+ mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca2+ mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca2+ mobilization, particularly antigen-mediated Ca2+ release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion. PMID:23847603

  5. Adiponectin Inhibits Insulin Function in Primary Trophoblasts by PPARα-Mediated Ceramide Synthesis

    PubMed Central

    Gao, Xiaoli; Weintraub, Susan T.; Jansson, Thomas; Powell, Theresa L.

    2014-01-01

    Maternal adiponectin (ADN) levels are inversely correlated with birth weight, and ADN infusion in pregnant mice down-regulates placental nutrient transporters and decreases fetal growth. In contrast to the insulin-sensitizing effects in adipose tissue and muscle, ADN inhibits insulin signaling in the placenta. However, the molecular mechanisms involved are unknown. We hypothesized that ADN inhibits insulin signaling and insulin-stimulated amino acid transport in primary human trophoblasts by peroxisome proliferator-activated receptor-α (PPARα)-mediated ceramide synthesis. Primary human term trophoblast cells were treated with ADN and/or insulin. ADN increased the phosphorylation of p38 MAPK and PPARα. ADN inhibited insulin signaling and insulin-stimulated amino acid transport. This effect was dependent on PPARα, because activation of PPARα with an agonist (GW7647) inhibited insulin signaling and function, whereas PPARα-small interfering RNA reversed the effects of ADN on the insulin response. ADN increased ceramide synthase expression and stimulated ceramide production. C2-ceramide inhibited insulin signaling and function, whereas inhibition of ceramide synthase (with Fumonisin B1) reversed the effects of ADN on insulin signaling and amino acid transport. These findings are consistent with the model that maternal ADN limits fetal growth mediated by activation of placental PPARα and ceramide synthesis, which inhibits placental insulin signaling and amino acid transport, resulting in reduced fetal nutrient availability. PMID:24606127

  6. Inhibition of xanthine oxidase-xanthine-iron mediated lipid peroxidation by eugenol in liposomes.

    PubMed

    Nagababu, E; Lakshmaiah, N

    1997-01-01

    The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe+3-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 microm concentration and hence formation of O2.- also. However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O2.- generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe2+ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe+3 ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. PMID:9046022

  7. Type I interferons mediate pancreatic toxicities of PERK inhibition.

    PubMed

    Yu, Qiujing; Zhao, Bin; Gui, Jun; Katlinski, Kanstantsin V; Brice, Angela; Gao, Yan; Li, ChangHong; Kushner, Jake A; Koumenis, Constantinos; Diehl, J Alan; Fuchs, Serge Y

    2015-12-15

    The great preclinical promise of the pancreatic endoplasmic reticulum kinase (PERK) inhibitors in neurodegenerative disorders and cancers is marred by pancreatic injury and diabetic syndrome observed in PERK knockout mice and humans lacking PERK function and suffering from Wolcott-Rallison syndrome. PERK mediates many of the unfolded protein response (UPR)-induced events, including degradation of the type 1 interferon (IFN) receptor IFNAR1 in vitro. Here we report that whole-body or pancreas-specific Perk ablation in mice leads to an increase in IFNAR1 protein levels and signaling in pancreatic tissues. Concurrent IFNAR1 deletion attenuated the loss of PERK-deficient exocrine and endocrine pancreatic tissues and prevented the development of diabetes. Experiments using pancreas-specific Perk knockouts, bone marrow transplantation, and cultured pancreatic islets demonstrated that stabilization of IFNAR1 and the ensuing increased IFN signaling in pancreatic tissues represents a major driver of injury triggered by Perk loss. Neutralization of IFNAR1 prevented pancreatic toxicity of PERK inhibitor, indicating that blocking the IFN pathway can mitigate human genetic disorders associated with PERK deficiency and help the clinical use of PERK inhibitors. PMID:26627716

  8. RNAi mediated Tiam1 gene knockdown inhibits invasion of retinoblastoma.

    PubMed

    Subramanian, Nithya; Navaneethakrishnan, Saranya; Biswas, Jyotirmay; Kanwar, Rupinder K; Kanwar, Jagat R; Krishnakumar, Subramanian

    2013-01-01

    T lymphoma invasion and metastasis protein (Tiam1) is up-regulated in variety of cancers and its expression level is related to metastatic potential of the type of cancer. Earlier, Tiam1 was shown to be overexpressed in retinoblastoma (RB) and we hypothesized that it was involved in invasiveness of RB. This was tested by silencing Tiam1 in RB cell lines (Y79 and Weri-Rb1) using siRNA pool, targeting different regions of Tiam1 mRNA. The cDNA microarray of Tiam1 silenced cells showed gene regulations altered by Tiam1 were predominantly on the actin cytoskeleton interacting proteins, apoptotic initiators and tumorogenic potential targets. The silenced phenotype resulted in decreased growth and increased apoptosis with non-invasive characteristics. Transfection of full length and N-terminal truncated construct (C1199) clearly revealed membrane localization of Tiam1 and not in the case of C580 construct. F-actin staining showed the interaction of Tiam1 with actin in the membrane edges that leads to ruffling, and also imparts varying invasive potential to the cell. The results obtained from our study show for the first time that Tiam1 modulates the cell invasion, mediated by actin cytoskeleton remodeling in RB. PMID:23950931

  9. Mosla dianthera inhibits mast cell-mediated allergic reactions through the inhibition of histamine release and inflammatory cytokine production

    SciTech Connect

    Lee, Dong-Hee; Kim, Sang-Hyun . E-mail: shkim72@knu.ac.kr; Eun, Jae-Soon; Shin, Tae-Yong . E-mail: tyshin@woosuk.ac.kr

    2006-11-01

    In this study, we investigated the effect of the aqueous extract of Mosla dianthera (Maxim.) (AEMD) on the mast cell-mediated allergy model and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases such as asthma, sinusitis and rheumatoid arthritis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. AEMD inhibited compound 48/80-induced systemic reactions in mice. AEMD decreased immunoglobulin E-mediated local allergic reactions, passive cutaneous anaphylaxis. AEMD attenuated intracellular calcium level and release of histamine from rat peritoneal mast cells activated by compound 48/80. Furthermore, AEMD attenuated the phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated TNF-{alpha}, IL-8 and IL-6 secretion in human mast cells. The inhibitory effect of AEMD on the pro-inflammatory cytokines was nuclear factor-{kappa}B (NF-{kappa}B) dependent. AEMD decreased PMA and A23187-induced degradation of I{kappa}B{alpha} and nuclear translocation of NF-{kappa}B. Our findings provide evidence that AEMD inhibits mast cell-derived immediate-type allergic reactions and involvement of pro-inflammatory cytokines and NF-{kappa}B in these effects.

  10. Retinal co-mediator acetylcholine evokes muscarinic inhibition of recurrent excitation in frog tectum column.

    PubMed

    Baginskas, Armantas; Kuras, Antanas

    2016-08-26

    Acetylcholine receptors contribute to the control of neuronal and neuronal network activity from insects to humans. We have investigated the action of acetylcholine receptors in the optic tectum of Rana temporaria (common frog). Our previous studies have demonstrated that acetylcholine activates presynaptic nicotinic receptors, when released into the frog optic tectum as a co-mediator during firing of a single retinal ganglion cell, and causes: a) potentiation of retinotectal synaptic transmission, and b) facilitation of transition of the tectum column to a higher level of activity. In the present study we have shown that endogenous acetylcholine also activates muscarinic receptors, leading to a delayed inhibition of recurrent excitatory synaptic transmission in the tectum column. The delay of muscarinic inhibition was evaluated to be of ∼80ms, with an extent of inhibition of ∼2 times. The inhibition of the recurrent excitation determines transition of the tectum column back to its resting state, giving a functional sense for the inhibition. PMID:27394688

  11. BRAF inhibition generates a host/tumor niche that mediates therapeutic escape

    PubMed Central

    Fedorenko, Inna V.; Wargo, Jennifer A.; Flaherty, Keith T.; Messina, Jane L.; Smalley, Keiran S.M.

    2015-01-01

    The current study defines a fibroblast-derived niche that facilitates the therapeutic escape of melanoma cells from BRAF inhibition. Vemurafenib treatment led to the release of TGF-β from the melanoma cells that increased the differentiation state of the fibroblasts; an affect associated with fibronectin deposition, increase in α-smooth muscle actin (α–SMA) expression and the release of neuregulin (NRG). At the same time, vemurafenib directly activated the fibroblasts through paradoxical stimulation of the MAPK pathway, causing them to secrete hepatocyte growth factor (HGF). Treatment with the BRAF/MEK inhibitor combination reversed the release of HGF. Adhesion of melanoma cells to fibronectin was critical in amplifying the fibroblast-derived NRG and HGF-mediated PI3K/AKT survival signaling in the melanoma cells following BRAF inhibition. In co-culture studies, combination treatment with inhibitors of BRAF/MET/HER kinase was ineffective at reversing the fibroblast-mediated therapeutic escape from BRAF inhibition. Instead, it was noted that combined BRAF/PI3K inhibition overcame fibroblast-mediated drug resistance in vitro and was associated with enhanced anti-tumor effects in an in vivo xenograft model. Thus, we show melanoma cells and fibroblasts remodel their microenvironment in response to BRAF inhibition and that these adaptations allow tumor cells to evade therapy through increased PI3K/AKT survival signaling. PMID:26302068

  12. BRAF Inhibition Generates a Host-Tumor Niche that Mediates Therapeutic Escape.

    PubMed

    Fedorenko, Inna V; Wargo, Jennifer A; Flaherty, Keith T; Messina, Jane L; Smalley, Keiran S M

    2015-12-01

    The current study defines a fibroblast-derived niche that facilitates the therapeutic escape of melanoma cells from BRAF inhibition. Vemurafenib treatment led to the release of transforming growth factor-β (TGF-β) from the melanoma cells that increased the differentiation state of the fibroblasts, an affect associated with fibronectin deposition, increase in α-smooth muscle actin expression, and the release of neuregulin (NRG). At the same time, vemurafenib directly activated the fibroblasts through paradoxical stimulation of the mitogen-activated protein kinase pathway, causing them to secrete hepatocyte growth factor (HGF). Treatment with the BRAF/MEK inhibitor combination reversed the release of HGF. Adhesion of melanoma cells to fibronectin was critical in amplifying the fibroblast-derived NRG- and HGF-mediated PI3K/AKT (phosphatidylinositol 3'-kinase/AKT) survival signaling in the melanoma cells following BRAF inhibition. In coculture studies, combination treatment with inhibitors of BRAF/MET/HER kinase was ineffective at reversing the fibroblast-mediated therapeutic escape from BRAF inhibition. Instead, it was noted that combined BRAF/PI3K inhibition overcame fibroblast-mediated drug resistance in vitro and was associated with enhanced antitumor effects in an in vivo xenograft model. Thus, we show that melanoma cells and fibroblasts remodel their microenvironment in response to BRAF inhibition and that these adaptations allow tumor cells to evade therapy through increased PI3K/AKT survival signaling. PMID:26302068

  13. Tempol inhibits neutrophil and hydrogen peroxide-mediated DNA damage.

    PubMed

    Hahn, S M; Mitchell, J B; Shacter, E

    1997-01-01

    Inflammatory conditions characterized by neutrophil activation are associated with a variety of chronic diseases. Reactive oxygen species are produced by activated neutrophils and produce DNA damage which may lead to tissue damage. Previous studies have shown that activated murine neutrophils induce DNA strand breaks in a target plasmacytoma cell, RIMPC 2394. We studied the effect of a water soluble nitroxide anti-oxidant, Tempol, on murine neutrophil induction of DNA strand breaks in this system. Murine neutrophils were isolated from the peritoneal cavity of BALB/cAn mice after an i.p. injection of pristane oil. Neutrophils were activated by the phorbol ester PMA and co-incubated with RIMPC 2394 cells. Control alkaline elution studies revealed progressive DNA strand breaks in RIMPC cells with time. The addition of Tempol to the incubation mixture prevented DNA damage in a dose dependent fashion. Five mM Tempol provided complete protection. Tempol protection against DNA strand breaks was similar for both stimulated neutrophils and exogenously added hydrogen peroxide. Measurement of hydrogen peroxide produced by stimulated neutrophils demonstrated that Tempol did not decrease hydrogen peroxide concentration. Oxidation of reduced metals, thereby interfering with the production of hydroxyl radical, is the most likely mechanism of nitroxide protection, although superoxide dismutase (SOD) like activity and scavenging of carbon-based free radicals may also account for a portion of the observed protection. The anti-oxidant activity of Tempol inhibited DNA damage by activated neutrophils. The nitroxides as a class of compounds may have a role in the investigation and modification of inflammatory conditions. PMID:9378367

  14. Imaging an optogenetic pH sensor reveals that protons mediate lateral inhibition in the retina

    PubMed Central

    Wang, Tzu-Ming; Holzhausen, Lars C.; Kramer, Richard H.

    2014-01-01

    The reciprocal synapse between photoreceptors and horizontal cells (HCs) underlies lateral inhibition and establishes the antagonistic center-surround receptive fields of retinal neurons, to enhance visual contrast. Despite decades of study, the signal mediating negative feedback from HCs to cones has remained controversial because the small, invaginated synaptic cleft has precluded measurement. Using zebrafish retinas, we show that light elicits a change in synaptic proton concentration with the correct magnitude, kinetics and spatial dependence to account for lateral inhibition. Light, which hyperpolarizes HCs, causes synaptic alkalinization, whereas activating an exogenously expressed ligand-gated Na+ channel, which depolarizes HCs, causes synaptic acidification. While acidification was prevented by blocking a proton pump, re-alkalinization was prevented by blocking proton-permeant ion channels, suggesting that distinct mechanisms underlie proton efflux and influx. These findings reveal that protons mediate lateral inhibition in the retina, raising the possibility that protons are unrecognized retrograde messengers elsewhere in the nervous system. PMID:24441679

  15. Inhibition of acetyl-CoA carboxylase by cystamine may mediate the hypotriglyceridemic activity of pantethine.

    PubMed

    McCarty, M F

    2001-03-01

    Pantethine is a versatile and well-tolerated hypolipidemic agent whose efficacy in this regard appears to be mediated by its catabolic product cystamine, a nucleophile which avidly attacks disulfide groups. An overview of pantethine research suggests that the hypotriglyceridemic activity of pantethine reflects cystamine-mediated inhibition of the hepatic acetyl-CoA carboxylase, which can be expected to activate hepatic fatty acid oxidation. Inhibition of HMG-CoA reductase as well as a more distal enzyme in the cholesterol synthetic pathway may account for pantethine's hypocholesterolemic effects. If pantethine does indeed effectively inhibit hepatic acetyl-CoA carboxylase, it may have adjuvant utility in the hepatothermic therapy of obesity. As a safe and effective compound of natural origin, pantethine merits broader use in the management of hyperlipidemias. PMID:11359352

  16. Inhibition of M current in sensory neurons by exogenous proteases: a signaling pathway mediating inflammatory nociception.

    PubMed

    Linley, John E; Rose, Kirstin; Patil, Mayur; Robertson, Brian; Akopian, Armen N; Gamper, Nikita

    2008-10-29

    Inflammatory pain is thought to be mediated in part through the action of inflammatory mediators on membrane receptors of peripheral nerve terminals, however, the downstream signaling events which lead to pain are poorly understood. In this study we investigated the nociceptive pathways induced by activation of protease-activated receptor 2 (PAR-2) in damage-sensing (nociceptive) neurons from rat dorsal root ganglion (DRG). We found that activation of PAR-2 in these cells strongly inhibited M-type potassium currents (conducted by Kv7 potassium channels). Such inhibition caused depolarization of the neuronal resting membrane potential leading, ultimately, to nociception. Consistent with this mechanism, injection of the specific M channel blocker XE991 into rat paw induced nociception in a concentration-dependent manner. Injection of a PAR-2 agonist peptide also induced nociception but coinjection of XE991 and the PAR-2 agonist did not result in summation of nociception, suggesting that the action of both agents may share a similar mechanism. We also studied the signaling pathway of M current inhibition by PAR-2 using patch-clamp and fluorescence imaging of DRG neurons. These experiments revealed that the PAR-2 effect was mediated by phospholipase C (PLC). Further experiments demonstrated that M current inhibition required concurrent rises in cytosolic Ca(2+) concentration and depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We propose that PLC- and Ca(2+)/PIP(2)-mediated inhibition of M current in sensory neurons may represent one of the general mechanisms underlying pain produced by inflammatory mediators, and may therefore open up a new therapeutic window for treatment of this major clinical problem. PMID:18971466

  17. Investigation of the effect of the uneven distribution of CYP3A4 and P-glycoprotein in the intestine on the barrier function against xenobiotics: a simulation study.

    PubMed

    Watanabe, Takao; Maeda, Kazuya; Nakai, Chikako; Sugiyama, Yuichi

    2013-09-01

    CYP3A4 and P-glycoprotein (P-gp) have similar substrate specificities and work together to form an intestinal absorption barrier against xenobiotics. Previous reports have indicated that CYP3A4 expression decreases gradually, whereas P-gp expression increases, from the upper to lower small intestine. The physiological rationale for this uneven distribution of CYP3A4 and P-gp as a barrier against xenobiotics has not been determined. To clarify the effect of these distribution patterns on barrier function, we constructed a mathematical model that included passive membrane permeation, P-gp-mediated apical efflux, and CYP3A4-mediated metabolism, and we simulated the effects of these distribution patterns on the fraction absorbed of co-substrates without changing their overall activities. The simulation showed that the physiological distribution patterns of both CYP3A4 and P-gp result in the lowest fraction absorbed, but not for drugs with low CYP3A4 and high P-gp-mediated clearances. These results suggest that the distribution pattern of CYP3A4 is especially important for the barrier function. On the other hand, physiological distribution pattern of P-gp exerts the maximum barrier function for dual good substrates for P-gp and CYP3A4, but even distribution of P-gp mostly suppresses the intestinal absorption of good P-gp, but poor CYP3A4 substrates. PMID:23754337

  18. P-glycoprotein is responsible for the poor intestinal absorption and low toxicity of oral aconitine: In vitro, in situ, in vivo and in silico studies

    SciTech Connect

    Yang, Cuiping Zhang, Tianhong Li, Zheng Xu, Liang Liu, Fei Ruan, Jinxiu Liu, Keliang Zhang, Zhenqing

    2013-12-15

    Aconitine (AC) is a highly toxic alkaloid from bioactive plants of the genus Aconitum, some of which have been widely used as medicinal herbs for thousands of years. In this study, we systematically evaluated the potential role of P-glycoprotein (P-gp) in the mechanisms underlying the low and variable bioavailability of oral AC. First, the bidirectional transport of AC across Caco-2 and MDCKII-MDR1 cells was investigated. The efflux of AC across monolayers of these two cell lines was greater than its influx. Additionally, the P-gp inhibitors, verapamil and cyclosporin A, significantly decreased the efflux of AC. An in situ intestinal perfusion study in rats showed that verapamil co-perfusion caused a significant increase in the intestinal permeability of AC, from 0.22 × 10{sup −5} to 2.85 × 10{sup −5} cm/s. Then, the pharmacokinetic profile of orally administered AC with or without pre-treatment with verapamil was determined in rats. With pre-treatment of verapamil, the maximum plasma concentration (C{sub max}) of AC increased sharply, from 39.43 to 1490.7 ng/ml. Accordingly, a 6.7-fold increase in the area under the plasma concentration–time curve (AUC{sub 0–12} {sub h}) of AC was observed when co-administered with verapamil. In silico docking analyses suggested that AC and verapamil possess similar P-gp recognition mechanisms. This work demonstrated that P-gp is involved in limiting the intestinal absorption of AC and attenuating its toxicity to humans. Our data indicate that potential P-gp-mediated drug–drug interactions should be considered carefully in the clinical application of aconite and formulations containing AC. - Highlights: • Verapamil and cyclosporin A decreased the efflux of aconitine across Caco-2 cells. • Both inhibitors decreased the efflux of aconitine across MDCKII-MDR1 cells. • Co-perfusion with verapamil increased the intestinal permeability of aconitine. • Co-administration with verapamil sharply increased the C{sub max

  19. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    PubMed Central

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-01-01

    AIM: To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development. METHODS: Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1/Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of host-microbiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that

  20. Sorafenib induces apoptosis in HL60 cells by inhibiting Src kinase-mediated STAT3 phosphorylation.

    PubMed

    Zhao, Wei; Zhang, Tao; Qu, Bingqian; Wu, Xingxin; Zhu, Xu; Meng, Fanyu; Gu, Yanhong; Shu, Yongqian; Shen, Yan; Sun, Yang; Xu, Qiang

    2011-01-01

    Signal transducer and activator of transcription 3 (STAT3) is constitutively active in approximately 50% of acute myeloid leukemia (AML) cases and mediates multiple cellular processes including cell resistance to apoptosis. Inhibition of constitutively active STAT3 has been shown to induce AML cell apoptosis. Our aim was to ascertain if sorafenib, a multikinase inhibitor, may also inhibit STAT3 signaling and, therefore, be efficacious for AML. We found that sorafenib inhibited proliferation and induced apoptosis in human AML cell line (HL60) cells. In addition, sorafenib exposure reduced constitutive STAT3 phosphorylation in HL60 cells and repressed STAT3 DNA-binding activity and Mcl-1 and Bcl-2 expression. Similar results were obtained with the Src kinase inhibitor I, suggesting that sorafenib suppresses STAT3 phosphorylation by inhibiting Src-kinase activity. Furthermore, significant inhibition of Src kinase activity by sorafenib was observed in the kinase assay. In addition, Src could be co-immunoprecipitated with STAT3, and the phosphorylation of STAT3 was significantly inhibited by sorafenib only in cell lines in which phosphorylated Src is highly expressed. Taken together, our study indicates that sorafenib blocks Src kinase-mediated STAT3 phosphorylation and decreases the expression of apoptosis regulatory proteins Mcl-1 and Bcl-2, which are associated with increased apoptosis in HL60 cells. These findings provide a rationale for the treatment of human AML. PMID:20881478

  1. GSK-3 Inhibition Sensitizes Acute Myeloid Leukemia Cells to 1,25D-Mediated Differentiation.

    PubMed

    Gupta, Kalpana; Stefan, Tammy; Ignatz-Hoover, James; Moreton, Stephen; Parizher, Gary; Saunthararajah, Yogen; Wald, David N

    2016-05-01

    1,25-dihydroxyvitamin D3 (1,25D), the biologically active form of vitamin D, is widely considered a promising therapy for acute myeloid leukemia (AML) based on its ability to drive differentiation of leukemic cells. However, clinical trials have been disappointing in part to dose-limiting hypercalcemia. Here we show how inhibiting glycogen synthase kinase 3 (GSK3) can improve the differentiation response of AML cells to 1,25D-mediated differentiation. GSK3 inhibition in AML cells enhanced the differentiating effects of low concentrations of 1,25D. In addition, GSK3 inhibition augmented the ability of 1,25D to induce irreversible growth inhibition and slow the progression of AML in mouse models. Mechanistic studies revealed that GSK3 inhibition led to the hyperphosphorylation of the vitamin D receptor (VDR), enabling an interaction between VDR and the coactivator, SRC-3 (NCOA3), thereby increasing transcriptional activity. We also found that activation of JNK-mediated pathways in response to GSK3 inhibition contributed to the potentiation of 1,25D-induced differentiation. Taken together, our findings offer a preclinical rationale to explore the repositioning of GSK3 inhibitors to enhance differentiation-based therapy for AML treatment. Cancer Res; 76(9); 2743-53. ©2016 AACR. PMID:26964622

  2. Bone marrow-derived mesenchymal stromal cells inhibit Th2-mediated allergic airways inflammation in mice.

    PubMed

    Goodwin, Meagan; Sueblinvong, Viranuj; Eisenhauer, Philip; Ziats, Nicholas P; LeClair, Laurie; Poynter, Matthew E; Steele, Chad; Rincon, Mercedes; Weiss, Daniel J

    2011-07-01

    Bone marrow-derived mesenchymal stromal cells (BMSCs) mitigate inflammation in mouse models of acute lung injury. However, specific mechanisms of BMSC actions on CD4 T lymphocyte-mediated inflammation in vivo remain poorly understood. Limited data suggests promotion of Th2 phenotype in models of Th1-mediated diseases. However, whether this might alleviate or worsen Th2-mediated diseases such as allergic asthma is unknown. To ascertain the effects of systemic administration of BMSCs in a mouse model of Th2-mediated allergic airways inflammation, ovalbumin (OVA)-induced allergic airways inflammation was induced in wild-type C57BL/6 and BALB/c mice as well as in interferon-γ (IFNγ) receptor null mice. Effects of systemic administration during antigen sensitization of either syngeneic or allogeneic BMSC on airways hyperreactivity, lung inflammation, antigen-specific CD4 T lymphocytes, and serum immunoglobulins were assessed. Both syngeneic and allogeneic BMSCs inhibited airways hyperreactivity and lung inflammation through a mechanism partly dependent on IFNγ. However, contrary to existing data, BMSCs did not affect antigen-specific CD4 T lymphocyte proliferation but rather promoted Th1 phenotype in vivo as assessed by both OVA-specific CD4 T lymphocyte cytokine production and OVA-specific circulating immunoglobulins. BMSCs treated to prevent release of soluble mediators and a control cell population of primary dermal skin fibroblasts only partly mimicked the BMSC effects and in some cases worsened inflammation. In conclusion, BMSCs inhibit Th2-mediated allergic airways inflammation by influencing antigen-specific CD4 T lymphocyte differentiation. Promotion of a Th1 phenotype in antigen-specific CD4 T lymphocytes by BMSCs is sufficient to inhibit Th2-mediated allergic airways inflammation through an IFNγ-dependent process. PMID:21544902

  3. Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody.

    PubMed

    Gutay-Tóth, Zsuzsanna; Fenyvesi, Ferenc; Bársony, Orsolya; Szente, Lajos; Goda, Katalin; Szabó, Gábor; Bacsó, Zsolt

    2016-03-01

    The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb. PMID:26704667

  4. Identification of residues in the drug translocation pathway of the human multidrug resistance P-glycoprotein by arginine mutagenesis.

    PubMed

    Loo, Tip W; Bartlett, M Claire; Clarke, David M

    2009-09-01

    P-glycoprotein (P-gp, ATP-binding cassette B1) is a drug pump that extracts toxic drug substrates from the plasma membrane and catalyzes their ATP-dependent efflux. To map the residues in the drug translocation pathway, we performed arginine-scanning mutagenesis on all transmembrane (TM) segments (total = 237 residues) of a P-gp processing mutant (G251V) defective in folding (15% maturation efficiency) (glycosylation state used to monitor folding). The rationale was that arginines introduced into the drug-binding sites would mimic drug rescue and enhance maturation of wild-type or processing mutants of P-gp. It was found that 38 of the 89 mutants that matured had enhanced maturation. Enhancer mutations were found in 11 of the 12 TM segments with the largest number found in TMs 6 and 12 (seven in each), TMs that are critical for P-gp-drug substrate interactions. Modeling of the TM segments showed that the enhancer arginines were found on the hydrophilic face, whereas inhibitory arginines were located on a hydrophobic face that may be in contact with the lipid bilayer. It was found that many of the enhancer arginines caused large alterations in P-gp-drug interactions in ATPase assays. For example, mutants A302R (TM5), L339R (TM6), G872R (TM10), F942R (TM11), Q946R (TM11), V982R (TM12), and S993R (TM12) reduced the apparent affinity for verapamil by approximately 10-fold, whereas the F336R (TM6) and M986R (TM12) mutations caused at least a 10-fold increase in apparent affinity for rhodamine B. The results suggest that P-gp contains a large aqueous-filled drug translocation pathway with multiple drug-binding sites that can accommodate the bulky arginine side chains to promote folding of the protein. PMID:19581304

  5. P-Glycoprotein (ABCB1) limits the brain distribution of YQA-14, a novel dopamine D3 receptor antagonist.

    PubMed

    Liu, Fei; Wang, Xiaoqing; Li, Zheng; Li, Jin; Zhuang, Xiaomei; Zhang, Zhenqing

    2015-01-01

    YQA-14 is a promising agent for treating addiction to cocaine and opioids. However, previous studies have showed there is marked contrast between the relatively small differences in pharmacological action in vivo and the large differences in their respective receptor binding properties in vitro. We hypothesized that the conflict between the in vivo and in vitro outcomes was attributable to poor brain exposure to YQA-14 caused by drug efflux transporters. To address this issue, we investigated the directional flux of YQA-14 across Caco-2 cells at 37°C or 4°C and the bidirectional transport in the presence and absence of transporter chemical inhibitors. These phenomena were further investigated by an in vivo determination of the brain and blood pharmacokinetics (PK) profile of YQA-14 following intraperitoneal administration with and without inhibitor. The efflux ratio of YQA-14 on Caco-2 cell monolayers was 2.39 and the efflux was temperature-dependent. When co-incubated with GF120918 or LY335979, the efflux of YQA-14 was markedly decreased. However, there was no significant difference in the permeability of YQA-14 when the cells were treated with Ko143. In vivo experiments showed that the brain-to-plasma ratio increased by more than 75-fold and 20-fold with co-administration of GF120918 and LY335979, respectively. Use of Ko143 did not change the brain-to-blood ratio of YQA-14. The results indicate that the brain distribution of YQA-14 was restricted because of active efflux transport at the blood brain barrier. In addition, P-glycoprotein (P-gp) played a dominant role in limiting the distribution of YQA-14 to the brain. PMID:26133067

  6. Induction of Expression and Functional Activity of P-glycoprotein Efflux Transporter by Bioactive Plant Natural Products

    PubMed Central

    Abuznait, Alaa H.; Qosa, Hisham; O’Connell, Nicholas D.; Akbarian-Tefaghi, Jessica; Sylvester, Paul W.; El Sayed, Khalid A.; Kaddoumi, Amal

    2011-01-01

    The effect of bioactive plant natural products on the expression and functional activity of P-glycoprotein (P-gp) is poorly understood. Interactions of bioactive plant-based food and dietary supplements with P-gp can cause significant alteration of pharmacokinetic properties of P-gp substrate drugs when used in combination. This can augment toxicity and/or interfere with the drug’s therapeutic outcomes. This study investigated the effects of diverse commonly used plant natural products on the expression and activity of P-gp in human adenocarcinoma cells (LS-180). These natural products included the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (cembratriene), the palm oil-derived γ-tocotrienol, the extra-virgin olive oil-derived secoiridoid oleocanthal, and the triterpene acid asiatic acid derived from Melaleuca ericifolia and abundant in several other common plant dietary supplements. Treatment with 25 μM of cembratriene, oleocanthal, γ-tocotrienol, or asiatic acid showed 2.3-3.0-fold increase in P-gp expression as demonstrated by Western blotting. These results were consistent with those obtained by quantitative analysis of fluorescent micrographs for P-gp. Accumulation studies demonstrated 31-38% decrease in rhodamine 123 intracellular levels when LS-180 cells were treated with the investigated compounds as a result of P-gp induction. Bioactive natural products can up-regulate the P-gp expression and functionality, which may induce herb/food-drug interactions when concomitantly used with P-gp substrate drugs. PMID:21851848

  7. Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

    PubMed

    Swartz, Douglas J; Mok, Leo; Botta, Sri K; Singh, Anukriti; Altenberg, Guillermo A; Urbatsch, Ina L

    2014-01-01

    Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful. PMID:24825346

  8. P-Glycoprotein Limits Oral Availability, Brain Penetration, and Toxicity of an Anionic Drug, the Antibiotic Salinomycin▿

    PubMed Central

    Lagas, Jurjen S.; Sparidans, Rolf W.; van Waterschoot, Robert A. B.; Wagenaar, Els; Beijnen, Jos H.; Schinkel, Alfred H.

    2008-01-01

    Salinomycin is a polyether organic anion that is extensively used as a coccidiostatic antibiotic in poultry and commonly fed to ruminant animals to improve feed efficiency. However, salinomycin also causes severe toxicity when accidentally fed to animals in high doses. In addition, humans are highly sensitive to salinomycin and severe toxicity has been reported. Multidrug efflux transporters like P-glycoprotein (P-gp), BCRP, and MRP2 are highly expressed in the intestine and can restrict the oral uptake and tissue penetration of xenobiotics. The purpose of this study was to investigate whether the anionic drug salinomycin is a substrate for one or more of these efflux pumps. Salinomycin was actively transported by human MDR1 P-gp expressed in polarized MDCK-II monolayers but not by the known organic anion transporters human MRP2 and murine Bcrp1. Using P-gp-deficient mice, we found a marked increase in plasma salinomycin concentrations after oral administration and decreased plasma clearance after intravenous administration. Furthermore, absence of P-gp resulted in significantly increased brain penetration. P-gp-deficient mice also displayed clearly increased susceptibility to salinomycin toxicity. Thus far, P-gp was thought to affect mainly hydrophobic, positively charged or neutral drugs in vivo. Our data show that P-gp can also be a major determinant of the pharmacokinetic behavior and toxicity of an organic anionic drug. Variation in P-gp activity might thus directly affect the effective exposure to salinomycin and possibly to other anionic drugs and toxin substrates. Individuals with reduced or absent P-gp activity could therefore be more susceptible to salinomycin toxicity. PMID:18195061

  9. Turning the gun on cancer: Utilizing lysosomal P-glycoprotein as a new strategy to overcome multi-drug resistance.

    PubMed

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

    2016-07-01

    Oxidative stress plays a role in the development of drug resistance in cancer cells. Cancer cells must constantly and rapidly adapt to changes in the tumor microenvironment, due to alterations in the availability of nutrients, such as glucose, oxygen and key transition metals (e.g., iron and copper). This nutrient flux is typically a consequence of rapid growth, poor vascularization and necrosis. It has been demonstrated that stress factors, such as hypoxia and glucose deprivation up-regulate master transcription factors, namely hypoxia inducible factor-1α (HIF-1α), which transcriptionally regulate the multi-drug resistance (MDR), transmembrane drug efflux transporter, P-glycoprotein (Pgp). Interestingly, in addition to the established role of plasma membrane Pgp in MDR, a new paradigm of intracellular resistance has emerged that is premised on the ability of lysosomal Pgp to transport cytotoxic agents into this organelle. This mechanism is enabled by the topological inversion of Pgp via endocytosis resulting in the transporter actively pumping agents into the lysosome. In this way, classical Pgp substrates, such as doxorubicin (DOX), can be actively transported into this organelle. Within the lysosome, DOX becomes protonated upon acidification of the lysosomal lumen, causing its accumulation. This mechanism efficiently traps DOX, preventing its cytotoxic interaction with nuclear DNA. This review discusses these effects and highlights a novel mechanism by which redox-active and protonatable Pgp substrates can utilize lysosomal Pgp to gain access to this compartment, resulting in catastrophic lysosomal membrane permeabilization and cell death. Hence, a key MDR mechanism that utilizes Pgp (the "gun") to sequester protonatable drug substrates safely within lysosomes can be "turned on" MDR cancer cells to destroy them from within. PMID:27154979

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

    PubMed

    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

  11. Michaelis-Menten kinetic analysis of drugs of abuse to estimate their affinity to human P-glycoprotein.

    PubMed

    Meyer, Markus R; Orschiedt, Tina; Maurer, Hans H

    2013-02-27

    The pharmacokinetics of various important drugs are known to be significantly influenced by the human ABC transporter P-glycoprotein (P-gp), which may lead to clinically relevant drug-drug interactions. In contrast to therapeutic drugs, emerging drugs of abuse (DOA) are sold and consumed without any safety pharmacology testing. Only some studies on their metabolism were published, but none about their affinity to the transporter systems. Therefore, 47 DOAs from various classes were tested for their P-gp affinity using human P-gp (hP-gp) to predict possible drug-drug interactions. DOAs were initially screened for general hP-gp affinity and further characterized by modeling classic Michaelis-Menten kinetics and assessing their K(m) and V(max) values. Among the tested drugs, 12 showed a stimulation of ATPase activity. The most intensive stimulating DOAs were further investigated and compared with the known P-gp model substrates sertraline and verapamil. ATPase stimulation kinetics coul