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Sample records for p-glycoprotein inhibition cell

  1. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

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    Hiroshi Fujise

    2004-01-01

    Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

  2. P-glycoprotein inhibition of drug resistant cell lines by nanoparticles.

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    Singh, Manu Smriti; Lamprecht, Alf

    2016-01-01

    Several pharmaceutical excipients are known for their ability to interact with cell membrane lipids and reverse the phenomenon of multidrug resistance (MDR) in cancer. Interestingly, many excipients act as stabilizers and are key ingredients in a variety of nano-formulations. In this study, representatives of ionic and non-ionic excipients were used as surface active agents in nanoparticle (NP) formulations to utilize their MDR reversing potential. In-vitro assays were performed to elucidate particle-cell interaction and accumulation of P-glycoprotein (P-gp) substrates-rhodamine-123 and calcein AM, in highly drug resistant glioma cell lines. Chemosensitization achieved using NPs and their equivalent dose of free excipients was assessed with the co-administered anti-cancer drug doxorubicin. Among the excipients used, non-ionic surfactant, Cremophor® EL, and cationic surfactant, cetyltrimethylammonuium bromide (CTAB), demonstrated highest P-gp modulatory activity in both free solution form (up to 7-fold lower IC50) and as a formulation (up to 4.7-fold lower IC50) as compared to doxorubicin treatment alone. Solutol® HS15 and Tween® 80 exhibited considerable chemosensitization as free solution but not when incorporated into a formulation. Sodium dodecyl sulphate (SDS)-based nanocarriers resulted in slightly improved cytotoxicity. Overall, the results highlight and envisage the usage of excipient in nano-formulations in a bid to improve chemosensitization of drug resistant cancer cells towards anti-cancer drugs.

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

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

    2017-01-15

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

  4. Ursodeoxycholic acid inhibits overexpression of P-glycoprotein induced by doxorubicin in HepG2 cells.

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    Komori, Yuki; Arisawa, Sakiko; Takai, Miho; Yokoyama, Kunihiro; Honda, Minako; Hayashi, Kazuhiko; Ishigami, Masatoshi; Katano, Yoshiaki; Goto, Hidemi; Ueyama, Jun; Ishikawa, Tetsuya; Wakusawa, Shinya

    2014-02-05

    The hepatoprotective action of ursodeoxycholic acid (UDCA) was previously suggested to be partially dependent on its antioxidative effect. Doxorubicin (DOX) and reactive oxygen species have also been implicated in the overexpression of P-glycoprotein (P-gp), which is encoded by the MDR1 gene and causes antitumor multidrug resistance. In the present study, we assessed the effects of UDCA on the expression of MDR1 mRNA, P-gp, and intracellular reactive oxygen species levels in DOX-treated HepG2 cells and compared them to those of other bile acids. DOX-induced increases in reactive oxygen species levels and the expression of MDR1 mRNA were inhibited by N-acetylcysteine, an antioxidant, and the DOX-induced increase in reactive oxygen species levels and DOX-induced overexpression of MDR1 mRNA and P-gp were inhibited by UDCA. Cells treated with UDCA showed improved rhodamine 123 uptake, which was decreased in cells treated with DOX alone. Moreover, cells exposed to DOX for 24h combined with UDCA accumulated more DOX than that of cells treated with DOX alone. Thus, UDCA may have inhibited the overexpression of P-gp by suppressing DOX-induced reactive oxygen species production. Chenodeoxycholic acid (CDCA) also exhibited these effects, whereas deoxycholic acid and litocholic acid were ineffective. In conclusion, UDCA and CDCA had an inhibitory effect on the induction of P-gp expression and reactive oxygen species by DOX in HepG2 cells. The administration of UDCA may be beneficial due to its ability to prevent the overexpression of reactive oxygen species and acquisition of multidrug resistance in hepatocellular carcinoma cells. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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    Jianfang Chen

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

  6. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

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

    2014-02-01

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

  7. Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein.

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    Villar, Victor Hugo; Vögler, Oliver; Barceló, Francisca; Gómez-Florit, Manuel; Martínez-Serra, Jordi; Obrador-Hevia, Antònia; Martín-Broto, Javier; Ruiz-Gutiérrez, Valentina; Alemany, Regina

    2014-04-01

    The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10-100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3 ± 1.11% and 68.8 ± 1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5-10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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    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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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

    2018-01-01

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

  10. Algerian Propolis Potentiates Doxorubicin Mediated Anticancer Effect against Human Pancreatic PANC-1 Cancer Cell Line through Cell Cycle Arrest, Apoptosis Induction and P-Glycoprotein Inhibition.

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    Rouibah, Hassiba; Mesbah, Lahouel; Kebsa, Wided; Zihlif, Malek; Ahram, Mamoun; Aburmeleih, Bachaer; Mostafa, Ibtihal; El Amir, Hemzeh

    2018-01-10

    Pancreatic cancer is one of the most aggressive and lethal cancer, with poor prognosis and high resistant to current chemotherapeutic agents. Therefore, new therapeutic strategies and targets are underscored. Propolis has been reported to exhibit a broad spectrum of biological activities including anticancer activity. This study was carried out to assess the possible efficacy of Algerian propolis on the antitumor effect of doxorubicin on human pancreatic cancer cell line (PANC-1). Modifications in cell viability, apoptosis and cell cycle progression, Pgp activity and intracellular accumulation of DOX were monitored to study the synergistic effect of Algerian propolis on the antitumor effects of DOX in PANC-1 cell line. Both propolis and its combination with doxorubicin inhibited cell growth in a dose-dependent manner by inducing cell cycle arrest and apoptosis. In the presence of 100 µg/ml of propolis, the IC50 of DOX against PANC-1 cells decreased by 10.9-fold. Propolis combined with DOX increased after 48h, the number of cells in the G0G1 phase with dramatical increase in sub-G1 phase to reach 47% of total cells, corresponding to an increase of senescence or apoptotic state of the cells. Dead cell assay with annexinV/PI staining demonstrated that propolis and propolis-DOX treatment resulted in a remarkable induction of apoptosis as detected by flow cytometry. It was interesting to note that propolis at its 5IC50 was found as the most potent inducer of apoptosis. Our finding revealed that induced apoptosis in our conditions was caspase-3 and caspase-9 dependent. Flow cytometry showed that propolis increased the accumulation of doxorubicin within PANC-1 cells. Moreover, fluorescent intensity detection revealed that propolis remarkably increased the retention of rhodamine-123, 7-fold compared to 3-fold of verapamil, the most effective P-gp inhibitor. In conclusion, propolis sensitize pancreatic cancer cells to DOX via enhancing the intracellular retention of DOX

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

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    Hongping Min

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

  12. Inhibition of the multidrug resistance P-glycoprotein: time for a change of strategy?

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    Callaghan, Richard; Luk, Frederick; Bebawy, Mary

    2014-04-01

    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.

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

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    Max K Leong

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

  14. Inhibitory Effects of Neochamaejasmin B on P-Glycoprotein in MDCK-hMDR1 Cells and Molecular Docking of NCB Binding in P-Glycoprotein

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    Lanying Pan

    2015-02-01

    Full Text Available 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-hydroxyphenylchroman-4-one.

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

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

    2009-09-01

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

  16. Effects of P-Glycoprotein and Its Inhibitors on Apoptosis in K562 Cells

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    Yaqiong Zu

    2014-08-01

    Full Text Available P-glycoprotein (P-gp is a major factor in multidrug resistance (MDR which is a serious obstacle in chemotherapy. P-gp has also been implicated in causing apoptosis of tumor cells, which was shown to be another important mechanism of MDR recently. To study the influence of P-gp in tumor cell apoptosis, K562/A cells (P-gp+ and K562/S cells (P-gp− were subjected to doxorubicin (Dox, serum withdrawal, or independent co-incubation with multiple P-gp inhibitors, including valspodar (PSC833, verapamil (Ver and H108 to induce apoptosis. Apoptosis was simultaneously detected by apoptotic rate, cell cycle by flow cytometry and cysteine aspartic acid-specific protease 3 (caspase 3 activity by immunoassay. Cytotoxicity and apoptosis induced by PSC833 were evaluated through an MTT method and apoptosis rate, and cell cycle combined with caspase 3 activity, respectively. The results show that K562/A cells are more resistant to apoptosis and cell cycle arrest than K562/S cells after treatment with Dox or serum deprivation. The apoptosis of K562/A cells increased after co-incubation with each of the inhibitors of P-gp. P-gp inhibitors also enhanced cell cycle arrest in K562/A cell. PSC833 most strikingly decreased viability and led to apoptosis and S phase arrest of cell cycle in K562/A cells. Our study demonstrates that P-gp inhibits the apoptosis of tumor cells in addition to participating in the efflux of intracellular chemotherapy drugs. The results of the caspase 3 activity assay also suggest that the role of P-gp in apoptosis avoidance is caspase-related.

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

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    Jennifer Pasquier

    2015-06-01

    Full Text Available 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.

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

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    Shengpeng Wang

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Xing Hua Tang

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

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

    Science.gov (United States)

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

    2010-08-27

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

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

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2010-08-01

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

  3. Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

    Science.gov (United States)

    Gartzke, Dominik; Delzer, Jürgen; Laplanche, Loic; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Sydor, Jens; Fricker, Gert

    2015-06-01

    To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

  4. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

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    Weibin Zha

    Full Text Available HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages.Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

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

    Directory of Open Access Journals (Sweden)

    Balaguer Trinidad

    2012-07-01

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

  6. The Role of Turmerones on Curcumin Transportation and P-Glycoprotein Activities in Intestinal Caco-2 Cells

    Science.gov (United States)

    Yue, Grace G.L.; Cheng, Sau-Wan; Yu, Hua; Xu, Zi-Sheng; Lee, Julia K.M.; Hon, Po-Ming; Lee, Mavis Y.H.; Kennelly, Edward J.; Deng, Gary; Yeung, Simon K.; Cassileth, Barrie R.; Fung, Kwok-Pui; Leung, Ping-Chung

    2012-01-01

    Abstract The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases. PMID:22181075

  7. The role of turmerones on curcumin transportation and P-glycoprotein activities in intestinal Caco-2 cells.

    Science.gov (United States)

    Yue, Grace G L; Cheng, Sau-Wan; Yu, Hua; Xu, Zi-Sheng; Lee, Julia K M; Hon, Po-Ming; Lee, Mavis Y H; Kennelly, Edward J; Deng, Gary; Yeung, Simon K; Cassileth, Barrie R; Fung, Kwok-Pui; Leung, Ping-Chung; Lau, Clara B S

    2012-03-01

    The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  9. ABCB1 (P-glycoprotein) reduces bacterial attachment to human gastrointestinal LS174T epithelial cells.

    Science.gov (United States)

    Crowe, Andrew; Bebawy, Mary

    2012-08-15

    The aim of this project was to show elevated P-glycoprotein (P-gp) expression decreasing bacterial association with LS174T human gastrointestinal cells, and that this effect could be reversed upon blocking functional P-gp efflux. Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Lactobacillus acidophilus and numerous strains of Escherichia coli, from commensal to enteropathogenic and enterohaemorrhagic strains (O157:H7) were fluorescently labelled and incubated on LS174T cultures either with or without P-gp amplification using rifampicin. PSC-833 was used as a potent functional P-gp blocking agent. Staphylococcus and Pseudomonas displayed the greatest association with the LS174T cells. Surprisingly, lactobacilli retained more fluorescence than enteropathogenic-E. coli in this system. Irrespective of attachment differences between the bacterial species, the increase in P-gp protein expression decreased bacterial fluorescence by 25-30%. This included the GFP-labelled E. coli, and enterohaemorrhagic E. coli (O157:H7). Blocking P-gp function through the co-administration of PSC-833 increased the amount of bacteria associated with P-gp expressing LS174T cells back to control levels. As most bacteria were affected to the same degree, irrespective of pathogenicity, it is unlikely that P-gp has a direct influence on adhesion of bacteria, and instead P-gp may be playing an indirect role by secreting a bank of endogenous factors or changing the local environment to one less suited to bacterial growth in general. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  10. Expression of the MDR1 gene and P-glycoprotein in canine mast cell tumor cell lines

    OpenAIRE

    NAKAICHI, Munekazu; TAKESHITA, Yoko; OKUDA, Masaru; NAKAMOTO, Yuya; ITAMOTO, Kazuhito; UNE, Satoshi; SASAKI, Nobuo; KADOSAWA, Tsuyoshi; TAKAHASHI, Tomoko; TAURA, Yasuho

    2007-01-01

    Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, origin...

  11. P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity.

    Science.gov (United States)

    Silva, Renata; Palmeira, Andreia; Carmo, Helena; Barbosa, Daniel José; Gameiro, Mariline; Gomes, Ana; Paiva, Ana Mafalda; Sousa, Emília; Pinto, Madalena; Bastos, Maria de Lourdes; Remião, Fernando

    2015-10-01

    The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20 µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation.

  12. Intercellular transfer of P-glycoprotein from the drug resistant human bladder cancer cell line BIU-87 does not require cell-to-cell contact.

    Science.gov (United States)

    Zhou, Hui-liang; Zheng, Yong-jun; Cheng, Xiao-zhi; Lv, Yi-song; Gao, Rui; Mao, Hou-ping; Chen, Qin

    2013-09-01

    The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2006-01-01

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

  14. P-glycoprotein Inhibition Increases the Brain Distribution and Antidepressant-Like Activity of Escitalopram in Rodents

    Science.gov (United States)

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

    2013-01-01

    Despite the clinical prevalence of the antidepressant escitalopram, over 30% of escitalopram-treated patients fail to respond to treatment. Recent gene association studies have highlighted a potential link between the drug efflux transporter P-glycoprotein (P-gp) and response to escitalopram. The present studies investigated pharmacokinetic and pharmacodynamic interactions between P-gp and escitalopram. In vitro bidirectional transport studies revealed that escitalopram is a transported substrate of human P-gp. Microdialysis-based pharmacokinetic studies demonstrated that administration of the P-gp inhibitor cyclosporin A resulted in increased brain levels of escitalopram without altering plasma escitalopram levels in the rat, thereby showing that P-gp restricts escitalopram transport across the blood–brain barrier (BBB) in vivo. The tail suspension test (TST) was carried out to elucidate the pharmacodynamic impact of P-gp inhibition on escitalopram effect in a mouse model of antidepressant activity. Pre-treatment with the P-gp inhibitor verapamil enhanced the response to escitalopram in the TST. Taken together, these data indicate that P-gp may restrict the BBB transport of escitalopram in humans, potentially resulting in subtherapeutic brain concentrations in certain patients. Moreover, by verifying that increasing escitalopram delivery to the brain by P-gp inhibition results in enhanced antidepressant-like activity, we suggest that adjunctive treatment with a P-gp inhibitor may represent a beneficial approach to augment escitalopram therapy in depression. PMID:23670590

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

    Science.gov (United States)

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

    2005-12-01

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

  16. [Small interfering RNA-mediated COX-2 gene silencing enhances chemosensitivity of KB/VCR cells by suppressing MDR-1 gene expression and P-glycoprotein activity].

    Science.gov (United States)

    Mo, Xianchao; Li, Weizhong

    2014-05-01

    To investigate the effect of small interfering RNA (siRNA)-mediated COX-2 gene silencing in enhancing the chemosensitivity of KB/VCR cell lines. KB/VCR cells were trasnfected with COX-2 siRNA were examined for expressions of COX-2 and MDR-1 mRNAs with RT-PCR and for Rho-123 accumulation using flow cytometry. MTT assay was used to analyze the proliferation of the transfected KB/VCR cells. Compared with the negative and blank control groups, COX-2 siRNA transfection resulted in significant growth inhibition of KB/VCR cells exposed to vincristine (PKB/VCR cells. COX-2 gene silencing can enhance the chemosensitivity of KB/VCR cells to vincristine, the mechanism of which may involve down-regulated MDR-1 gene expression and inhibition of P-glycoprotein activity.

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

    Science.gov (United States)

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

    2016-09-01

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

  18. Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Weber, J M; Sircar, S; Horvath, J; Dion, P

    1989-11-01

    Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

  19. Overcoming of P-glycoprotein mediated vincristine resistance of L1210/VCR mouse leukemic cells could be induced by pentoxifylline but not by theophylline and caffeine

    International Nuclear Information System (INIS)

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

    1996-01-01

    Effects of xanthine derivatives (pentoxifylline (PTX), caffeine, theophylline, 1-methyl-3-isobutylxanthine) on P-glycoprotein mediated vincristine resistance of L1210/VCR mouse leukemic cell sub-line were studied. From the applied xanthines only PTX was found to reverse the vincristine resistance of the above cells. Moreover, only PTX, but not other xanthine, increased the accumulation of [ 3 H]vincristine by L1210/VCR cells. Thus it may be concluded that PTX-induced reversal of vincristine (VCR) resistance could not be explained from the point of known pharmacological effects of PTX that are common for other xanthines such as inhibition of phosphodiesterase activity, calcium mobilizing effect, inhibition of tumor necrosis factor α (TNF), etc. (author)

  20. Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein.

    Science.gov (United States)

    Kim, Mi Kyoung; Kim, Yunyoung; Choo, Hyunah; Chong, Youhoon

    2017-02-01

    Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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...

  2. The H2 receptor antagonist nizatidine is a P-glycoprotein substrate: characterization of its intestinal epithelial cell efflux transport.

    Science.gov (United States)

    Dahan, Arik; Sabit, Hairat; Amidon, Gordon L

    2009-06-01

    The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H(2) receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05-10 mM in both apical-basolateral (AP-BL) and BL-AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC(50) of verapamil on nizatidine P-gp secretion was 1.2 x 10(-2) mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J(max) = 5.7 x 10(-3) nmol cm(-2) s(-1) and K(m) = 2.2 mM) and one nonsaturable component (K(d) = 7 x 10(-4) microL cm(-2) s(-1)). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V(max) and K(m) estimated for nizatidine P-gp-mediated secretion were 4 x 10(-3) nmol cm(-2) s(-1) and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug-drug interactions.

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

    Directory of Open Access Journals (Sweden)

    Viera Bohacova

    2018-05-01

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

  4. P-glycoprotein Inhibition Increases the Brain Distribution and Antidepressant-Like Activity of Escitalopram in Rodents

    OpenAIRE

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

    2013-01-01

    Despite the clinical prevalence of the antidepressant escitalopram, over 30% of escitalopram-treated patients fail to respond to treatment. Recent gene association studies have highlighted a potential link between the drug efflux transporter P-glycoprotein (P-gp) and response to escitalopram. The present studies investigated pharmacokinetic and pharmacodynamic interactions between P-gp and escitalopram. In vitro bidirectional transport studies revealed that escitalopram is a transported subst...

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

    DEFF Research Database (Denmark)

    Nielsen, D; Maare, C; Eriksen, J

    2001-01-01

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

  6. Natural lignans from Arctium lappa modulate P-glycoprotein efflux function in multidrug resistant cancer cells.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier GmbH. All rights reserved.

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

    Science.gov (United States)

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

    2018-06-01

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

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

    International Nuclear Information System (INIS)

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

    1994-01-01

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

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

    Science.gov (United States)

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

    2018-03-01

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

  10. Different regulation of P-glycoprotein function between Caco-2 and Caki-1 cells by ezrin, radixin and moesin proteins.

    Science.gov (United States)

    Yano, Kentaro; Otsuka, Kyoma; Kato, Yuko; Kawabata, Hideaki; Ohmori, Shinya; Arakawa, Hiroshi; Ogihara, Takuo

    2016-03-01

    P-glycoprotein (P-gp) mediates efflux of many xenobiotics, including therapeutic drugs, from normal and tumour tissues, and its functional localization on the plasma membrane of cells is regulated by scaffold proteins, such as ezrin, radixin and moesin (ERM proteins). We previously reported that radixin is involved in post-translational regulation of P-gp in hepatocellular carcinoma HepG2 cells and mouse small intestine, but not in mouse kidney. Here, we investigated whether the role of ERM proteins in regulation of P-gp transport activity in cancers is the same as that in the corresponding normal tissues, using human colon adenocarcinoma (Caco-2) cells and renal carcinoma (Caki-1) cells. In Caco-2 cells, radixin silencing alone reduced the P-gp-mediated intracellular accumulation of rhodamine123 (Rho123), while the mRNA level of P-gp was unchanged. Thus, it appears that only radixin among the ERMs regulates P-gp activity in Caco-2 cells. On the other hand, none of the ERM proteins influenced P-gp activity in Caki-1 cells. The regulation of P-gp by ERM proteins is different between Caco-2 and Caki-1 cells. Moreover, these regulatory properties are the same as those of the corresponding normal tissues, and suggest that tissue-specific differences in the regulation of P-gp by ERM proteins are retained in cancerous tissues. © 2016 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

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

    International Nuclear Information System (INIS)

    Wang Ruoyu; Wang Hui; Fan Kai; Lv Shen

    2007-01-01

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

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

  14. Pumping of drugs by P-glycoprotein

    DEFF Research Database (Denmark)

    Litman, Thomas; Skovsgaard, Torben; Stein, Wilfred D

    2003-01-01

    The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different...... levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows...... but rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin...

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

    International Nuclear Information System (INIS)

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

    2001-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Anna Maria Bianucci

    2012-06-01

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

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

    Science.gov (United States)

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

    2018-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  19. P-glycoprotein targeted nanoscale drug carriers

    KAUST Repository

    Li, Wengang; Abu Samra, Dina Bashir Kamil; Merzaban, Jasmeen; Khashab, Niveen M.

    2013-01-01

    Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug

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

    Directory of Open Access Journals (Sweden)

    Mahsa Mohseni

    2016-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-15

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2004-10-01

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

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

    DEFF Research Database (Denmark)

    Brodin, Birger; Ozgür, Burak; Saaby, Lasse

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tomono, Takumi [Laboratory of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Kajita, Masahiro [Laboratory of Molecular Pharmaceutics and Technology, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Yano, Kentaro [Laboratory of Biopharmaceutics, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Ogihara, Takuo, E-mail: togihara@takasaki-u.ac.jp [Laboratory of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan)

    2016-08-05

    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. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

  7. Influence of P-Glycoprotein Inhibition or Deficiency at the Blood-Brain Barrier on (18)F-2-Fluoro-2-Deoxy-D-glucose ( (18)F-FDG) Brain Kinetics.

    Science.gov (United States)

    Tournier, Nicolas; Saba, Wadad; Goutal, Sébastien; Gervais, Philippe; Valette, Héric; Scherrmann, Jean-Michel; Bottlaender, Michel; Cisternino, Salvatore

    2015-05-01

    The fluorinated D-glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG) is the most prevalent radiopharmaceutical for positron emission tomography (PET) imaging. P-Glycoprotein's (P-gp, MDR1, and ABCB1) function in various cancer cell lines and tumors was shown to impact (18)F-FDG incorporation, suggesting that P-gp function at the blood-brain barrier may also modulate (18)F-FDG brain kinetics. We tested the influence of P-gp inhibition using the cyclosporine analog valspodar (PSC833; 5 μM) on the uptake of (18)F-FDG in standardized human P-gp-overexpressing cells (MDCKII-MDR1). Consequences for (18)F-FDG brain kinetics were then assessed using (i) (18)F-FDG PET imaging and suitable kinetic modelling in baboons without or with P-gp inhibition by intravenous cyclosporine infusion (15 mg kg(-1) h(-1)) and (ii) in situ brain perfusion in wild-type and P-gp/Bcrp (breast cancer resistance protein) knockout mice and controlled D-glucose exposure to the brain. In vitro, the time course of (18)F-FDG uptake in MDR1 cells was influenced by the presence of valspodar in the absence of D-glucose but not in the presence of high D-glucose concentration. PET analysis revealed that P-gp inhibition had no significant impact on estimated brain kinetics parameters K 1, k 2, k 3, V T , and CMRGlc. The lack of P-gp effect on in vivo (18)F-FDG brain distribution was confirmed in P-gp/Bcrp-deficient mice. P-gp inhibition indirectly modulates (18)F-FDG uptake into P-gp-overexpressing cells, possibly through differences in the energetic cell level state. (18)F-FDG is not a P-gp substrate at the BBB and (18)F-FDG brain kinetics as well as estimated brain glucose metabolism are influenced by neither P-gp inhibition nor P-gp/Bcrp deficiencies in baboon and mice, respectively.

  8. IPEC-J2 MDR1, a Novel High-Resistance Cell Line with Functional Expression of Human P-glycoprotein (ABCB1) for Drug Screening Studies

    DEFF Research Database (Denmark)

    Saaby, Lasse; Helms, Hans Christian Cederberg; Brodin, Birger

    2016-01-01

    The P-glycoprotein (P-gp) efflux pump has been shown to affect drug distribution and absorption in various organs and to cause drug resistance in cancer therapy. The aim of this work was to develop a cell line to serve as a screening system for potential substrates of P-gp. This requires a cell...... line with high paracellular tightness, low expression of nonhuman ABC transporters, and high expression of functional human P-gp (ABCB1). The porcine intestinal epithelial cell line, IPEC-J2, was selected as a transfection host, due to its ability to form extremely high-resistance monolayers (>10,000 Ω......·cm(2)) and its low endogenous expression of ABC-type efflux transporters. The IPEC-J2 cells were transfected with a plasmid that contained the sequence of the human MDR1 gene, which encodes P-gp, followed by a selection of successfully transfected cells with geneticin and puromycin. The resulting cell...

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

    Science.gov (United States)

    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 30%) in resistant cells, when compared to untreated control cells (ptheobromine showed to be an effective Pgp inhibitor with the lowest toxicity.

  10. Changes of Tc-99m sestamibi uptake in P-glycoprotein expressing leukaemia cells treated in vivo with antisense oligodeoxynucleotide complementary to mdr1 mRNA

    International Nuclear Information System (INIS)

    Kinuya, S.; Yokoyama, K; Fukuoka, M.; Michigishi, T.; Tonami, N.; Shiba, K.; Mori, H.; Watanabe, N.; Shuke, N.

    2006-01-01

    We examined the feasibility of Tc-99m sestamibi to monitor changes of mRNA expression of MDRl/P-glycoprotein (Pgp) following antisense oligodeoxynucleotide (AS-ODN) treatment in vivo. Three days after the intraperitoneal inoculation of murine leukaemia P388/R cells expressing MDR1/P-gp in CDFI mice, 15-mer phosphorothioate ASODN to the initiation codon of mouse mdr1 mRNA was administered intraperitoneally at 10 mg/kg daily for 3 or 4 days. Cells collected from ascites were suspended in medium for Tc-99m sestamibi uptake studies. To know the duration of antisense effects, cells were harvested 2 days later after the 3-day treatment. AS-ODN treatment increased Tc-99m sestamibi uptake. Effects of 3-day treatment and 4-day treatment were the same. Treatment effects were not detected when uptake was observed 2 days after 3-day treatment. Based on the results it was concluded that in vivo treatment with AS-ODN specific to the coding portion of mdr1 mRNA increased Tc-99m sestamibi uptake in leukaemia cells possessing MDR function. (author)

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

    Science.gov (United States)

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

    2017-01-01

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

  12. Molecular mechanisms involved in modulation of p-glycoprotein expression from squamous cell carcinoma by low dose fractionated radiation (LDFR)

    International Nuclear Information System (INIS)

    Shajahan; Shahin; Shareef, Mohammed M.; Sathishkumar, Sabapathi; Mohiuddin, Mohammed; Ahmed, Mansoor M.; Brown, Brandee C.; Jones, Raleigh; Spring, Paul M.

    2004-01-01

    In the present study, two squamous cell carcinoma oral cavity cells (SCCOC), T-167 (p53 wild type) and T-409 (p53 mutant), were exposed to either clinically relevant dose (2 Gy), high dose (7Gy) or fractionated low dose (LDFR) (0.5 Gy x 4) and the expression of Mdr1 gene was assessed by real time RT-PCR, semiquantitative 32 P RT-PCR and luciferase reporter assay

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

    DEFF Research Database (Denmark)

    Nielsen, D; Eriksen, J; Maare, C

    2000-01-01

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

  14. The effect of lycopene on cytochrome P450 isoenzymes and P-glycoprotein by using human liver microsomes and Caco-2 cell monolayer model.

    Science.gov (United States)

    Kong, Lingti; Song, Chunli; Ye, Linhu; Xu, Jian; Guo, Daohua; Shi, Qingping

    2018-01-11

    Lycopene is widely used as a dietary supplement. However, the effects of lycopene on cytochrome P450 (CYP) enzymes or P-glycoprotein (P-gp) are not comprehensive. The present study was performed to investigate the effects of lycopene on the CYP enzymes and P-gp activity. A cocktail method was used to evaluate the activities of CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Caco-2 cell monolayer model was carried out to assay lycopene on P-gp activity. The results indicated that lycopene had a moderate inhibitory effect on CYP2E1, with IC50 value of 43.65 μM, whereas no inhibitory effects on CYP3A4, CYP2C19, CYP2D6 and CYP2E1, with IC50 values all over 100 μM. In addition, lycopene showed almost no inhibitory effect on rhodamine-123 efflux and uptake (p > .05), indicated no effects on P-gp activity. In conclusion, there should be required attention when lycopene are coadministered with other drugs that are metabolised by CYP2E1.

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-02-15

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

  17. Sodium butyrate attenuates soybean oil-based lipid emulsion-induced increase in intestinal permeability of lipopolysaccharide by modulation of P-glycoprotein in Caco-2 cells

    International Nuclear Information System (INIS)

    Yan, Jun-Kai; Gong, Zi-Zhen; Zhang, Tian; Cai, Wei

    2017-01-01

    Down-regulation of intestinal P-glycoprotein (P-gp) by soybean oil-based lipid emulsion (SOLE) may cause elevated intestinal permeability of lipopolysaccharide (LPS) in patients with total parenteral nutrition, but the appropriate preventative treatment is currently limited. Recently, sodium butyrate (NaBut) has been demonstrated to regulate the expression of P-gp. Therefore, this study aimed to address whether treatment with NaBut could attenuate SOLE-induced increase in intestinal permeability of LPS by modulation of P-gp in vitro. Caco-2 cells were exposed to SOLE with or without NaBut. SOLE-induced down-regulation of P-gp was significantly attenuated by co-incubation with NaBut. Nuclear recruitment of FOXO 3a in response to NaBut was involved in P-gp regulation. Transport studies revealed that SOLE-induced increase in permeability of LPS was significantly attenuated by co-incubation with NaBut. Collectively, our results suggested that NaBut may be a potentially useful medication to prevent SOLE-induced increase in intestinal permeability of LPS. - Highlights: • Caco-2 cells were used as models for studying parenteral nutrition in vitro. • NaBut restored SOLE-induced down-regulation of P-gp in Caco-2 cells. • Regulation of P-gp by NaBut was mediated via nuclear recruitment of FOXO 3a. • NaBut modulated the permeability of LPS by P-gp function, not barrier function.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  20. Tariquidar-induced P-glycoprotein inhibition at the rat blood-brain barrier studied with (R)-11C-verapamil and PET.

    Science.gov (United States)

    Bankstahl, Jens P; Kuntner, Claudia; Abrahim, Aiman; Karch, Rudolf; Stanek, Johann; Wanek, Thomas; Wadsak, Wolfgang; Kletter, Kurt; Müller, Markus; Löscher, Wolfgang; Langer, Oliver

    2008-08-01

    The multidrug efflux transporter P-glycoprotein (P-gp) is expressed in high concentrations at the blood-brain barrier (BBB) and is believed to be implicated in resistance to central nervous system drugs. We used small-animal PET and (R)-11C-verapamil together with tariquidar, a new-generation P-gp modulator, to study the functional activity of P-gp at the BBB of rats. To enable a comparison with human PET data, we performed kinetic modeling to estimate the rate constants of radiotracer transport across the rat BBB. A group of 7 Wistar Unilever rats underwent paired (R)-11C-verapamil PET scans at an interval of 3 h: 1 baseline scan and 1 scan after intravenous injection of tariquidar (15 mg/kg, n = 5) or vehicle (n = 2). After tariquidar administration, the distribution volume (DV) of (R)-11C-verapamil was 12-fold higher than baseline (3.68 +/- 0.81 vs. 0.30 +/- 0.08; P = 0.0007, paired t test), whereas the DVs were essentially the same when only vehicle was administered. The increase in DV could be attributed mainly to an increased influx rate constant (K1) of (R)-11C-verapamil into the brain, which was about 8-fold higher after tariquidar. A dose-response assessment with tariquidar provided an estimated half-maximum effect dose of 8.4 +/- 9.5 mg/kg. Our data demonstrate that (R)-11C-verapamil PET combined with tariquidar administration is a promising approach to measure P-gp function at the BBB.

  1. Annotating Human P-Glycoprotein Bioassay Data.

    Science.gov (United States)

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

    2012-08-01

    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.

  2. Inhibition of P-glycoprotein and multidrug resistance-associated protein 2 regulates the hepatobiliary excretion and plasma exposure of thienorphine and its glucuronide conjugate

    Directory of Open Access Journals (Sweden)

    Ling-Lei Kong

    2016-08-01

    Full Text Available 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.

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sanja Perkovic

    2012-09-01

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

  5. P-glycoprotein targeted nanoscale drug carriers

    KAUST Repository

    Li, Wengang

    2013-02-01

    Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug carrying processes that shuttle the drugs out of tumor cells. Thus, P -gp inhibitors have attracted a lot of attention as they can stop cancer drugs from being pumped out of target cells with the consumption of ATP. Using quantitive structure activity relationship (QSAR), we have successfully synthesized a series of novel P -gp inhibitors. The obtained dihydropyrroloquinoxalines series were fully characterized and then tested against bacterial and tumor assays with over-expressed P -gps. All compounds were bioactive especially compound 1c that had enhanced antibacterial activity. Furthermore, these compounds were utilized as targeting vectors to direct drug delivery vehicles such as silica nanoparticles (SNPs) to cancerous Hela cells with over expressed P -gps. Cell uptake studies showed a successful accumulation of these decorated SNPs in tumor cells compared to undecorated SNPs. The results obtained show that dihydropyrroloquinoxalines constitute a promising drug candidate for targeting cancers with MDR. Copyright © 2013 American Scientific Publishers All rights reserved.

  6. Efflux of rhodamine from CD56+ cells as a surrogate marker for reversal of P-glycoprotein-mediated drug efflux by PSC 833

    DEFF Research Database (Denmark)

    Robey, R; Bakke, S; Stein, W

    1999-01-01

    minutes. A dose-response relationship was shown between the concentration of PSC 833 in the blood and the inhibition of rhodamine efflux, with an apparent plateau of the inhibition of rhodamine efflux at approximately 1,000 ng/mL. The Ki, defined as the concentration required for half-maximal inhibition...... of Pgp-mediated rhodamine efflux, was determined to be in the range of 29 to 181 ng/mL; although results in two patients were distinctly different, with Ki values of 914 and 916 ng/mL. MRK-16 staining was similar among all patients. We conclude that measurement of rhodamine efflux from CD56(+) cells...

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

    DEFF Research Database (Denmark)

    Stein, Ulrike; Lage, Hermann; Jordan, Andreas

    2002-01-01

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

  8. Determination of P-Glycoprotein Expression by Flow Cytometry in Hematological Malignancies

    Directory of Open Access Journals (Sweden)

    Berkay Saraymen

    2016-03-01

    Full Text Available Objective: Determination the expression of P-glycoprotein is especially problematic for normal tissues because immuno­logical methods are limited in terms of sensitivity. We aimed to determine the expression of P-glycoprotein and CD34 by flow cytometry, and to evaluate the level of expression of P-glycoprotein and CD34 with unresponsive to treatment in pa­tients diagnosed with hematologic malignancy. Methods: Our study included fifty patients diagnosed with acute myeloblastic leukemia and acute lymphoblastic leuke­mia, and twenty healthy controls who were admitted to Erci­yes University Hematology-Oncology Hospital. The suspend­ed cells from bone marrow samples of patients and the pe­ripheral blood samples of healthy people were marked with P-glycoprotein phycoerythrin and CD34 FITC or PerCP Cy 5.5; and then surface expression was measured by means of flow cytometry. Results: In 6 of 30 acute myeloblastic leukemia patients P-glycoprotein and CD34 expression, in 6 of 20 acute lympho­blastic leukemia patients P-glycoprotein, in 5 of them CD34 expression were determined. A significant relation between P-glycoprotein and CD34 expressions in acute myeloblas­tic leukemia and acute lymphoblastic leukemia bone marrow samples was reported. Conclusion: Our data indicate that flow cytometry is more reliable, precise and faster than molecular methods for mea­suring P-glycoprotein expression and suggests the pos­sibility of a significant relationship between P-glycoprotein and CD34 expressions in acute myeloblastic leukemia and acute lymphoblastic leukemia bone marrow samples. The blast cells expressing CD34 on their surface along with P-glycoprotein simultaneously show that multi drug resistance 1 gene is mostly active in immature cells.

  9. 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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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)-[ 11 C]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. 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 six rats, paired VPM PET scans were performed before and after administration of 3 mg/kg tariquidar. Inhibitor administration resulted in an up to 11-fold increase in VPM brain distribution volumes (DV) with half-maximum effective dose (ED 50 ) 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. 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 three 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. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Chen HH

    2015-08-01

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  12. P-glycoprotein activity and biological response

    International Nuclear Information System (INIS)

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

    2005-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-08-01

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

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

    International Nuclear Information System (INIS)

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

    1998-01-01

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

  15. Multiple Drug Transport Pathways through Human P-Glycoprotein.

    Science.gov (United States)

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

    2015-07-21

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

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

    Science.gov (United States)

    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

  17. 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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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......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...

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

    Directory of Open Access Journals (Sweden)

    Aoranit Somno

    2016-01-01

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

  19. P-glycoprotein in autoimmune rheumatic diseases.

    Science.gov (United States)

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

    2015-07-01

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

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

    Science.gov (United States)

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

    2011-03-01

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

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

    Science.gov (United States)

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

    2013-02-01

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

  2. Irradiation of rat brain reduces P-glycoprotein expression and function

    NARCIS (Netherlands)

    Bart, J.; Nagengast, W. B.; Coppes, R. P.; Wegman, T. D.; van der Graaf, W. T. A.; Groen, H. J. M.; Vaalburg, W.; de Vries, E. G. E.; Hendrikse, N. H.

    2007-01-01

    The blood - brain barrier ( BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P- glycoprotein ( P- gp), expressed on brain capillary endothelial cells, is part of the BBB. P- gp expression on capillary endothelium decreases 5 days after brain

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-04-01

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

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

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    Li-Li Liu

    2017-01-01

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

  6. Zosuquidar restores drug sensitivity in P-glycoprotein expressing acute myeloid leukemia (AML)

    International Nuclear Information System (INIS)

    Tang, Ruoping; Faussat, Anne-Marie; Perrot, Jean-Yves; Marjanovic, Zora; Cohen, Simy; Storme, Thomas; Morjani, Hamid; Legrand, Ollivier; Marie, Jean-Pierre

    2008-01-01

    Chemotherapeutic drug efflux via the P-glycoprotein (P-gp) transporter encoded by the MDR1/ABCB1 gene is a significant cause of drug resistance in numerous malignancies, including acute leukemias, especially in older patients with acute myeloid leukemia (AML). Therefore, the P-gp modulators that block P-gp-mediated drug efflux have been developed, and used in combination with standard chemotherapy. In this paper, the capacity of zosuquidar, a specific P-gp modulator, to reverse chemoresistance was examined in both leukemia cell lines and primary AML blasts. The transporter protein expressions were analyzed by flow cytometry using their specific antibodies. The protein functionalities were assessed by the uptake of their fluorescence substrates in presence or absence their specific modulators. The drug cytotoxicity was evaluated by MTT test. Zosuquidar completely or partially restored drug sensitivity in all P-gp-expressing leukemia cell lines tested and enhanced the cytotoxicity of anthracyclines (daunorubicin, idarubicin, mitoxantrone) and gemtuzumab ozogamicin (Mylotarg) in primary AML blasts with active P-gp. In addition, P-gp inhibition by zosuquidar was found to be more potent than cyclosporine A in cells with highly active P-gp. These in vitro studies suggest that zosuquidar may be an effective adjunct to cytotoxic chemotherapy for AML patients whose blasts express P-gp, especially for older patients

  7. Effect of zolpidem on human cytochrome P450 activity, and on transport mediated by P-glycoprotein.

    Science.gov (United States)

    von Moltke, Lisa L; Weemhoff, James L; Perloff, Michael D; Hesse, Leah M; Harmatz, Jerold S; Roth-Schechter, Barbara F; Greenblatt, David J

    2002-12-01

    The influence of high concentrations of zolpidem (100 microM, corresponding to approximately 200 times maximum therapeutic concentrations) on the activity of six human Cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Zolpidem produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. Transport of rhodamine 123, presumed to be mediated mainly by the energy-dependent efflux transport protein P-glycoprotein, was studied in a cell culture system using a human intestinal cell line. High concentrations of zolpidem (100 microM), exceeding the usual therapeutic range by more than 100-fold, produced only modest impairment of rhodamine 123 transport. The findings indicate that zolpidem is very unlikely to cause clinical drug interactions attributable to impairment of CYP activity or P-gp mediated transport. Copyright 2002 John Wiley & Sons, Ltd.

  8. Synthesis and characterization of macromolecular rhodamine tethers and their interactions with P-glycoprotein.

    Science.gov (United States)

    Crawford, Lindsey; Putnam, David

    2014-08-20

    Rhodamine dyes are well-known P-glycoprotein (P-gp) substrates that have played an important role in the detection of inhibitors and other substrates of P-gp, as well as in the understanding of P-gp function. Macromolecular conjugates of rhodamines could prove useful as tethers for further probing of P-gp structure and function. Two macromolecular derivatives of rhodamine, methoxypolyethylene glycol-rhodamine6G and methoxypolyethylene glycol-rhodamine123, were synthesized through the 2'-position of rhodamine6G and rhodamine123, thoroughly characterized, and then evaluated by inhibition with verapamil for their ability to interact with P-gp and to act as efflux substrates. To put the results into context, the P-gp interactions of the new conjugates were compared to the commercially available methoxypolyethylene glycol-rhodamineB. FACS analysis confirmed that macromolecular tethers of rhodamine6G, rhodamine123, and rhodamineB were accumulated in P-gp expressing cells 5.2 ± 0.3%, 26.2 ± 4%, and 64.2 ± 6%, respectively, compared to a sensitive cell line that does not overexpress P-gp. Along with confocal imaging, the efflux analysis confirmed that the macromolecular rhodamine tethers remain P-gp substrates. These results open potential avenues for new ways to probe the function of P-gp both in vitro and in vivo.

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

    Directory of Open Access Journals (Sweden)

    Cara Andrea

    2007-03-01

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

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

    Science.gov (United States)

    Efferth, Thomas; Volm, Manfred

    2017-07-01

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

  11. Evaluation of pharmacokinetic and pharmacodynamic interaction between repaglinide and atazanavir in healthy, diabetic and hepatic impaired rats: possible inhibition of CYP3A, OATP, and P-glycoprotein transporters

    Directory of Open Access Journals (Sweden)

    Thirumal Eswara Goud

    2016-10-01

    Full Text Available The metabolic syndrome in HIV infected patients is particularly associated with the use protease inhibitors. Atazanavir is an inhibitor of the cytochrome P 450 (CYP system, in particular CYP3A4 and CYP2C9 which can affect the metabolism of several drugs. To treat metabolic syndrome in HIV patients repaglinide is used and it is a short acting insulin secretagogues undergoing metabolism with CYP 3A4 and CYP 2C8 enzyme system. The purpose of this study was to assess the possible pharmacokinetic and pharmacodynamic drug interaction of repaglinide and atazanavir in healthy, diabetic and impaired hepatic function rats. Human oral therapeutic doses of atazanavir and repaglinide were extrapolated to rats based on the body surface area. The pharmacokinetic parameters and blood glucose concentrations of repaglinide were determined after oral administration of repaglinide alone (0.5 mg/kg and in the presence of atazanavir (36 mg/kg in normal, diabetic and hepatic impaired rats. The pharmacokinetics (PK and blood glucose concentrations of repaglinide were significantly altered in the presence of atazanavir. The peak plasma concentration (Cmax, area under the plasma concentration time profile (AUC and elimination half-life of repaglinide were significantly (P<0.0001 increased. The repaglinide clearance (CL was significantly (P<0.0001 decreased in the presence of atazanavir treatment. In the presence of atazanavir, repaglinide hypoglycaemic activity was increased significantly (P<0.0001 when compared with the repaglinide control group. The present study demonstrated the significant difference in the PK/PD changes due to the enhanced bioavailability and decreased total body clearance of repaglinide may be due to the inhibition of the CYP P450 metabolic system, OATP and P-gp transporters by atazanavir.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

  13. MDR1 P-glycoprotein transports endogenous opioid peptides

    NARCIS (Netherlands)

    Oude Elferink, R. P.; Zadina, J.

    2001-01-01

    MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore

  14. Comparison of In Vitro Assays in Selecting Radiotracers for In Vivo P-Glycoprotein PET Imaging

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    Renske M. Raaphorst

    2017-09-01

    Full Text Available Positron emission tomography (PET imaging of P-glycoprotein (P-gp in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer´s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R-N-[18F]fluoroethylverapamil, (R-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM, P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands.

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

    Science.gov (United States)

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

    2014-12-01

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

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    NARCIS (Netherlands)

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

    1997-01-01

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

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

    Science.gov (United States)

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

    2016-05-15

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

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

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    Lee Stephen D

    2008-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Vanessa Lopes-Rodrigues

    2016-11-01

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

  1. In vitro and in vivo evaluations of the P-glycoprotein-mediated efflux of dibenzoylhydrazines

    Energy Technology Data Exchange (ETDEWEB)

    Miyata, Ken-ichi, E-mail: Miyata.Kenichi@otsuka.jp [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan); Otsuka Pharmaceutical Co., Ltd., Tokushima 771-0182 (Japan); Nakagawa, Yoshiaki; Kimura, Yasuhisa [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan); Ueda, Kazumitsu [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8502 (Japan); Akamatsu, Miki, E-mail: akamatsu@kais.kyoto-u.ac.jp [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan)

    2016-05-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter family. It actively transports a wide variety of compounds out of cells to protect humans from xenobiotics. Thus, determining whether chemicals are substrates and/or inhibitors of P-gp is important in risk assessments of pharmacokinetic interactions among chemicals because P-gp-mediated transport processes play a significant role in their absorption and disposition. We previously reported that dibenzoylhydrazines (DBHs) such as tebufenozide and methoxyfenozide (agrochemicals) stimulated P-gp ATPase activity. However, it currently remains unclear whether these derivatives are transport substrates of P-gp and inhibit transport of other chemicals by P-gp. In the present study, in order to evaluate the interactions of DBHs with other chemicals in humans, we determined whether DBHs are P-gp transport substrates using both the in vitro bidirectional transport assay and the in vivo study of rats. In the in vivo study, we investigated the influence of P-gp inhibitors on the brain to plasma ratio of methoxyfenozide in rats. We also examined the inhibitory effects of DBHs on quinidine (a P-gp substrate) transport by P-gp in order to ascertain whether these derivatives are inhibitors of P-gp. Based on the results, DBHs were concluded to be weak P-gp transport substrates and moderate P-gp inhibitors. However, the risk of DBHs caused by interaction with other chemicals including drugs was considered to be low by considering the DBHs' potential as the substrates and inhibitors of P-gp as well as their plasma concentrations as long as DBHs are properly used. - Highlights: • Transport of DBHs by P-gp was not detected in in vitro bidirectional transport assay. • DBHs were weak P-gp transport substrates based on in vivo studies in rats. • The in vivo studies are useful methods for evaluating P-gp transport substrates. • DBHs inhibit quinidine transport by P-gp in in vitro bidirectional transport

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2001-01-01

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

  4. Serotonin transporter protein (SERT) and P-glycoprotein (P-gp) binding activity of montanine and coccinine from three species of Haemanthus L. (Amaryllidaceae)

    DEFF Research Database (Denmark)

    Stafford, Gary Ivan; Birer, C.; Brodin, Birger

    2013-01-01

    The alkaloid rich extracts from an acid/base extraction of bulb material of Haemanthus coccineus L., H. montanus Baker and H. sanguineus Jacq. revealed that two montanine type Amaryllidaceae alkaloids, montanine (1) and coccinine (2) were the major alkaloid constituents. Together these two...... to the relative proportions of coccinine and montanine in the extracts and thus are likely to be due to more potent unidentified minor constituents. Both alkaloids exhibited low binding affinity to P-glycoprotein (P-gp) as demonstrated by low inhibition of calcein-AM efflux in the MDCK-MDR1 cell line....... This indicates that P-gp efflux will not be limiting for blood-brain-barrier passage of the alkaloids....

  5. Effects of sertraline and fluoxetine on p-glycoprotein at barrier sites: in vivo and in vitro approaches.

    Directory of Open Access Journals (Sweden)

    Amita Kapoor

    Full Text Available Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.The P-gp substrate, tritiated digoxin ([(3H] digoxin, was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [(3H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.

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

    Science.gov (United States)

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

    2001-10-01

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

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

    NARCIS (Netherlands)

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

    1993-01-01

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

  8. Inhibitory Effects of Daiokanzoto (Da-Huang-Gan-Cao-Tang on P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Yuka Watanabe

    2012-01-01

    Full Text Available We have studied the effects of various Kampo medicines on P-glycoprotein (P-gp, a drug transporter, in vitro. The present study focused on Daiokanzoto (Da-Huang-Gan-Cao-Tang, which shows the most potent inhibitory effects on P-gp among the 50 Kampo medicines studied, and investigated the P-gp inhibitory effects of Daiokanzoto herbal ingredients (rhubarb and licorice root and their components by an ATPase assay using human P-gp membrane. Both rhubarb and licorice root significantly inhibited ATPase activity, and the effects of rhubarb were more potent than those of licorice root. The content of rhubarb in Daiokanzoto is double that in licorice root, and the inhibition patterns of Daiokanzoto and rhubarb involve both competitive and noncompetitive inhibition, suggesting that the inhibitory effects of Daiokanzoto are mainly due to rhubarb. Concerning the components of rhubarb, concentration-dependent inhibitory effects were observed for (−-catechin gallate, (−-epicatechin gallate, and (−-epigallocatechin gallate. In conclusion, rhubarb may cause changes in the drug dispositions of P-gp substrates through the inhibition of P-gp. It appears that attention should be given to the interactions between these drugs and Kampo medicines containing rhubarb as an herbal ingredient.

  9. Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

    International Nuclear Information System (INIS)

    Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner . E-mail; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria

    2008-01-01

    The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo- and radiotherapy, particularly when radiation exposure precedes chemical treatment. (author)

  10. Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear]. E-mail; neylisantos@yahoo.com.br; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria [Fundacao de Hematologia e Hemoterapia de Pernambuco, Recife, PE (Brazil). Unidade de Laboratorios Especializados. Lab. de Imunofenotipagem

    2008-12-15

    The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo- and radiotherapy, particularly when radiation exposure precedes chemical treatment. (author)

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Erica Lynn Bradshaw-Pierce

    2013-03-01

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

  13. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier.

    Directory of Open Access Journals (Sweden)

    Yangfang Li

    Full Text Available 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.

  14. THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

    Directory of Open Access Journals (Sweden)

    A. V. Shulkin

    2015-09-01

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

  15. Overview of P-glycoprotein inhibitors: a rational outlook

    Directory of Open Access Journals (Sweden)

    Kale Mohana Raghava Srivalli

    2012-09-01

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

  16. Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

    Science.gov (United States)

    Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

    2008-01-01

    Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

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

    Science.gov (United States)

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

    2016-05-24

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

  18. Tumor P-Glycoprotein Correlates with Efficacy of PF-3758309 in in vitro and in vivo Models of Colorectal Cancer

    OpenAIRE

    Bradshaw-Pierce, Erica Lynn; Pitts, Todd M.; Tan, Aik-Choon; McPhillips, Kelly; West, Mark; Gustafson, Daniel L.; Halsey, Charles; Nguyen, Leslie; Lee, Nathan V.; Kan, Julie L. C.; Murray, Brion William; Eckhardt, S. Gail

    2013-01-01

    P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309), a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB1...

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

    OpenAIRE

    Erica Lynn Bradshaw-Pierce; Erica Lynn Bradshaw-Pierce; Todd M Pitts; Aik-Choon eTan; Kelly eMcPhillips; Mark eWest; Daniel L Gustafson; Charles eHalsey; Leslie eNguyen; Nathan V Lee; Julie LC Kan; Brion William Murray; S. Gail eEckhardt

    2013-01-01

    P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309), a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB...

  20. Murine P-glycoprotein deficiency alters intestinal injury repair and blunts lipopolysaccharide-induced radioprotection.

    Science.gov (United States)

    Staley, Elizabeth M; Yarbrough, Vanisha R; Schoeb, Trenton R; Daft, Joseph G; Tanner, Scott M; Steverson, Dennis; Lorenz, Robin G

    2012-09-01

    P-glycoprotein (P-gp) has been reported to increase stem cell proliferation and regulate apoptosis. Absence of P-gp results in decreased repair of intestinal epithelial cells after chemical injury. To further explore the mechanisms involved in the effects of P-gp on intestinal injury and repair, we used the well-characterized radiation injury model. In this model, injury repair is mediated by production of prostaglandins (PGE(2)) and lipopolysaccharide (LPS) has been shown to confer radioprotection. B6.mdr1a(-/-) mice and wild-type controls were subjected to 12 Gy total body X-ray irradiation and surviving crypts in the proximal jejunum and distal colon were evaluated 3.5 days after irradiation. B6.mdr1a(-/-) mice exhibited normal baseline stem cell proliferation and COX dependent crypt regeneration after irradiation. However, radiation induced apoptosis was increased and LPS-induced radioprotection was blunted in the C57BL6.mdr1a(-/-) distal colon, compared to B6 wild-type controls. The LPS treatment induced gene expression of the radioprotective cytokine IL-1α, in B6 wild-type controls but not in B6.mdr1a(-/-) animals. Lipopolysaccharid-induced radioprotection was absent in IL-1R1(-/-) animals, indicating a role for IL-1α in radioprotection, and demonstrating that P-gp deficiency interferes with IL-1α gene expression in response to systemic exposure to LPS.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

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

    1995-01-01

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

  3. Mucoadhesive properties and interaction with P-glycoprotein (P-gp) of thiolated-chitosans and -glycol chitosans and corresponding parent polymers: a comparative study.

    Science.gov (United States)

    Trapani, Adriana; Palazzo, Claudio; Contino, Marialessandra; Perrone, Maria Grazia; Cioffi, Nicola; Ditaranto, Nicoletta; Colabufo, Nicola Antonio; Conese, Massimo; Trapani, Giuseppe; Puglisi, Giovanni

    2014-03-10

    The aim of the present work was to compare the mucoadhesive and efflux pump P-glycoprotein (P-gp) interacting properties of chitosan (CS)- and glycolchitosan (GCS)-based thiomers and corresponding unmodified parent polymers. For this purpose, the glycol chitosan-N-acetyl-cysteine (GCS-NAC) and glycol chitosan-glutathione (GCS-GSH) thiomers were prepared under simple and mild conditions. Their mucoadhesive characteristics were studied by turbidimetric and zeta potential measurements. The P-gp interacting properties were evaluated measuring the effects of thiolated- and unmodified-polymers on the bidirectional transport (BA/AB) of rhodamine-123 across Caco-2 cells as well as in the calcein-AM and ATPase activity assays. Although all the thiomers and unmodified polymers showed optimal-excellent mucoadhesive properties, the best mucoadhesive performances have been obtained by CS and CS-based thiomers. Moreover, it was found that the pretreatment of Caco-2 cell monolayer with GCS-NAC or GCS restores Rho-123 cell entrance by inhibiting P-gp activity. Hence, GCS-NAC and GCS may constitute new biomaterials useful for improving the bioavailability of P-gp substrates.

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

    DEFF Research Database (Denmark)

    Saaby, Lasse; Brodin, Birger

    2017-01-01

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

  5. Pharmacokinetic compatibility of ginsenosides and Schisandra Lignans in Shengmai-san: from the perspective of p-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Yan Liang

    Full Text Available 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.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.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 Rb1 significantly.

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

    Directory of Open Access Journals (Sweden)

    Gao W

    2014-07-01

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

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

    Directory of Open Access Journals (Sweden)

    К. Yu. Khristenko

    2016-01-01

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

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

    Science.gov (United States)

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

    2002-02-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  10. In vivo comparison of various polymeric and low molecular mass inhibitors of intestinal P-glycoprotein.

    Science.gov (United States)

    Föger, Florian; Hoyer, Herbert; Kafedjiiski, Krum; Thaurer, Michael; Bernkop-Schnürch, Andreas

    2006-12-01

    Several polymers have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a direct in vivo comparison of delivery systems based on Pluronic P85, Myrj 52 and chitosan-4-thiobutylamidine (Ch-TBA) in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. Furthermore, the postulated low molecular mass P-gp inhibitors 6-mercaptopurine and reduced glutathione (GSH) were evaluated in vitro and in vivo. In vitro, the permeation enhancing effect of 6-mercaptopurine, GSH, Pluronic P85, Myrj 52, and the combination of Ch-TBA with GSH was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type diffusion chambers. In comparison to buffer only, Rho-123 transport in presence of 100 microm 6-mercaptopurine, 0.5% (w/v) GSH, 0.5% (w/v) Pluronic P85, 0.5% (w/v) Myrj 52 and the combination of 0.5% (w/v) Ch-TBA/ 0.5% (w/v) GSH, was 2.1, 1.6, 1.9, 1.8, 3.0-fold improved, respectively. In vivo in rat, enteric-coated tablets based on Pluronic P85, Myrj 52 or Ch-TBA/GSH increased the area under the plasma concentration time curve (AUC(0-12)) of Rho-123 1.6-fold, 2.4-fold, 4.3-fold, respectively, in comparison to control only. Contrariwise, the low molecular mass excipients 6-mercaptopurine and GSH showed no significant effect in vivo at all. This in vivo study showed that polymeric P-gp inhibitors and especially the delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

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

    NARCIS (Netherlands)

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

    2007-01-01

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

  15. St. John's Wort constituents modulate P-glycoprotein transport activity at the blood-brain barrier.

    NARCIS (Netherlands)

    Ott, M.; Huls, M.; Cornelius, M.G.; Fricker, G.

    2010-01-01

    PURPOSE: The purpose of this study was to investigate the short-term signaling effects of St. John's Wort (SJW) extract and selected SJW constituents on the blood-brain barrier transporter P-glycoprotein and to describe the role of PKC in the signaling. METHODS: Cultured porcine brain capillary

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

    NARCIS (Netherlands)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Wise, John G

    2012-06-26

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

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

    Science.gov (United States)

    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.

  20. Evaluation of the P-glycoprotein- and breast cancer resistance protein-mediated brain penetration of 11C-labeled topotecan using small-animal positron emission tomography

    International Nuclear Information System (INIS)

    Yamasaki, Tomoteru; Fujinaga, Masayuki; Kawamura, Kazunori; Hatori, Akiko; Yui, Joji; Nengaki, Nobuki; Ogawa, Masanao; Yoshida, Yuichiro; Wakizaka, Hidekatsu; Yanamoto, Kazuhiko; Fukumura, Toshimitsu; Zhang Mingrong

    2011-01-01

    Introduction: Topotecan (TPT) is a camptothecin derivative and is an anticancer drug working as a topoisomerase-I-specific inhibitor. But TPT cannot penetrate through the blood-brain barrier. In this study, we synthesized a new positron emission tomography (PET) probe, [ 11 C]TPT, to evaluate the P-glycoprotein (Pgp)- and breast cancer resistance protein (BCRP)-mediated brain penetration of [ 11 C]TPT using small-animal PET. Methods: [ 11 C]TPT was synthesized by the reaction of a desmethyl precursor with [ 11 C]CH 3 I. In vitro study using [ 11 C]TPT was carried out in MES-SA and doxorubicin-resistant MES-SA/Dx5 cells in the presence or absence of elacridar, a specific inhibitor for Pgp and BCRP. The biodistribution of [ 11 C]TPT was determined using small-animal PET and the dissection method in mice. Results: The transport of [ 11 C]TPT to the extracellular side was determined in MES-SA/Dx5 cells exhibiting the expressions of Pgp and BCRP at high levels. This transport was inhibited by coincubation with elacridar. In Mdr1a/b -/- Bcrp1 -/- mice, PET results indicated that the brain uptake of [ 11 C]TPT was about two times higher than that in wild-type mice. Similarly, the brain penetration of [ 11 C]TPT in wild-type mice was increased by treatment with elacridar. The radioactivity in the brain of elacridar-treated mice was maintained at a certain level after the injection of [ 11 C]TPT, although the radioactivity in the blood decreased with time. Conclusions: We demonstrated the increase of brain penetration of [ 11 C]TPT by deficiency and inhibition of Pgp and BCRP functions using small-animal PET in mice.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Andre Luis Dias Araujo Mazzari

    2016-08-01

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

  3. The complexity of roles of P-glycoprotein in refractory epilepsy: Pharmacoresistance, epileptogenesis, SUDEP and relapsing marker after surgical treatment

    Directory of Open Access Journals (Sweden)

    Alberto Lazarowski

    2015-07-01

    Full Text Available As described initially from clinical and experimental studies, P-glycoprotein (P-gp plays a central role in the pharmacoresistance of epilepsy, acting by efflux of AEDs mainly at blood brain barrier (BBB level. However, repetitive seizures can produce both brain and heart P-gp overexpression. Because P-gp activity induces membrane depolarization, its neuronal expression could be acting in the intrinsic mechanism of epileptogenesis, and its heart expression, can be a high risk factor of death, after severe-continuo convulsive stresses as in  fatal status epilepticus or in SUDEP. Additionally, because P-gp is also a stem cell marker, we suggests that its constitutive overexpression in dysplastic neurons from brain epileptogenic areas observed in patients with refractory epilepsies, should be addressed as a risk factor of seizures relapse after surgical treatment. Here we discuss these concepts, based on our own clinical and experimental experiences, and reviewing the current literature on these subjects.

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

    Science.gov (United States)

    Saaby, Lasse; Brodin, Birger

    2017-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Qian Liu

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

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

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

    NARCIS (Netherlands)

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

    1991-01-01

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

  8. Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr1a P-glycoprotein

    NARCIS (Netherlands)

    Mayer, U; Wagenaar, E; Beijnen, J.H; Smit, J.W; Meijer, D.K F; van Asperen, J.; Borst, P; Schinkel, A.H

    1 We have used mice with a disrupted mdrla P-glycoprotein gene (mdrIa (-/-) mice) to study the role of P-glycoprotein in the pharmacokinetics of digoxin, a model P-glycoprotein substrate. 2 [K-3]-digoxin at a dose of 0.2 mg kg(-1) was administered as a single i.v. or oral bolus injection. We

  9. MC225, a Novel Probe for P-glycoprotein PET Imaging at the Blood-brain Barrier: In Vitro Cardiovascular Safety Evaluation.

    Science.gov (United States)

    Fusi, Fabio; Durante, Miriam; Gorelli, Beatrice; Perrone, Maria Grazia; Colabufo, Nicola Antonio; Saponara, Simona

    2017-12-01

    The P-glycoprotein (P-gp) substrate MC225, at concentrations ≤10 nM, is a valuable radiotracer for positron emission tomography imaging of P-gp function in rats and mice. The aim of this study was to evaluate its potential toxic hazard toward the cardiovascular system through an in-depth analysis of its effects on rat aorta rings, on CaV1.2 channel current (ICa1.2) of A7r5 cells and on Langendorff-perfused rat heart. In aortic rings, MC225 relaxed phenylephrine-induced contraction in a concentration-dependent and endothelium-independent manner, with an IC50 value of about 1 μM. At concentrations ≥3 μM, it antagonized the response to cumulative concentrations of K. MC225, 1 and 10 μM, inhibited ICa1.2 by 15% and 31%, respectively, without affecting either current activation or inactivation kinetics. In Langendorff-perfused rat hearts, only 10 μM MC225 significantly decreased left ventricular pressure and increased coronary perfusion pressure while reducing heart rate and prolonging the cardiac cycle length as well as the atrioventricular conduction time (PQ interval) on the electrocardiogram. Lower concentrations of the drug were ineffective. These findings demonstrate that MC225-induced cardiovascular effects took place at concentrations that are at least 2 orders of magnitude higher than those allowing in vivo measurement of P-gp function. Therefore, MC225 represents a promising positron emission tomography tool for in vivo straightforward P-gp quantification.

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

    International Nuclear Information System (INIS)

    Decleves, Xavier.; Niel, Elisabeth; Debray, Marcel; Scherrmann, Jean-Michel

    2006-01-01

    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

  11. Substrate-Dependent Inhibition of P-glycoprotein Mediated Efflux Transport of Digoxin

    DEFF Research Database (Denmark)

    Saaby, Lasse; Ozgür, Burak; Brodin, Birger

    , distribution and excretion of a wide range of structurally diverse drug compounds and P-gp therefore constitutes a potential site for drug-drug interactions. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommend that all new investigational drugs be screened...... for interactions with P-gp among other transporters in vitro. This includes an evaluation of new drug compounds as inhibitors of P-gp, preferably in competition with a clinically relevant probe substrate such as digoxin. However, increasing evidence indicates that the binding pocket of P-gp contains several...... overlapping binding sites for substrates. This suggests that drug-drug interactions may be pairwise specific and observations of interaction from a single drug-drug combination may therefore not apply to a second drug combination. At present, it is not known which drug combinations should be considered...

  12. The Role of P-Glycoprotein in Transport of Danshensu across the Blood-Brain Barrier

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    Peng-Fei Yu

    2011-01-01

    Full Text Available Danshensu (3-(3, 4-dihydroxyphenyl lactic acid, a water-soluble active component isolated from the root of Salvia miltiorrhiza Bunge, is widely used for the treatment of cerebrovascular diseases. The present study aims to investigate the role of P-glycoprotein in transport of Danshensu across the blood-brain barrier. Sprague-Dawley rats were pretreated with verapamil at a dose of 20 mg kg−1 (verapamil group or the same volume of normal saline (control group. Ninety minutes later, the animals were administrated with Danshensu (15 mg kg−1 by intravenous injection. At 15 min, 30 min, and 60 min after Danshensu administration, the levels of Danshensu in the blood and brain were detected by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS. The results showed that Danshensu concentrations in the brain of the rats pretreated with verapamil were significantly increased. In addition, the brain-plasma ratios of the group pretreated with verapamil were much higher than that of the control group. There was no difference in Danshensu level in plasma between the verapamil group and control group. The findings indicated that Danshensu can pass the blood-brain barrier, and P-glycoprotein plays an important role in Danshensu transportation in brain.

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

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    Yue-Hong Zhang

    2017-07-01

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

  14. Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

    Science.gov (United States)

    Singh, Samsher; Kalia, Nitin P; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R; Kumar, Ashok; Bharate, Sandip B; Khan, Inshad A

    2017-01-01

    This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P -glycoprotein ( P -gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P -gp with half maximal inhibitory concentration (IC 50 ) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P -gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

  15. Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo

    International Nuclear Information System (INIS)

    Bošnjak, Ivana; Borra, Marco; Iamunno, Franco; Benvenuto, Giovanna; Ujević, Ivana; Bušelić, Ivana; Roje-Busatto, Romana; Mladineo, Ivona

    2014-01-01

    Highlights: • Effects of BPA on embryonic development of Paracentrotus lividus were determined. • Transport assay, intracellular BPA measurements and gene expression surveys were made. • Multidrug efflux transporter P-gp/ABCB1 is involved in BPA elimination. • Endocrine disruption is inferred by orphan steroid hormone receptor (shr2) upregulation. • BPA delayed mitosis, inducing aberrant karyokinesis and dysfunctional microfilaments. - Abstract: Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation

  16. Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo

    Energy Technology Data Exchange (ETDEWEB)

    Bošnjak, Ivana [Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, Zagreb (Croatia); Borra, Marco [Molecular Biology Service, Stazione Zoologica Anton Dohrn, Villa Comunale 80121, Napoli (Italy); Iamunno, Franco; Benvenuto, Giovanna [Electron Microscopy Service, Stazione Zoologica Anton Dohrn, Villa Comunale 80121, Napoli (Italy); Ujević, Ivana [Laboratory of Plankton and Shellfish Toxicity, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Bušelić, Ivana [Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Roje-Busatto, Romana [Laboratory of Plankton and Shellfish Toxicity, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Mladineo, Ivona, E-mail: mladineo@izor.hr [Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Assemble Marine Laboratory, Stazione Zoological Anton Dohrn, Villa Comunale, Naples (Italy)

    2014-11-15

    Highlights: • Effects of BPA on embryonic development of Paracentrotus lividus were determined. • Transport assay, intracellular BPA measurements and gene expression surveys were made. • Multidrug efflux transporter P-gp/ABCB1 is involved in BPA elimination. • Endocrine disruption is inferred by orphan steroid hormone receptor (shr2) upregulation. • BPA delayed mitosis, inducing aberrant karyokinesis and dysfunctional microfilaments. - Abstract: Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation.

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

    Directory of Open Access Journals (Sweden)

    Renu Singh Dhanikula

    2016-10-01

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

  18. Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo.

    Science.gov (United States)

    Bošnjak, Ivana; Borra, Marco; Iamunno, Franco; Benvenuto, Giovanna; Ujević, Ivana; Bušelić, Ivana; Roje-Busatto, Romana; Mladineo, Ivona

    2014-11-01

    Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100nM and 4μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. HMGB1 Contributes to the Expression of P-Glycoprotein in Mouse Epileptic Brain through Toll-Like Receptor 4 and Receptor for Advanced Glycation End Products.

    Directory of Open Access Journals (Sweden)

    Yan Chen

    Full Text Available The objective of the present study was to investigate the role of high-mobility group box-1 (HMGB1 in the seizure-induced P-glycoprotein (P-gp overexpression and the underlying mechanism. Kainic acid (KA-induced mouse seizure model was used for in vivo experiments. Male C57BL/6 mice were divided into four groups: normal saline control (NS group, KA-induced epileptic seizure (EP group, and EP group pretreated with HMGB1 (EP+HMGB1 group or BoxA (HMGB1 antagonist, EP+BoxA group. Compared to the NS group, increased levels of HMGB1 and P-gp in the brain were observed in the EP group. Injection of HMGB1 before the induction of KA further increased the expression of P-gp while pre-treatment with BoxA abolished this up-regulation. Next, the regulatory role of HMGB1 and its potential involved signal pathways were investigated in mouse microvascular endothelial bEnd.3 cells in vitro. Cells were treated with HMGB1, HMGB1 plus lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS [toll-like receptor 4 (TLR4 antagonist], HMGB1 plus FPS-ZM1 [receptor for advanced glycation end products (RAGE inhibitor], HMGB1 plus SN50 [nuclear factor-kappa B (NF-κB inhibitor], or vehicle. Treatment with HMGB1 increased the expression levels of P-gp, TLR4, RAGE and the activation of NF-κB in bEnd.3 cells. These effects were inhibited by the pre-treatment with either LPS-RS or FPS-ZM1, and were abolished by the pre-treatment of SN50 or a combination treatment of both LPS-RS and FPS-ZM1. Luciferase reporter assays showed that exogenous expression of NF-κB p65 increased the promoter activity of multidrug resistance 1a (P-gp-encoding gene in endothelial cells. These data indicate that HMGB1 contributes to the overexpression of P-gp in mouse epileptic brain tissues via activation of TLR4/RAGE receptors and the downstream transcription factor NF-κB in brain microvascular endothelial cells.

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

    International Nuclear Information System (INIS)

    Patel, Krupa J; Tannock, Ian F

    2009-01-01

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

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

    Science.gov (United States)

    Tsujimura, Shizuyo; Tanaka, Yoshiya

    2012-02-01

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

  2. Cytochrome P450 and P-Glycoprotein-Mediated Interactions Involving African Herbs Indicated for Common Noncommunicable Diseases

    Directory of Open Access Journals (Sweden)

    Gregory Ondieki

    2017-01-01

    Full Text Available Herbal remedies are regularly used to complement conventional therapies in the treatment of various illnesses in Africa. This may be because they are relatively cheap and easily accessible and are believed by many to be safe, cause fewer side effects, and are less likely to cause dependency. On the contrary, many herbs have been shown to alter the pharmacokinetics of coadministered allopathic medicines and can either synergize or antagonize therapeutic effects as well as altering the toxicity profiles of these drugs. Current disease burden data point towards epidemiological transitions characterised by increasing urbanization and changing lifestyles, risk factors for chronic diseases like hypertension, diabetes, and cancer which often present as multimorbidities. As a result, we highlight African herb-drug interactions (HDIs modulated via cytochrome P450 enzyme family (CYP and P-glycoprotein (P-gp and the consequences thereof in relation to antihypertensive, antidiabetic, and anticancer drugs. CYPs are enzymes which account for to up to 70% of drug metabolism while P-gp is an efflux pump that extrudes drug substrates out of cells. Consequently, regulation of the relative activity of both CYP and P-gp by African herbs influences the effective drug concentration at the site of action and modifies therapeutic outcomes.

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

    Science.gov (United States)

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

    1999-05-01

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

  4. Multi-drug resistance (MDR1 gene and P-glycoprotein influence on pharmacokinetic and pharmacodymanic of therapeutic drugs

    Directory of Open Access Journals (Sweden)

    Linardi Renata Lehn

    2006-01-01

    Full Text Available (MDR1 gene expressed in tumor cells and also in several normal tissues, such as intestine, liver, kidney, blood-brain barrier, spinal cord, and placenta. P-gp has been identified in mice, rat, bovine, monkey, rodents, and human beings and has been receiving a particular clinical relevance because this protein expression limits brain access and intestinal absorption of many drugs. This protein plays a role as a protective barrier against a wide variety of substrates, avoiding drug entry into the central nervous system. P-glycoprotein also interferes with drug bioavailability and disposition, including absorption, distribution, metabolization, and excretion, influencing pharmacokinetic and pharmacodynamic of drugs. Modulation of P-gp may help the efficacy of treatment of several diseases and can explain some adverse central nervous system effects induced by drugs after intravenous administration and the poor response of oral administration in patients. Alteration in P-gp expression or function has been associated with several diseases susceptibility in humans and animals. Furthermore, additional studies relating MDR1 and P-gp expression has an important clinical implication also in terms of treatment efficacy.

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

    Directory of Open Access Journals (Sweden)

    Doublier Sophie

    2012-01-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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 1 [20(S)-GF 1 ] exhibited significant inhibition (> 50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC 50 = 9.42 μM), followed by 18β-GA (IC 50 = 21.78 μM), 20(S)-GF 1 (IC 50 = 76.08 μM) and DAG (IC 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 m and V 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 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 0−t and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF 1 and Rh 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, 18β-GA, DAG, and 20(S)-GF 1 significantly inhibited P-gp in vitro

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xue, E-mail: lixue@imm.ac.cn; Hu, Jinping, E-mail: hujp@imm.ac.cn; Wang, Baolian, E-mail: wangbaolian@imm.ac.cn; Sheng, Li, E-mail: shengli@imm.ac.cn; Liu, Zhihao, E-mail: liuzhihao@imm.ac.cn; Yang, Shuang, E-mail: yangsh@imm.ac.cn; Li, Yan, E-mail: yanli@imm.ac.cn

    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

  8. Acute Effects of Viral Exposure on P-Glycoprotein Function in the Mouse Fetal Blood-Brain Barrier

    Directory of Open Access Journals (Sweden)

    Enrrico Bloise

    2017-02-01

    Full Text Available Background/Aims: Viral infection during pregnancy is known to affect the fetal brain. The toll-like receptor (TLR-3 is a pattern recognition receptor activated by viruses known to elicit adverse fetal neurological outcomes. The P-glycoprotein (P-gp efflux transporter protects the developing fetus by limiting the transfer of substrates across both the placenta and the fetal blood-brain barrier (BBB. As such, inhibition of P-gp at these blood-barrier sites may result in increased exposure of the developing fetus to environmental toxins and xenobiotics present in the maternal circulation. We hypothesized that viral exposure during pregnancy would impair P-gp function in the placenta and in the developing BBB. Here we investigated whether the TLR-3 ligand, polyinosinic:polycytidylic acid (PolyI:C, increased accumulation of one P-gp substrate in the fetus and in the developing fetal brain. Methods: Pregnant C57BL/6 mice (GD15.5 were injected (i.p. with PolyI:C (5 mg/kg or 10 mg/kg or vehicle (saline. [3H]digoxin (P-gp substrate was injected (i.v. 3 or 23h post-treatment and animals were euthanized 1h later. Maternal plasma, ‘fetal-units’ (fetal membranes, amniotic fluid and whole fetus, and fetal brains were collected. Results: PolyI:C exposure (4h significantly elevated maternal plasma IL-6 (P<0.001 and increased [3H]digoxin accumulation in the fetal brain (P<0.05. In contrast, 24h after PolyI:C exposure, no effect on IL-6 or fetal brain accumulation of P-gp substrate was observed. Conclusion: Viral infection modeled by PolyI:C causes acute increases in fetal brain accumulation of P-gp substrates and by doing so, may increase fetal brain exposure to xenobiotics and environmental toxins present in the maternal circulation.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  11. 7-Piperazinethylchrysin inhibits melanoma cell proliferation by ...

    African Journals Online (AJOL)

    In B16F10 and A375 cells, treatment with PEC caused the inhibition ... Conclusion: PEC inhibited melanoma cell proliferation, apparently by blocking the cell cycle at G0/G1 .... all statistical analyses. .... Financial support from the Department of.

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

    Directory of Open Access Journals (Sweden)

    Frances Jane Sharom

    2014-03-01

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

  13. ATP-binding cassette transporters P-glycoprotein and breast cancer related protein are reduced in capillary cerebral amyloid angiopathy

    NARCIS (Netherlands)

    Carrano, A.; Snkhchyan, H.; Kooij, G.; van der Pol, S.; van Horssen, J.; Veerhuis, R.; Hoozemans, J.J.M.; Rozemuller, A.J.M.; de Vries, H.E.

    2014-01-01

    Alzheimer's disease (AD) is the most common form of dementia and marked by deposition of amyloid-β (Aβ) within the brain. Alterations of Aβ transporters at the neurovasculature may play a role in the disease process. We investigated the expression of ABC transporters P-glycoprotein (P-gp) and breast

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

    NARCIS (Netherlands)

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

    1999-01-01

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

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

    NARCIS (Netherlands)

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

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

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

    NARCIS (Netherlands)

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

    2016-01-01

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

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

    NARCIS (Netherlands)

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

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

  18. Verapamil inhibits tumor progression of chemotherapy-resistant pancreatic cancer side population cells

    Science.gov (United States)

    ZHAO, LU; ZHAO, YUE; SCHWARZ, BETTINA; MYSLIWIETZ, JOSEF; HARTIG, ROLAND; CAMAJ, PETER; BAO, QI; JAUCH, KARL-WALTER; GUBA, MAKUS; ELLWART, JOACHIM WALTER; NELSON, PETER JON; BRUNS, CHRISTIANE JOSEPHINE

    2016-01-01

    Tumor side population (SP) cells display stem-like properties that can be modulated by treatment with the calcium channel blocker verapamil. Verapamil can enhance the cytotoxic effects of chemotherapeutic drugs and multi-drug resistance by targeting the transport function of the P-glycoprotein (P-gp). This study focused on the therapeutic potential of verapamil on stem-like SP tumor cells, and further investigated its chemosensitizing effects using L3.6pl and AsPC-1 pancreatic carcinoma models. As compared to parental L3.6pl cells (0.9±0.22%), L3.6pl gemcitabine-resistant cells (L3.6plGres) showed a significantly higher percentage of SP cells (5.38±0.99%) as detected by Hoechst 33342/FACS assays. The L3.6plGres SP cells showed stable gemcitabine resistance, enhanced colony formation ability and increased tumorigenicity. Verapamil effectively inhibited L3.6plGres and AsPC-1 SP cell proliferation in vitro. A pro-apoptotic effect of verapamil was observed in L3.6pl cells, but not in L3.6plGres cells, which was linked to their differential expression of P-gp and equilibrative nucleoside transporter-1 (ENT-1). In an orthotopic pancreatic cancer mouse model, both low and high dose verapamil was shown to substantially reduce L3.6plGres-SP cell tumor growth and metastasis, enhance tumor apoptosis, and reduce microvascular density. PMID:27177126

  19. Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

    Science.gov (United States)

    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

  20. Knockout of a P-glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua.

    Science.gov (United States)

    Zuo, Y-Y; Huang, J-L; Wang, J; Feng, Y; Han, T-T; Wu, Y-D; Yang, Y-H

    2018-02-01

    P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua. © 2017 The Royal Entomological Society.

  1. Rhodamine-123: a p-glycoprotein marker complex with sodium lauryl sulfate.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2008-08-27

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

  3. Etanercept overcomes P-glycoprotein-induced drug resistance in lymphocytes of patients with intractable rheumatoid arthritis.

    Science.gov (United States)

    Tsujimura, Shizuyo; Saito, Kazuyoshi; Nakayamada, Shingo; Tanaka, Yoshiya

    2010-04-01

    P-glycoprotein (P-gp) on activated lymphocytes is an adenosine triphosphate (ATP)-binding cassette transporter that causes drug resistance by exclusion of intracellular drugs in patients with active rheumatoid arthritis (RA). However, infliximab with methotrexate (MTX) can overcome P-gp-mediated drug resistance. We encounter patients who cannot continue infliximab or MTX. Here we tested how etanercept affected P-gp-mediated drug resistance in such intractable RA patients. Peripheral lymphocytes of 11 RA patients (3 switched from infliximab and 8 who could not be treated with MTX) were analyzed for P-gp expression by flow cytometry and for drug exclusion using radioisotope-labeled dexamethasone. Activated lymphocytes of RA patients overexpressed P-gp and coexpressed CD69. Incubation of these lymphocytes with dexamethasone in vitro reduced intracellular dexamethasone levels. Two-week etanercept therapy significantly reduced P-gp expression and eliminated such P-gp- and CD69-high-expressing subgroup. The reduction in P-gp resulted in recovery of intracellular dexamethasone levels in lymphocytes and improvement of disease activity, thus allowing tapering of corticosteroids. None of the patients experienced any severe adverse effects. Etanercept is useful for overcoming P-gp-mediated treatment resistance in intractable RA patients who have to discontinue infliximab or are intolerant to MTX.

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

    Directory of Open Access Journals (Sweden)

    Raquel C. Maia

    2018-01-01

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

  5. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-02-19

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

  7. Evaluation of the P-glycoprotein- and breast cancer resistance protein-mediated brain penetration of {sup 11}C-labeled topotecan using small-animal positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Yamasaki, Tomoteru; Fujinaga, Masayuki; Kawamura, Kazunori; Hatori, Akiko; Yui, Joji [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Nengaki, Nobuki; Ogawa, Masanao; Yoshida, Yuichiro [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); SHI Accelerator Service, Ltd., Tokyo 141-8686 (Japan); Wakizaka, Hidekatsu [Department of Biophysics, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Yanamoto, Kazuhiko [Division of Health Sciences, Graduate School of Medicine, Osaka University, Osaka 565-0871 (Japan); Fukumura, Toshimitsu [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Zhang Mingrong, E-mail: zhang@nirs.go.jp [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan)

    2011-07-15

    Introduction: Topotecan (TPT) is a camptothecin derivative and is an anticancer drug working as a topoisomerase-I-specific inhibitor. But TPT cannot penetrate through the blood-brain barrier. In this study, we synthesized a new positron emission tomography (PET) probe, [{sup 11}C]TPT, to evaluate the P-glycoprotein (Pgp)- and breast cancer resistance protein (BCRP)-mediated brain penetration of [{sup 11}C]TPT using small-animal PET. Methods: [{sup 11}C]TPT was synthesized by the reaction of a desmethyl precursor with [{sup 11}C]CH{sub 3}I. In vitro study using [{sup 11}C]TPT was carried out in MES-SA and doxorubicin-resistant MES-SA/Dx5 cells in the presence or absence of elacridar, a specific inhibitor for Pgp and BCRP. The biodistribution of [{sup 11}C]TPT was determined using small-animal PET and the dissection method in mice. Results: The transport of [{sup 11}C]TPT to the extracellular side was determined in MES-SA/Dx5 cells exhibiting the expressions of Pgp and BCRP at high levels. This transport was inhibited by coincubation with elacridar. In Mdr1a/b{sup -/-}Bcrp1{sup -/-} mice, PET results indicated that the brain uptake of [{sup 11}C]TPT was about two times higher than that in wild-type mice. Similarly, the brain penetration of [{sup 11}C]TPT in wild-type mice was increased by treatment with elacridar. The radioactivity in the brain of elacridar-treated mice was maintained at a certain level after the injection of [{sup 11}C]TPT, although the radioactivity in the blood decreased with time. Conclusions: We demonstrated the increase of brain penetration of [{sup 11}C]TPT by deficiency and inhibition of Pgp and BCRP functions using small-animal PET in mice.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-06-02

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

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

    International Nuclear Information System (INIS)

    Ivanina, Anna V.; Sokolova, Inna M.

    2008-01-01

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

  11. Exhaustive sampling of docking poses reveals binding hypotheses for propafenone type inhibitors of P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Freya Klepsch

    2011-05-01

    Full Text Available Overexpression of the xenotoxin transporter P-glycoprotein (P-gp represents one major reason for the development of multidrug resistance (MDR, leading to the failure of antibiotic and cancer therapies. Inhibitors of P-gp have thus been advocated as promising candidates for overcoming the problem of MDR. However, due to lack of a high-resolution structure the concrete mode of interaction of both substrates and inhibitors is still not known. Therefore, structure-based design studies have to rely on protein homology models. In order to identify binding hypotheses for propafenone-type P-gp inhibitors, five different propafenone derivatives with known structure-activity relationship (SAR pattern were docked into homology models of the apo and the nucleotide-bound conformation of the transporter. To circumvent the uncertainty of scoring functions, we exhaustively sampled the pose space and analyzed the poses by combining information retrieved from SAR studies with common scaffold clustering. The results suggest propafenone binding at the transmembrane helices 5, 6, 7 and 8 in both models, with the amino acid residue Y307 playing a crucial role. The identified binding site in the non-energized state is overlapping with, but not identical to, known binding areas of cyclic P-gp inhibitors and verapamil. These findings support the idea of several small binding sites forming one large binding cavity. Furthermore, the binding hypotheses for both catalytic states were analyzed and showed only small differences in their protein-ligand interaction fingerprints, which indicates only small movements of the ligand during the catalytic cycle.

  12. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    Science.gov (United States)

    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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Interaction between Red Yeast Rice and CYP450 Enzymes/P-Glycoprotein and Its Implication for the Clinical Pharmacokinetics of Lovastatin

    Directory of Open Access Journals (Sweden)

    Chia-Hao Chen

    2012-01-01

    Full Text Available Red yeast rice (RYR can reduce cholesterol through its active component, lovastatin. This study was to investigate the pharmacokinetic properties of lovastatin in RYR products and potential RYR-drug interactions. Extracts of three registered RYR products (LipoCol Forte, Cholestin, and Xuezhikang were more effective than pure lovastatin in inhibiting the activities of cytochrome P450 enzymes and P-glycoprotein. Among CYP450 enzymes, RYR showed the highest inhibition on CYP1A2 and CYP2C19, with comparable inhibitory potencies to the corresponding typical inhibitors. In healthy volunteers taking the RYR product LipoCol Forte, the pharmacokinetic properties of lovastatin and lovastatin acid were linear in the dose range of 1 to 4 capsules taken as a single dose and no significant accumulation was observed after multiple dosing. Concomitant use of one LipoCol Forte capsule with nifedipine did not change the pharmacokinetics of nifedipine. Yet, concomitant use of gemfibrozil with LipoCol Forte resulted in a significant increase in the plasma concentration of lovastatin acid. These findings suggest that the use of RYR products may not have effects on the pharmacokinetics of concomitant comedications despite their effects to inhibit the activities of CYP450 enzymes and P-gp, whereas gemfibrozil affects the pharmacokinetics of lovastatin acid when used concomitantly with RYR products.

  14. Activation of P-glycoprotein and CYP 3A by Coptidis Rhizoma in vivo: Using cyclosporine as a probe substrate in rats

    Directory of Open Access Journals (Sweden)

    Chung-Ping Yu

    2018-04-01

    Full Text Available Coptidis Rhizoma (CR, the rhizome of Coptis chinensis FRANCH, is a popular Chinese herb. CR contains plenty of isoquinoline alkaloids such as berberine, coptisine and palmatine. Cyclosporine (CSP, an important immunosuppressant with narrow therapeutic window, is employed as a probe substrate of P-glycoprotein (P-gp and CYP3A4 in order to investigate the in vivo modulation effect of CR on P-gp and CYP3A4. Three groups of rats were orally administered CSP without and with single dose or repeated dosing of CR in a parallel design. Blood samples were collected at specific time points and the blood CSP concentration was determined by a specific monoclonal fluorescence polarization immunoassay. The results showed that a single dose (1.0 g/kg and the 7th dose (1.0 g/kg of CR significantly decreased the Cmax of CSP by 56.9% and 70.4%, and reduced the AUC0-540 by 56.4% and 68.7%, respectively. Cell study indicated that CR decoction, berberine, coptisine, palmatine all activated the efflux transport of P-gp. Ex-vivo study showed that the serum metabolites of CR activated CYP 3A4. In conclusion, through using CSP as an in vivo probe substrate, we have verified that oral intake of CR activated the functions of P-gp and CYP3A based on in vivo and in vitro studies. Keywords: Cyclosporine, P-glycoprotein, Cytochrome P450 3A, Herb–drug interactions, Pharmacokinetics

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

    Science.gov (United States)

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

    2016-12-01

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

  16. (R)-[11C]Emopamil as a novel tracer for imaging enhanced P-glycoprotein function

    International Nuclear Information System (INIS)

    Toyohara, Jun; Okamoto, Mayumi; Aramaki, Hiroki; Zaitsu, Yuto; Shimizu, Isao; Ishiwata, Kiichi

    2016-01-01

    Introduction: 2-Isopropyl-5-[methyl-(2-phenylethyl)amino]-2-phenylpentanenitrile (emopamil; EMP) is a calcium channel blocker of the phenylalkylamine class, with weak substrate properties for P-glycoprotein (P-gp). A weak substrate for P-gp would be suitable for measuring enhanced P-gp function. This study was performed to synthesise (R)- and (S)-[ 11 C]EMP and characterise their properties as P-gp tracers. Methods: We synthesised (R)- and (S)-[ 11 C]EMP and compared their biodistribution, peripheral metabolism, and effects of the P-gp inhibitor cyclosporine A (CsA, 50 mg/kg). We compared the brain pharmacokinetics of (R)-[ 11 C]EMP and (R)-[ 11 C]verapamil [(R)-[ 11 C]VER] at baseline and CsA pretreatment with small animal positron emission tomography (PET). Results: (R)- and (S)-[ 11 C]EMP were synthesised from (R)- and (S)-noremopamil, respectively, by methylation with [ 11 C]methyl triflate in the presence of NaOH at room temperature. (R)- and (S)-[ 11 C]EMP yields were ~ 30%, with specific activity > 74 GBq/μmol and radiochemical purity > 99%. (R)-[ 11 C]EMP showed significantly greater uptake in the mouse brain than (S)-[ 11 C]EMP. Both showed homogeneous non-stereoselective regional brain distributions. (R)- and (S)-[ 11 C]EMP were rapidly metabolised to hydrophilic metabolites. Unchanged plasma (S)-[ 11 C]EMP level was significantly lower than that of (R)-[ 11 C]EMP 15 minutes post-injection, whilst > 88% of radioactivity in the brain was intact at 15 minutes post-injection and was non-stereoselective. CsA pretreatment increased brain activity ~ 3-fold in mice, but was non-stereoselective. The baseline area-under-the-curve (AUC) of brain radioactivity (0–60 minutes) of (R)-[ 11 C]EMP was 2-fold higher than that of (R)-[ 11 C]VER, but their AUCs after CsA pretreatment were comparable. Conclusions: (R)-[ 11 C]EMP is a novel tracer for imaging P-gp function with higher baseline uptake than (R)-[ 11 C]VER.

  17. P-glycoprotein recognition of substrates and circumvention through rational drug design.

    Science.gov (United States)

    Raub, Thomas J

    2006-01-01

    It is now well recognized that membrane efflux transporters, especially P-glycoprotein (P-gp; ABCB1), play a role in determining the absorption, distribution, metabolism, excretion, and toxicology behaviors of some drugs and molecules in development. An investment in screening structure-activity relationship (SAR) is warranted in early discovery when exposure and/or target activity in an in vivo efficacy model is not achieved and P-gp efflux is identified as a rate-limiting factor. However, the amount of investment in SAR must be placed into perspective by assessing the risks associated with the intended therapeutic target, the potency and margin of safety of the compound, the intended patient population(s), and the market competition. The task of rationally designing a chemistry strategy for circumventing a limiting P-gp interaction can be daunting. The necessity of retaining biological potency and metabolic stability places restrictions on what can be done, and the factors for P-gp recognition of substrates are complicated and poorly understood. The parameters within the assays that affect overall pump efficiency or net efflux, such as passive diffusion, membrane partitioning, and molecular interaction between pump and substrate, should be understood when interpreting data sets associated with chemistry around a scaffold. No single, functional group alone is often the cause, but one group can accentuate the recognition points existing within a scaffold. This can be likened to a rheostat, rather than an on/off switch, where addition or removal of a key group can increase or decrease the pumping efficiency. The most practical approach to de-emphasize the limiting effects of P-gp on a particular scaffold is to increase passive diffusion. Efflux pumping efficiency may be overcome when passive diffusion is fast enough. Eliminating, or substituting with fewer, groups that solvate in water, or decreasing their hydrogen bonding capacity, and adding halogen groups can

  18. Saturable active efflux by p-glycoprotein and breast cancer resistance protein at the blood-brain barrier leads to nonlinear distribution of elacridar to the central nervous system.

    Science.gov (United States)

    Sane, Ramola; Agarwal, Sagar; Mittapalli, Rajendar K; Elmquist, William F

    2013-04-01

    The study objective was to investigate factors that affect the central nervous system (CNS) distribution of elacridar. Elacridar inhibits transport mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and has been used to study the influence of transporters on brain distribution of chemotherapeutics. Adequate distribution of elacridar across the blood-brain barrier (BBB) and into the brain parenchyma is necessary to target tumor cells in the brain that overexpress transporters and reside behind an intact BBB. We examined the role of P-gp and Bcrp on brain penetration of elacridar using Friend leukemia virus strain B wild-type, Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice. Initially, the mice were administered 2.5 mg/kg of elacridar intravenously, and the plasma and brain concentrations were determined. The brain-to-plasma partition coefficient of elacridar in the wild-type mice was 0.82, as compared with 3.5 in Mdr1a/b(-/-) mice, 6.6 in Bcrp1(-/-) mice, and 15 in Mdr1a/b(-/-)Bcrp1(-/-) mice, indicating that both P-gp and Bcrp limit the brain distribution of elacridar. The four genotypes were then administered increasing doses of elacridar, and the CNS distribution of elacridar was determined. The observed and model predicted maximum brain-to-plasma ratios (Emax) at the highest dose were not significantly different in all genotypes. However, the ED50 was lower for Mdr1a/b(-/-) mice compared with Bcrp1(-/-) mice. These findings correlate with the relative expression of P-gp and Bcrp at the BBB in these mice and demonstrate the quantitative enhancement in elacridar CNS distribution as a function of its dose. Overall, this study provides useful concepts for future applications of elacridar as an adjuvant therapy to improve targeting of chemotherapeutic agents to tumor cells in the brain parenchyma.

  19. Ligand Promiscuity between the Efflux Pumps Human P-Glycoprotein and S. aureus NorA.

    Science.gov (United States)

    Brincat, Jean Pierre; Broccatelli, Fabio; Sabatini, Stefano; Frosini, Maria; Neri, Annalisa; Kaatz, Glenn W; Cruciani, Gabriele; Carosati, Emanuele

    2012-03-08

    Thirty-two diverse compounds were evaluated for their ability to inhibit both Pgp-mediated efflux in mouse T-lymphoma L5178 MDR1 and NorA-mediated efflux in S. aureus SA-1199B. Only four compounds were strong inhibitors of both efflux pumps. Three compounds were found to inhibit Pgp exclusively and strongly, while seven compounds inhibited only NorA. These results demonstrate that Pgp and NorA inhibitors do not necessarily overlap, opening the way to safer therapeutic use of effective NorA inhibitors.

  20. Effects of resveratrol on P-glycoprotein and cytochrome P450 3A in vitro and on pharmacokinetics of oral saquinavir in rats

    Directory of Open Access Journals (Sweden)

    Li JP

    2016-11-01

    Full Text Available Jiapeng Li,1,2 Yang Liu,2 Jingru Zhang,1,2 Xiaotong Yu,1,2 Xiaoling Wang,1 Libo Zhao11Department of Pharmacy, Beijing Children’s Hospital, Capital Medical University, 2Department of Pharmacy Administration and Clinical Pharmacy, School of Pharmaceutical Sciences, Peking University, Beijing, People’s Republic of China Background: The intestinal cytochrome P450 3A (CYP 3A and P-glycoprotein (P-gp present a barrier to the oral absorption of saquinavir (SQV. Resveratrol (RESV has been indicated to have modulatory effects on P-gp and CYP 3A. Therefore, this study was to investigate the effects of RESV on P-gp and CYP 3A activities in vitro and in vivo on oral SQV pharmacokinetics in rats.Methods: In vitro, intestinal microsomes were used to evaluate RESV effect on CYP 3A-mediated metabolism of SQV; MDR1-expressing Madin–Darby canine kidney (MDCKII-MDR1 cells were employed to assess the impact of RESV on P-gp-mediated efflux of SQV. In vivo effects were studied using 10 rats randomly assigned to receive oral SQV (30 mg/kg with or without RESV (20 mg/kg. Serial blood samples were obtained over the following 24 h. Concentrations of SQV in samples were ascertained using high-performance liquid chromatography-tandem mass spectrometry analysis.Results: RESV (1–100 µM enhanced residual SQV (% of control in a dose-dependent manner after incubation with intestinal microsomes. RESV (1–100 µM reduced the accumulation of SQV in MDCKII-MDR1 cells in a concentration-dependent manner. A double peaking phenomenon was observed in the plasma SQV profiles in rats. The first peak of plasma SQV concentration was increased, but the second peak was reduced by coadministration with RESV. The mean AUC0–∞ of SQV was slightly decreased, with no statistical significance probably due to the high individual variation.Conclusion: RESV can alter the plasma SQV concentration profiles, shorten the Tmax of SQV. RESV might also cause a slight decrease tendency in the

  1. Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

    Science.gov (United States)

    Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

    2009-07-01

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

  2. Cell Cycle Inhibition To Treat Sleeping Sickness

    Directory of Open Access Journals (Sweden)

    Conrad L. Epting

    2017-09-01

    Full Text Available African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei. During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR, which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo. These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.

  3. Screening for the P-glycoprotein inhibitory pump activity of plant ...

    African Journals Online (AJOL)

    The results revealed that out of the 45 plant extracts tested, 3 extracts i.e. Bauhinia thoningii, Clerodendrum myricoides and Rhus natalensis exhibited pronounced activity at the concentration of 100 µg/ml. In comparison to the negative control, B. thoningii, C. myricoides and R. natalensis extracts inhibited the pump by a ...

  4. Capsaicin pretreatment enhanced the bioavailability of fexofenadine in rats by P-glycoprotein modulation: in vitro, in situ and in vivo evaluation.

    Science.gov (United States)

    Bedada, Satish Kumar; Appani, Ramgopal; Boga, Praveen Kumar

    2017-06-01

    Capsaicin is the main pungent principle present in chili peppers has been found to possess P-glycoprotein (P-gp) inhibition activity in vitro, which may have the potential to modulate bioavailability of P-gp substrates. Therefore, purpose of this study was to evaluate the effect of capsaicin on intestinal absorption and bioavailability of fexofenadine, a P-gp substrate in rats. The mechanistic evaluation was determined by non-everted sac and intestinal perfusion studies to explore the intestinal absorption of fexofenadine. These results were confirmed by an in vivo pharmacokinetic study of oral administered fexofenadine in rats. The intestinal transport and apparent permeability (P app ) of fexofenadine were increased significantly by 2.8 and 2.6 fold, respectively, in ileum of capsaicin treated rats when compared to control group. Similarly, absorption rate constant (K a ), fraction absorbed (F ab ) and effective permeability (P eff ) of fexofenadine were increased significantly by 2.8, 2.9 and 3.4 fold, respectively, in ileum of rats pretreated with capsaicin when compared to control group. In addition, maximum plasma concentration (C max ) and area under the concentration-time curve (AUC) were increased significantly by 2.3 and 2.4 fold, respectively, in rats pretreated with capsaicin as compared to control group. Furthermore, obtained results in rats pretreated with capsaicin were comparable to verapamil (positive control) treated rats. Capsaicin pretreatment significantly enhanced the intestinal absorption and bioavailability of fexofenadine in rats likely by inhibition of P-gp mediated cellular efflux, suggesting that the combined use of capsaicin with P-gp substrates may require close monitoring for potential drug interactions.

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

    Directory of Open Access Journals (Sweden)

    Mary Dyszlewski

    2002-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-07-27

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

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

    Science.gov (United States)

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

    2006-01-01

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

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

    International Nuclear Information System (INIS)

    Meng Zili; Shen Zhenya

    2002-01-01

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

  9. Natural Products based P-glycoprotein Activators for Improved β-amyloid Clearance in Alzheimer's Disease: An in silico Approach.

    Science.gov (United States)

    Shinde, Pravin; Vidyasagar, Nikhil; Dhulap, Sivakami; Dhulap, Abhijeet; Hirwani, Raj

    2015-01-01

    Alzheimer's disease is an age related disorder and is defined to be progressive, irreversible neurodegenerative disease. The potential targets which are associated with the Alzheimer's disease are cholinesterases, N-methyl-D-aspartate receptor, Beta secretase 1, Pregnane X receptor (PXR) and P-glycoprotein (Pgp). P-glycoprotein is a member of the ATP binding cassette (ABC) transporter family, which is an important integral of the blood-brain, blood-cerebrospinal fluid and the blood-testis barrier. Reports from the literature provide evidences that the up-regulation of the efflux pump is liable for a decrease in β -amyloid intracellular accumulation and is an important hallmark in Alzheimer's disease (AD). Thus, targeting β-amyloid clearance by stimulating Pgp could be a useful strategy to prevent Alzheimer's advancement. Currently available drugs provide limited effectiveness and do not assure to cure Alzheimer's disease completely. On the other hand, the current research is now directed towards the development of synthetic or natural based therapeutics which can delay the onset or progression of Alzheimer's disease. Since ancient time medicinal plants such as Withania somnifera, Bacopa monieri, Nerium indicum have been used to prevent neurological disorders including Alzheimer's disease. Till today around 125 Indian medicinal plants have been screened on the basis of ethnopharmacology for their activity against neurological disorders. In this paper, we report bioactives from natural sources which show binding affinity towards the Pgp receptor using ligand based pharmacophore development, virtual screening, molecular docking and molecular dynamics simulation studies for the bioactives possessing acceptable ADME properties. These bioactives can thus be useful to treat Alzheimer's disease.

  10. Genetic deletion of P-glycoprotein alters stress responsivity and increases depression-like behavior, social withdrawal and microglial activation in the hippocampus of female mice.

    Science.gov (United States)

    Brzozowska, Natalia I; Smith, Kristie L; Zhou, Cilla; Waters, Peter M; Cavalcante, Ligia Menezes; Abelev, Sarah V; Kuligowski, Michael; Clarke, David J; Todd, Stephanie M; Arnold, Jonathon C

    2017-10-01

    P-glycoprotein (P-gp) is an ABC transporter expressed at the blood brain barrier and regulates the brain uptake of various xenobiotics and endogenous mediators including glucocorticoid hormones which are critically important to the stress response. Moreover, P-gp is expressed on microglia, the brain's immune cells, which are activated by stressors and have an emerging role in psychiatric disorders. We therefore hypothesised that germline P-gp deletion in mice might alter the behavioral and microglial response to stressors. Female P-gp knockout mice displayed an unusual, frantic anxiety response to intraperitoneal injection stress in the light-dark test. They also tended to display reduced conditioned fear responses compared to wild-type (WT) mice in a paradigm where a single electric foot-shock stressor was paired to a context. Foot-shock stress reduced social interaction and decreased microglia cell density in the amygdala which was not varied by P-gp genotype. Independently of stressor exposure, female P-gp deficient mice displayed increased depression-like behavior, idiosyncratic darting behavior, age-related social withdrawal and hyperactivity, facilitated sensorimotor gating and altered startle reactivity. In addition, P-gp deletion increased microglia cell density in the CA3 region of the hippocampus, and the microglial cells exhibited a reactive, hypo-ramified morphology. Further, female P-gp KO mice displayed increased glucocorticoid receptor (GR) expression in the hippocampus. In conclusion, this research shows that germline P-gp deletion affected various behaviors of relevance to psychiatric conditions, and that altered microglial cell activity and enhanced GR expression in the hippocampus may play a role in mediating these behaviors. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2012-12-18

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

  12. The effect of P-glycoprotein on 18F-FDG uptake in vitro

    International Nuclear Information System (INIS)

    Yu Chunjing; Zhang Bin; Deng Shengming; Wan Weixing; Wu Yiwei

    2013-01-01

    Objective: To evaluate the effect of P-gp inhibitors of verapamil (VER) and GF120918 on 18 F-FDG uptake in Bcap37 and Bcap37/multidrug resistance (MDR)1 cell lines in vitro, and to explore the relationship between 18 F-FDG uptake and P-gp expression at cellular level. Methods: Bcap37 and Bcap37/MDR1 cells were seeded into 6-well plates at a density of 1 × 10 6 per well. Three days later,37 kBq/ml 18 F-FDG, or 37 kBq/ml 18 F-FDG + 100 μmol/L VER, or 37 kBq/ml 18 F-FDG + 50 μmol/L GF120918 were added into each well. After incubated for 10, 30, 60 and 120 min at 37 ℃ and in 5% CO 2 , the medium was removed and the cells were washed three times with 1 ml ice-cold PBS immediately. The radioactivity of 18 F-FDG was measured using a gamma counter. The uptake of 18 F-FDG was expressed as the ratio of 18 F-FDG radioactivity in Bcap37 or Bcap37/MDR1 cells and the overall radioactivity added to the cells in each well.The t test was used for statistical analysis. Results: 18 F-FDG uptake was higher in Bcap37/MDR1 cells than that in Bcap37 cells after incubated for 10 min. The uptake rate was (1.88 ±0.19) % in Bcap37/MDR1 cells and (1.37 ± 0.18) % in Bcap37 cells (t=7.832, P<0.05). On the contrary, 18 F-FDG uptake was significantly higher in Bcap37 cells than that in Bcap37/MDR1 cells after incubated for 60 and 120 min. The uptake rates were (2.29 ±0.23)% and (2.34 ±0.15)% in Bcap37 cells, (1.47 ±0.14)% and (1.53 ±0.22)% in Bcap37/MDR1 cells (t=8.437, 8.283, both P<0.05). 18 F-FDG uptake was significantly higher with VER or GF120918 in Bcap37/MDR1 cells than that without VER or GF120918 after the incubation of 60 and 120 min (t=9.032, 9.243 and 8.765, 8.803, all P<0.05). The uptake rates with VER or GF120918 were (2.45 ±0.21)% and (2.46 ±0.25)%, (2.50 ±0.24)% and (2.48 ±0.27)%. There was no significant difference of 18 F-FDG uptake in Bcap37 cells with or without VER or GF120918. Conclusions: 18 F-FDG is a substrate of P-gp at cellular level. P-gp may act as an

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

    Science.gov (United States)

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

    2014-11-01

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

  14. Platelets Inhibit Migration of Canine Osteosarcoma Cells.

    Science.gov (United States)

    Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

    2017-01-01

    The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2017-07-01

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

  16. Assessment of cerebral P-glycoprotein expression and function with PET by combined [11C]inhibitor and [11C]substrate scans in rats

    International Nuclear Information System (INIS)

    Müllauer, Julia; Karch, Rudolf; Bankstahl, Jens P.; Bankstahl, Marion; Stanek, Johann; Wanek, Thomas; Mairinger, Severin; Müller, Markus; Löscher, Wolfgang; Langer, Oliver; Kuntner, Claudia

    2013-01-01

    Introduction: The adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) protects the brain from accumulation of lipophilic compounds by active efflux transport across the blood–brain barrier. Changes in Pgp function/expression may occur in neurological disorders, such as epilepsy, Alzheimer’s or Parkinson’s disease. In this work we investigated the suitability of the radiolabeled Pgp inhibitors [ 11 C]elacridar and [ 11 C]tariquidar to visualize Pgp density in rat brain with PET. Methods: Rats underwent a first PET scan with [ 11 C]elacridar (n = 5) or [ 11 C]tariquidar (n = 6) followed by a second scan with the Pgp substrate (R)-[ 11 C]verapamil after administration of unlabeled tariquidar at a dose which half-maximally inhibits cerebral Pgp (3 mg/kg). Compartmental modeling using an arterial input function and Logan graphical analysis were used to estimate rate constants and volumes of distribution (V T ) of radiotracers in different brain regions. Results: Brain PET signals of [ 11 C]elacridar and [ 11 C]tariquidar were very low (∼ 0.5 standardized uptake value, SUV). There was a significant negative correlation between V T and K 1 (i.e. influx rate constant from plasma into brain) values of [ 11 C]elacridar or [ 11 C]tariquidar and V T and K 1 values of (R)-[ 11 C]verapamil in different brain regions which was consistent with binding of [ 11 C]inhibitors to Pgp and efflux of (R)-[ 11 C]verapamil by Pgp. Conclusion: The small Pgp binding signals obtained with [ 11 C]elacridar and [ 11 C]tariquidar limit the applicability of these tracers to measure cerebral Pgp density. PET tracers with higher (i.e. subnanomolar) binding affinities will be needed to visualize the low density of Pgp in brain

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

    Directory of Open Access Journals (Sweden)

    Long Wu

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Abdelfatah, Sara A A; Efferth, Thomas

    2015-02-15

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

  20. Multiple efflux pumps are involved in the transepithelial transport of colchicine: combined effect of p-glycoprotein and multidrug resistance-associated protein 2 leads to decreased intestinal absorption throughout the entire small intestine.

    Science.gov (United States)

    Dahan, Arik; Sabit, Hairat; Amidon, Gordon L

    2009-10-01

    The purpose of this study was to thoroughly characterize the efflux transporters involved in the intestinal permeability of the oral microtubule polymerization inhibitor colchicine and to evaluate the role of these transporters in limiting its oral absorption. The effects of P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on colchicine bidirectional permeability were studied across Caco-2 cell monolayers, inhibiting one versus multiple transporters simultaneously. Colchicine permeability was then investigated in different regions of the rat small intestine by in situ single-pass perfusion. Correlation with the P-gp/MRP2 expression level throughout different intestinal segments was investigated by immunoblotting. P-gp inhibitors [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), verapamil, and quinidine], and MRP2 inhibitors [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), indomethacin, and p-aminohippuric acid (p-AH)] significantly increased apical (AP)-basolateral (BL) and decreased BL-AP Caco-2 transport in a concentration-dependent manner. No effect was obtained by the BCRP inhibitors fumitremorgin C (FTC) and pantoprazole. P-gp/MRP2 inhibitors combinations greatly reduced colchicine mucosal secretion, including complete abolishment of efflux (GF120918/MK571). Colchicine displayed low (versus metoprolol) and constant permeability along the rat small-intestine. GF120918 significantly increased colchicine permeability in the ileum with no effect in the jejunum, whereas MK571 augmented jejunal permeability without changing the ileal transport. The GF120918/MK571 combination caused an effect similar to that of MK571 alone in the jejunum and to that of GF120918 alone in the ileum. P-gp expression followed a gradient increasing from

  1. Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug- Drug Interaction Risk Assessment Decision Criterias

    NARCIS (Netherlands)

    Bentz, J.; O'Connor, M.P.; Bednarczyk, D.; Coleman, J.; Lee, C.; Palm, J.; Pak, Y.A.; Perloff, E.S.; Reyner, E.; Balimane, P.; Brännström, M.; Chu, X.; Funk, C.; Guo, A.; Hanna, I.; Herédi-Szabó, K.; Hillgren, K.; Li, L.; Hollnack-Pusch, E.; Jamei, M.; Lin, X.; Mason, A.K.; Neuhoff, S.; Patel, A.; Podila, L.; Plise, E.; Rajaraman, G.; Salphati, L.; Sands, E.; Taub, M.E.; Taur, J.-S.; Weitz, D.; Wortelboer, H.M.; Xia, C.Q.; Xiao, G.; Yabut, J.; Yamagata, T.; Zhang, L.; Ellens, H.

    2013-01-01

    A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC 50 determinations. Each laboratory followed its in-house protocol to determine in vitro

  2. Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Akt signaling pathway in ER-negative human gastric cancer cells.

    Science.gov (United States)

    Mao, Zonglei; Zhou, Jin; Luan, Junwei; Sheng, Weihua; Shen, Xiaochun; Dong, Xiaoqiang

    2014-03-01

    Multidrug resistance (MDR), mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem limiting successful chemotherapy of gastric cancer. Tamoxifen (TAM), a triphenylethylene nonsteroidal antiestrogen agent, shows broad-spectrum antitumor properties. Emerging studies demonstrated that TAM could significantly reduce the MDR in a variety of human cancers. Here we investigated the effects and possible underlying mechanisms of action of TAM on the reversion of MDR in ER-negative human gastric cancer cells. Our results demonstrated that in MDR phenotype SGC7901/CDDP gastric cancer cells TAM dramatically lowered the IC50 of CDDP, 5-FU and ADM, increased the intracellular Rhodamine123 accumulation and induced G0/G1 phase arrest, while G2/M phase decreased accordingly. Furthermore, at the molecular level, TAM substantially decreased the expression of P-gp, p-Akt and the Akt-regulated downstream effectors such as p-GSK-3β, p-BAD, Bcl-XL and cyclinD1 proteins without affecting the expression of t-Akt, t-GSK-3β, t-BAD proteins in SGC7901/CDDP cells. Thus, our findings demonstrate that TAM reverses P-gp-mediated gastric cancer cell MDR via inhibiting the PI3K/Akt signaling pathway. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. Activation of P-glycoprotein and CYP 3A by Coptidis Rhizoma in vivo: Using cyclosporine as a probe substrate in rats.

    Science.gov (United States)

    Yu, Chung-Ping; Huang, Ching-Ya; Lin, Shiuan-Pey; Hou, Yu-Chi

    2018-04-01

    Coptidis Rhizoma (CR), the rhizome of Coptis chinensis FRANCH, is a popular Chinese herb. CR contains plenty of isoquinoline alkaloids such as berberine, coptisine and palmatine. Cyclosporine (CSP), an important immunosuppressant with narrow therapeutic window, is employed as a probe substrate of P-glycoprotein (P-gp) and CYP3A4 in order to investigate the in vivo modulation effect of CR on P-gp and CYP3A4. Three groups of rats were orally administered CSP without and with single dose or repeated dosing of CR in a parallel design. Blood samples were collected at specific time points and the blood CSP concentration was determined by a specific monoclonal fluorescence polarization immunoassay. The results showed that a single dose (1.0 g/kg) and the 7th dose (1.0 g/kg) of CR significantly decreased the C max of CSP by 56.9% and 70.4%, and reduced the AUC 0-540 by 56.4% and 68.7%, respectively. Cell study indicated that CR decoction, berberine, coptisine, palmatine all activated the efflux transport of P-gp. Ex-vivo study showed that the serum metabolites of CR activated CYP 3A4. In conclusion, through using CSP as an in vivo probe substrate, we have verified that oral intake of CR activated the functions of P-gp and CYP3A based on in vivo and in vitro studies. Copyright © 2017. Published by Elsevier B.V.

  4. The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer

    Directory of Open Access Journals (Sweden)

    Li Yan

    2009-11-01

    Full Text Available Abstract Background To detect the expression of multidrug resistance molecules P-glycoprotein (P-gp, Lung resistnce protein (LRP and Multidrug resistance-associated protein (MRP and analyze the relationship between them and the clinico-pathological features. Methods The expressions of P-gp, LRP and MRP in formalin-fixed paraffin-embedded tissue sections from 59 gastric cancer patients were determined by a labbelled Streptavidin-Peroxidase (SP immunohistochemical technique, and the results were analyzed in correlation with clinicopathological data. None of these patients received chemotherapy prior to surgery. Results The positive rates of P-gp, LRP, MRP were 86.4%, 84.7% and 27.1%, respectively. The difference between the positive rate of P-gp and MRP was significant statistically, as well as the difference between the expression of MRP and LRP. No significant difference was observed between P-gp and LRP, but the positively correlation between the expression of P-gp and LRP had been found. No significant correlation between the expression of P-gp, LRP, MRP and the grade of differentiation were observed. The expression of P-gp was correlated with clinical stages positively (r = 0.742, but the difference with the expression of P-gp in different stages was not significant. Conclusion The expressions of P-gp, LRP and MRP in patients with gastric cancer without prior chemotherapy are high, indicating that innate drug resistance may exist in gastric cancer.

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

    Directory of Open Access Journals (Sweden)

    Adena S Spiro

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

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

    Science.gov (United States)

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

    2013-01-01

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

  7. Altered brain concentrations of citalopram and escitalopram in P-glycoprotein deficient mice after acute and chronic treatment.

    Science.gov (United States)

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

    2013-11-01

    According to both in vitro and in vivo data P-glycoprotein (P-gp) may restrict the uptake of several antidepressants into the brain, thus contributing to the poor success rate of current antidepressant therapies. The therapeutic activity of citalopram resides in the S-enantiomer, whereas the R-enantiomer is practically devoid of serotonin reuptake potency. To date, no in vivo data are available that address whether the enantiomers of citalopram and its metabolites are substrates of P-gp. P-gp knockout (abcb1ab (-/-)) and wild-type (abcb1ab (+/+)) mice underwent acute (single-dose) and chronic (two daily doses for 10 days) treatment with citalopram (10mg/kg) or escitalopram (5mg/kg) Serum and brain samples were collected 1-6h after the first or last i.p. injection for subsequent drug analysis by an enantioselective HPLC method. In brain, 3-fold higher concentrations of S- and R-citalopram, and its metabolites, were found in abcb1ab (-/-) mice than in abcb1ab (+/+) mice after both acute and chronic citalopram treatments. After escitalopram treatment, the S-citalopram brain concentration was 3-5 times higher in the knockout mice than in controls. The results provide novel evidence that the enantiomers of citalopram are substrates of P-gp. Possible clinical and toxicological implications of this finding need to be further elucidated. Copyright © 2013 Elsevier B.V. and ECNP. All rights reserved.

  8. P-glycoprotein interactions of novel psychoactive substances - stimulation of ATP consumption and transport across Caco-2 monolayers.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Anja Brenn

    2011-01-01

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

  10. Inhibiting cancer cell hallmark features through nuclear export inhibition.

    Science.gov (United States)

    Sun, Qingxiang; Chen, Xueqin; Zhou, Qiao; Burstein, Ezra; Yang, Shengyong; Jia, Da

    2016-01-01

    Treating cancer through inhibition of nuclear export is one of the best examples of basic research translation into clinical application. Nuclear export factor chromosomal region maintenance 1 (CRM1; Xpo1 and exportin-1) controls cellular localization and function of numerous proteins that are critical for the development of many cancer hallmarks. The diverse actions of CRM1 are likely to explain the broad ranging anti-cancer potency of CRM1 inhibitors observed in pre-clinical studies and/or clinical trials (phase I-III) on both advanced-stage solid and hematological tumors. In this review, we compare and contrast the mechanisms of action of different CRM1 inhibitors, and discuss the potential benefit of unexplored non-covalent CRM1 inhibitors. This emerging field has uncovered that nuclear export inhibition is well poised as an attractive target towards low-toxicity broad-spectrum potent anti-cancer therapy.

  11. Menadione inhibits MIBG uptake in two neuroendocrine cell lines

    NARCIS (Netherlands)

    Cornelissen, J.; Tytgat, G. A.; van den Brug, M.; van Kuilenburg, A. B.; Voûte, P. A.; van Gennip, A. H.

    1997-01-01

    In this paper we report on our studies of the effect of menadione on the uptake of MIBG in the neuroendocrine cell lines PC12 and SK-N-SH. Menadione inhibits the uptake of MIBG in both cell lines in a dose-dependent manner. Inhibition of MIBG uptake is most pronounced in the PC12 cell line.

  12. 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

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Cuiping, E-mail: yangsophia76@hotmail.com; Zhang, Tianhong, E-mail: wdzth@sina.com; Li, Zheng, E-mail: lizh2524@126.com; Xu, Liang, E-mail: wj24998@163.com; Liu, Fei, E-mail: liufeipharm@163.com; Ruan, Jinxiu, E-mail: ruanjx1936@yahoo.com.cn; Liu, Keliang, E-mail: keliangliu55@126.com; Zhang, Zhenqing, E-mail: zhangzhenqingpharm@163.com

    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

  13. 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

    International Nuclear Information System (INIS)

    Yang, Cuiping; Zhang, Tianhong; Li, Zheng; Xu, Liang; Liu, Fei; Ruan, Jinxiu; Liu, Keliang; Zhang, Zhenqing

    2013-01-01

    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 −5 to 2.85 × 10 −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 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 0–12 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 max and AUC of aconitine. • P

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

    International Nuclear Information System (INIS)

    Cheng Bing; Liu Baoping; Han Xingmin

    2003-01-01

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

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

    International Nuclear Information System (INIS)

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

    2002-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-08-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

  18. Primary breast cancer imaging with technetium-99m sestamibi and its relation with P-glycoprotein overexpression

    Energy Technology Data Exchange (ETDEWEB)

    Moretti, J.L. [Medicine Nucleaire, CHU Bobigny, Paris (France); Azaloux, H. [Medicine Nucleaire Oncologie, Hopital P. Zobda Quitman, Fort de France (France); Boisseron, D. [Medicine Nucleaire Oncologie, Hopital P. Zobda Quitman, Fort de France (France); Kouyoumdjian, J.C. [Service de Biochemie, Hopital Henri Mondor, Creteil (France); Vilcoq, J. [Service de Cancerologie-Radiotherapie, Inst. Curie, Paris (France)

    1996-08-01

    The aim of this preliminary study was to evaluate retrospectively sestamibi scintigraphy in relation to the presence of the 170-kDa P-glycoprotein (Pgp), which represents an expression of multidrug resistance in patients with primary breast cancer. Fifteen women (age range 37-76 years) were referred for technetium-99m sestamibi scintigraphy because of suspicious breast lesions detected by mammography and ultrasonography, and subsequently assessed by fine-needle aspiration. Scintigraphy was performed 30 min following the injection of 500 MBq {sup 99m}Tc-sestamibi. Three planar anterior and oblique images were obtained with the patient in the supine position. Excised tumours were assessed for cytosolic CA 15.3, oestrogen (OR) and progesterone (PR) receptors and c-erb B2 neu oncogene. Pathology revealed that only 13 of the 15 patients had malignant tumours. The two benign tumours were sestamibi-negative and Pgp-positive. Sestamibi scintigraphy was positive in 10 of the 13 malignant lesions (including nine of ten infiltrating ductal carcinomas). Two of the three lesions with false-negative scintigraphy were Pgp-negative; in one of these cases histology revealed an invasive lobular carcinoma and in the other, mucinous adenocarcinoma. The third false-negative lesion was a Pgp-positive infiltrating ductal carcinoma which was c-erb B2 neu-negative but CA 15.3-, OR- and PR-positive. This preliminary study confirms that the resistance to chemotherapy which may occur in patients with primary breast cancer can be a cause of negative sestamibi scintigraphy. (orig.)

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

    Science.gov (United States)

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

    2018-04-01

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

  20. Technetium-99m sestamibi imaging in paediatric neuroblastoma and ganglioneuroma and its relation to P-glycoprotein

    International Nuclear Information System (INIS)

    Moerloose, B. de; Dhooge, C.; Benoit, Y.; Laureys, G.; Wiele, C. van de; Dierckx, R.A.; Philippe, J.; Speleman, F.

    1999-01-01

    Imaging with technetium-99m sestamibi offers a non-invasive approach to detect the presence of functional P-glycoprotein (Pgp), one of the major causes of multidrug resistance, in human malignancies. A clinical role for Pgp has been suggested in the subpopulation of primary neuroblastoma without amplification of the proto-oncogene MYCN. We wanted to evaluate the usefulness of 99m Tc-sestamibi scintigraphy in the screening of neural crest tumours for the presence of Pgp. In ten children suffering from MYCN-negative neuroblastoma, ganglioneuroblastoma or ganglioneuroma, 99m Tc-sestamibi imaging was performed at initial diagnosis. All patients underwent planar imaging 20-30 min and 3.5-4 h after intravenous injection of 740 MBq/1.73 m 2 99m Tc-sestamibi. Tumour to normal tissue ratios, as well as washout rates, were determined and compared with in vitro flow cytometric analysis of Pgp expression and function. Pgp expression was analysed flow cytometrically with the monoclonal antibodies 4E3 and MRK16, and Pgp function was evaluated by means of rhodamine 123 uptake and efflux either in the absence or in the presence of the Pgp inhibitor verapamil. In nine of ten patients, we found that the intratumoral 99m Tc-sestamibi activity was comparable to the background activity, which might be suggestive of Pgp presence. This was confirmed flow cytometrically in all but one patient. 99m Tc-sestamibi enhancement was seen in the primary tumour and the bone marrow metastases of one of the ten patients, and this result was concordant with a negative Pgp status. The findings presented suggest that 99m Tc-sestamibi imaging results might correlate with the presence of functional Pgp in neural crest tumours without MYCN amplification. (orig.)

  1. Technetium-99m sestamibi imaging in paediatric neuroblastoma and ganglioneuroma and its relation to P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Moerloose, B. de; Dhooge, C.; Benoit, Y.; Laureys, G. [Department of Pediatrics, University Hospital Gent (Belgium); Wiele, C. van de; Dierckx, R.A. [Department of Nuclear Medicine, University Hospital Gent (Belgium); Philippe, J. [Department of Clinical Chemistry, Microbiology and Immunology, University Hospital Gent (Belgium); Speleman, F. [Department of Medical Genetics, University Hospital Gent (Belgium)

    1999-04-29

    Imaging with technetium-99m sestamibi offers a non-invasive approach to detect the presence of functional P-glycoprotein (Pgp), one of the major causes of multidrug resistance, in human malignancies. A clinical role for Pgp has been suggested in the subpopulation of primary neuroblastoma without amplification of the proto-oncogene MYCN. We wanted to evaluate the usefulness of {sup 99m}Tc-sestamibi scintigraphy in the screening of neural crest tumours for the presence of Pgp. In ten children suffering from MYCN-negative neuroblastoma, ganglioneuroblastoma or ganglioneuroma, {sup 99m}Tc-sestamibi imaging was performed at initial diagnosis. All patients underwent planar imaging 20-30 min and 3.5-4 h after intravenous injection of 740 MBq/1.73 m{sup 2} {sup 99m}Tc-sestamibi. Tumour to normal tissue ratios, as well as washout rates, were determined and compared with in vitro flow cytometric analysis of Pgp expression and function. Pgp expression was analysed flow cytometrically with the monoclonal antibodies 4E3 and MRK16, and Pgp function was evaluated by means of rhodamine 123 uptake and efflux either in the absence or in the presence of the Pgp inhibitor verapamil. In nine of ten patients, we found that the intratumoral {sup 99m}Tc-sestamibi activity was comparable to the background activity, which might be suggestive of Pgp presence. This was confirmed flow cytometrically in all but one patient. {sup 99m}Tc-sestamibi enhancement was seen in the primary tumour and the bone marrow metastases of one of the ten patients, and this result was concordant with a negative Pgp status. The findings presented suggest that {sup 99m}Tc-sestamibi imaging results might correlate with the presence of functional Pgp in neural crest tumours without MYCN amplification. (orig.) With 4 figs., 2 tabs., 44 refs.

  2. Effect of fermented red ginseng on cytochrome P450 and P-glycoprotein activity in healthy subjects, as evaluated using the cocktail approach.

    Science.gov (United States)

    Kim, Min-Gul; Kim, Yunjeong; Jeon, Ji-Young; Kim, Dal-Sik

    2016-12-01

    We assessed the drug interaction profile of fermented red ginseng with respect to the activity of major cytochrome (CYP) P450 enzymes and of a drug transporter protein, P-glycoprotein (P-gp), in healthy volunteers. This study was an open-label crossover study. The CYP probe cocktail drugs caffeine, losartan, dextromethorphan, omeprazole, midazolam and fexofenadine were administered before and after 2 weeks of fermented red ginseng administration. Plasma samples were collected, and tolerability was assessed. Pharmacokinetic parameters were calculated, and the 90% confidence intervals (CIs) of the geometric mean ratios of the parameters were determined from logarithmically transformed data. Values were compared between before and after fermented red ginseng administration using analysis of variance (anova). Fifteen healthy male subjects were evaluated, none of whom were genetically defined as a poor CYP2C9, CYP2C19 or CYP2D6 metabolizer based on genotyping. Before and after fermented red ginseng administration, the geometric least-square mean metabolic ratio (90% CI) was 0.901 (0.830-0.979) for caffeine (CYP1A2) to paraxanthine, 0.774 (0.720-0.831) for losartan (CYP2C9) to EXP3174, 1.052 (0.925-1.197) for omeprazole (CYP2C19) to 5-hydroxyomeprazole, 1.150 (0.860-1.538) for dextromethorphan (CYP2D6) to dextrorphan, and 0.816 (0.673-0.990) for midazolam (CYP3A4) to 1-hydroxymidazolam. The geometric mean ratio of the area under the curve of the last sampling time (AUC last ) for fexofenadine (P-gp) was 1.322 (1.112-1.571). No significantly different drug interactions were observed between fermented red ginseng and the CYP probe substrates following the two-week administration of concentrated fermented red ginseng. However, the inhibition of P-gp was significantly different between fermented red ginseng and the CYP probe substrates. The use of fermented red ginseng requires close attention due to the potential for increased systemic exposure when it is used in

  3. [18F]FDG is not transported by P-glycoprotein and breast cancer resistance protein at the rodent blood–brain barrier

    International Nuclear Information System (INIS)

    Wanek, Thomas; Traxl, Alexander; Bankstahl, Jens P.; Bankstahl, Marion; Sauberer, Michael; Langer, Oliver; Kuntner, Claudia

    2015-01-01

    Introduction: Transport of 2-[ 18 F]fluoro-2-deoxy-D-glucose ([ 18 F]FDG) by the multidrug efflux transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) at the blood–brain barrier (BBB) may confound the interpretation of [ 18 F]FDG brain PET data. Aim of this study was to assess the influence of ABCB1 and ABCG2 at the BBB on brain distribution of [ 18 F]FDG in vivo by performing [ 18 F]FDG PET scans in wild-type and transporter knockout mice and by evaluating changes in [ 18 F]FDG brain distribution after transporter inhibition. Methods: Dynamic small-animal PET experiments (60 min) were performed with [ 18 F]FDG in groups of wild-type and transporter knockout mice (Abcb1a/b (−/−) , Abcg2 (−/−) and Abcb1a/b (−/−) Abcg2 (−/−) ) and in wild-type rats without and with i.v. pretreatment with the known ABCB1 inhibitor tariquidar (15 mg/kg, given at 2 h before PET). Blood was sampled from animals from the orbital sinus vein at the end of the PET scans and measured in a gamma counter. Brain uptake of [ 18 F]FDG was expressed as the brain-to-blood radioactivity concentration ratio in the last PET time frame (K b,brain ). Results: K b,brain values of [ 18 F]FDG were not significantly different between different mouse types both without and with tariquidar pretreatment. The blood-to-brain transfer rate constant of [ 18 F]FDG was significantly lower in tariquidar-treated as compared with vehicle-treated rats (0.350 ± 0.025 mL/min/g versus 0.416 ± 0.024 mL/min/g, p = 0.026, paired t-test) but K b,brain values were not significantly different between both rat groups. Conclusion: Our results show that [ 18 F]FDG is not transported by Abcb1 at the mouse and rat BBB in vivo. In addition we found no evidence for Abcg2 transport of [ 18 F]FDG at the mouse BBB. Advances in knowledge and implications for patient care: Our findings imply that functional activity of ABCB1 and ABCG2 at the BBB does not need to be taken into account when

  4. In Silico and in Vitro Screening for P-Glycoprotein Interaction with Tenofovir, Darunavir, and Dapivirine: An Antiretroviral Drug Combination for Topical Prevention of Colorectal HIV Transmission.

    Science.gov (United States)

    Swedrowska, Magda; Jamshidi, Shirin; Kumar, Abhinav; Kelly, Charles; Rahman, Khondaker Miraz; Forbes, Ben

    2017-08-07

    The aim of the study was to use in silico and in vitro techniques to evaluate whether a triple formulation of antiretroviral drugs (tenofovir, darunavir, and dapivirine) interacted with P-glycoprotein (P-gp) or exhibited any other permeability-altering drug-drug interactions in the colorectal mucosa. Potential drug interactions with P-gp were screened initially using molecular docking, followed by molecular dynamics simulations to analyze the identified drug-transporter interaction more mechanistically. The transport of tenofovir, darunavir, and dapivirine was investigated in the Caco-2 cell models and colorectal tissue, and their apparent permeability coefficient (P app ), efflux ratio (ER), and the effect of transporter inhibitors were evaluated. In silico, dapivirine and darunavir showed strong affinity for P-gp with similar free energy of binding; dapivirine exhibiting a ΔG PB value -38.24 kcal/mol, darunavir a ΔG PB value -36.84 kcal/mol. The rank order of permeability of the compounds in vitro was tenofovir dapivirine. The P app for tenofovir in Caco-2 cell monolayers was 0.10 ± 0.02 × 10 -6 cm/s, ER = 1. For dapivirine, P app was 32.2 ± 3.7 × 10 -6 cm/s, but the ER = 1.3 was lower than anticipated based on the in silico findings. Neither tenofovir nor dapivirine transport was influenced by P-gp inhibitors. The absorptive permeability of darunavir (P app = 6.4 ± 0.9 × 10 -6 cm/s) was concentration dependent with ER = 6.3, which was reduced by verapamil to 1.2. Administration of the drugs in combination did not alter their permeability compared to administration as single agents. In conclusion, in silico modeling, cell culture, and tissue-based assays showed that tenofovir does not interact with P-gp and is poorly permeable, consistent with a paracellular transport mechanism. In silico modeling predicted that darunavir and dapivirine were P-gp substrates, but only darunavir showed P-gp-dependent permeability in the biological models, illustrating that

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

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

    2003-03-07

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

  7. Paeoniflorin inhibits cell growth and induces cell cycle arrest through inhibition of FoxM1 in colorectal cancer cells.

    Science.gov (United States)

    Yue, Meng; Li, Shiquan; Yan, Guoqiang; Li, Chenyao; Kang, Zhenhua

    2018-01-01

    Paeoniflorin (PF) exhibits tumor suppressive functions in a variety of human cancers. However, the function of PF and molecular mechanism in colorectal cancer are elusive. In the present study, we investigated whether PF could exert its antiproliferative activity, anti-migration, and anti-invasive function in colorectal cancer cells. We found that PF inhibited cell growth and induced apoptosis and blocked cell cycle progression in the G0/G1 phase in colorectal cancer cells. Moreover, we found that PF suppressed cell migration and invasion in colorectal cancer cells. FoxM1 has been reported to play an important oncogenic role in human cancers. We also determine whether PF inhibited the expression of FoxM1, leading to its anti-cancer activity. We found that PF treatment in colorectal cancer cells resulted in down-regulation of FoxM1. The rescue experiments showed that overexpression of FoxM1 abrogated the tumor suppressive function induced by PF treatment. Notably, depletion of FoxM1 promoted the anti-tumor activity of PF in colorectal cancer cells. Therefore, inhibition of FoxM1 could participate in the anti-tumor activity of PF in colorectal cancer cells.

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

    Science.gov (United States)

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

    2013-01-01

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

  9. Mullerian Inhibiting Substances (MIS) Augments IFN-gamma Mediated Inhibition of Breast Cancer Cell Growth

    National Research Council Canada - National Science Library

    Gupta, Vandana

    2006-01-01

    MIS is a member of the TGF family. The purpose of this study is to test the hypothesis that MIS and IFN-gamma might be more effective in the inhibition of breast cancer cell growth than either agent alone...

  10. Mullerian Inhibiting Substance (MIS) Augments IFN-gamma Mediated Inhibition of Breast Cancer Cell Growth

    National Research Council Canada - National Science Library

    Gupta, Vandana

    2004-01-01

    Mullerian Inhibiting Substance (MIS), a member of the TGFB family regulates growth, differentiation, and apoptosis in many cell types In the male embryo, MIS causes regression of the Mullerian duct...

  11. Grapefruit juice and its constituents augment colchicine intestinal absorption: potential hazardous interaction and the role of p-glycoprotein.

    Science.gov (United States)

    Dahan, Arik; Amidon, Gordon L

    2009-04-01

    To investigate the potential interaction between grapefruit juice (GFJ) and the oral microtubule polymerization inhibitor colchicine, a P-gp and CYP3A4 substrate. Colchicine intestinal epithelial transport was investigated across Caco-2 cell monolayers in both AP-BL and BL-AP directions, in the absence/presence of known P-gp inhibitors (verapamil and quinidine). The concentration-dependent effects of GFJ and its major constituents (6'-7'-dihydroxybergamottin, naringin and naringenin) on colchicine Caco-2 mucosal secretion were examined. The effect of GFJ on colchicine intestinal-permeability was then investigated in-situ in the rat perfusion model, in both jejunum and ileum. Colchicine exhibited 20-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion, which was reduced by verapamil/quinidine. Colchicine AP-BL permeability was increased and BL-AP was decreased by GFJ in a concentration-dependent manner (IC(50) values of 0.75% and 0.46% respectively), suggesting inhibition of efflux transport, rather than metabolizing enzyme. Similar effects obtained following pre-experiment incubation with GFJ, even though the juice was not present throughout the transepithelial study. 6'-7'-Dihydroxybergamottin, naringin and naringenin displayed concentration-dependent inhibition on colchicine BL-AP secretion (IC(50) values of 90, 592 and 11.6 microM respectively). Ten percent GFJ doubled colchicine rat in-situ ileal permeability, and increased 1.5-fold jejunal permeability. The data suggest that GFJ may augment colchicine oral bioavailability. Due to colchicine narrow therapeutic-index and severely toxic side-effects, awareness of this interaction is prudent.

  12. Ability of polymer-bound P-glycoprotein inhibitor ritonavir to overcome multidrug resistance in various resistant neuroblastoma cell lines

    Czech Academy of Sciences Publication Activity Database

    Koziolová, Eva; Chytil, Petr; Etrych, Tomáš; Janoušková, Olga

    2017-01-01

    Roč. 28, č. 10 (2017), s. 1126-1130 ISSN 0959-4973 R&D Projects: GA MŠk(CZ) LO1507 Institutional support: RVO:61389013 Keywords : drug-delivery polymers * multidrug resistance * N-(2-hydroxypropyl) methacrylamide Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 2.320, year: 2016

  13. Hyperoxia Inhibits T Cell Activation in Mice

    Science.gov (United States)

    Hughes-Fulford, M.; Meissler, J.; Aguayo, E. T.; Globus, R.; Aguado, J.; Candelario, T.

    2013-02-01

    , spleens were removed and the splenocytes were isolated and kept as individual biological samples. We have also examined transcription factors (JASPAR) and pathways of the immune system to help us understand the mechanism of regulation. Results: Our recent mouse immunology experiment aboard STS-131 suggests that the early T cell immune response was inhibited in animals that have been exposed to spaceflight, even 24 hours after return to earth. Moreover, recent experiments in hyperoxic mice show that many of the same genes involved in early T cell activation were altered. Specifically, expression of IL-2Rα, Cxcl2, TNFα, FGF2, LTA and BCL2 genes are dysregulated in mice exposed to hyperoxia. Conclusions: If these hyperoxia-induced changes of gene expression in early T cell activation are additive to the changes seen in the microgravity of spaceflight, there could be an increased infection risk to EVA astronauts, which should be addressed prior to conducting a Mars or other long-term mission.

  14. CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells-comparison with other conazole pesticides.

    Science.gov (United States)

    Sergent, Thérèse; Dupont, Isabelle; Jassogne, Coralie; Ribonnet, Laurence; van der Heiden, Edwige; Scippo, Marie-Louise; Muller, Marc; McAlister, Dan; Pussemier, Luc; Larondelle, Yvan; Schneider, Yves-Jacques

    2009-02-10

    Imazalil (IMA) is a widely used imidazole-antifungal pesticide and, therefore, a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ingested drugs and pollutants, we have investigated the effects of IMA, at realistic intestinal concentrations, on xenobiotic-metabolizing enzymes and efflux pumps by using Caco-2 cells, as a validated in vitro model of the human intestinal absorptive epithelium. For comparison, other conazole fungicides, i.e. ketoconazole, propiconazole and tebuconazole, were also studied. IMA induced cytochrome P450 (CYP) 1A1 activity to the same extent as benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in a dose- and time-dependent manner. Cell-free aryl hydrocarbon receptor (AhR) binding assay and reporter gene assay suggested that IMA is not an AhR-ligand, implying that IMA-mediated induction should involve an AhR-independent pathway. Moreover, IMA strongly inhibited the CYP3A4 activity in 1,25-vitamin D(3)-induced Caco-2 cells. The other fungicides had weak or nil effects on CYP activities. Study of the apical efflux pump activities revealed that ketoconazole inhibited both P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP-2) or breast cancer resistance protein (BCRP), whereas IMA and other fungicides did not. Our results imply that coingestion of IMA-contaminated food and CYP3A4- or CYP1A1-metabolizable drugs or chemicals could lead to drug bioavailability modulation or toxicological interactions, with possible adverse effects for human health.

  15. Agnus castus extracts inhibit prolactin secretion of rat pituitary cells.

    Science.gov (United States)

    Sliutz, G; Speiser, P; Schultz, A M; Spona, J; Zeillinger, R

    1993-05-01

    In our studies on prolactin inhibition by plant extracts we focused on the effects of extracts of Vitex agnus castus and its preparations on rat pituitary cells under basal and stimulated conditions in primary cell culture. Both extracts from Vitex agnus castus as well as synthetic dopamine agonists (Lisuride) significantly inhibit basal as well as TRH-stimulated prolactin secretion of rat pituitary cells in vitro and as a consequence inhibition of prolactin secretion could be blocked by adding a dopamine receptor blocker. Therefore because of its dopaminergic effect Agnus castus could be considered as an efficient alternative phytotherapeutic drug in the treatment of slight hyperprolactinaemia.

  16. Hili Inhibits HIV Replication in Activated T Cells.

    Science.gov (United States)

    Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

    2017-06-01

    P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4 + T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNA Arg (UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4 + T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements. Copyright © 2017 American Society for Microbiology.

  17. Inhibition of brain tumor cell proliferation by alternating electric fields

    International Nuclear Information System (INIS)

    Jeong, Hyesun; Oh, Seung-ick; Hong, Sunghoi; Sung, Jiwon; Jeong, Seonghoon; Yoon, Myonggeun; Koh, Eui Kwan

    2014-01-01

    This study was designed to investigate the mechanism by which electric fields affect cell function, and to determine the optimal conditions for electric field inhibition of cancer cell proliferation. Low-intensity (<2 V/cm) and intermediate-frequency (100–300 kHz) alternating electric fields were applied to glioblastoma cell lines. These electric fields inhibited cell proliferation by inducing cell cycle arrest and abnormal mitosis due to the malformation of microtubules. These effects were significantly dependent on the intensity and frequency of applied electric fields

  18. Inhibition of brain tumor cell proliferation by alternating electric fields

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Hyesun; Oh, Seung-ick; Hong, Sunghoi, E-mail: shong21@korea.ac.kr, E-mail: radioyoon@korea.ac.kr [School of Biosystem and Biomedical Science, Korea University, Seoul 136-703 (Korea, Republic of); Sung, Jiwon; Jeong, Seonghoon; Yoon, Myonggeun, E-mail: shong21@korea.ac.kr, E-mail: radioyoon@korea.ac.kr [Department of Bio-convergence Engineering, Korea University, Seoul 136-703 (Korea, Republic of); Koh, Eui Kwan [Seoul Center, Korea Basic Science Institute, Seoul 136-713 (Korea, Republic of)

    2014-11-17

    This study was designed to investigate the mechanism by which electric fields affect cell function, and to determine the optimal conditions for electric field inhibition of cancer cell proliferation. Low-intensity (<2 V/cm) and intermediate-frequency (100–300 kHz) alternating electric fields were applied to glioblastoma cell lines. These electric fields inhibited cell proliferation by inducing cell cycle arrest and abnormal mitosis due to the malformation of microtubules. These effects were significantly dependent on the intensity and frequency of applied electric fields.

  19. 7-Piperazinethylchrysin inhibits melanoma cell proliferation by ...

    African Journals Online (AJOL)

    PEC) on melanoma cell lines. Methods: Cell viability was analyzed by trypan blue exclusion assays and the cell cycle by flow cytometry using ModFit LT software. Specifically, cells were stained with propidium iodide (0.5 mg/mL) supplemented ...

  20. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis.

    Science.gov (United States)

    Li, Qingli; Lambrechts, Mark J; Zhang, Qiuyang; Liu, Sen; Ge, Dongxia; Yin, Rutie; Xi, Mingrong; You, Zongbing

    2013-01-01

    Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose) polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy.

  1. Thrombopoietin inhibits murine mast cell differentiation

    Science.gov (United States)

    Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

    2009-01-01

    We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

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

    Science.gov (United States)

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

    2010-04-01

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

  3. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind

    2015-01-01

    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...

  4. Activation of P-glycoprotein and CYP 3A by Coptidis Rhizoma in vivo: Using cyclosporine as a probe substrate in rats

    OpenAIRE

    Chung-Ping Yu; Ching-Ya Huang; Shiuan-Pey Lin; Yu-Chi Hou

    2018-01-01

    Coptidis Rhizoma (CR), the rhizome of Coptis chinensis FRANCH, is a popular Chinese herb. CR contains plenty of isoquinoline alkaloids such as berberine, coptisine and palmatine. Cyclosporine (CSP), an important immunosuppressant with narrow therapeutic window, is employed as a probe substrate of P-glycoprotein (P-gp) and CYP3A4 in order to investigate the in vivo modulation effect of CR on P-gp and CYP3A4. Three groups of rats were orally administered CSP without and with single dose or repe...

  5. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation.

    Directory of Open Access Journals (Sweden)

    José Manuel Tirado-Vélez

    Full Text Available Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40-70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.

  6. Playing with Opening and Closing of Heterocycles: Using the Cusmano-Ruccia Reaction to Develop a Novel Class of Oxadiazolothiazinones, Active as Calcium Channel Modulators and P-Glycoprotein Inhibitors

    Directory of Open Access Journals (Sweden)

    Domenico Spinelli

    2014-10-01

    Full Text Available As a result of the ring-into-ring conversion of nitrosoimidazole derivatives, we obtained a molecular scaffold that, when properly decorated, is able to decrease inotropy by blocking L-type calcium channels. Previously, we used this scaffold to develop a quantitative structure-activity relationship (QSAR model, and we used the most potent oxadiazolothiazinone as a template for ligand-based virtual screening. Here, we enlarge the diversity of chemical decorations, present the synthesis and in vitro data for 11 new derivatives, and develop a new 3D-QSAR model with recent in silico techniques. We observed a key role played by the oxadiazolone moiety: given the presence of positively charged calcium ions in the transmembrane channel protein, we hypothesize the formation of a ternary complex between the oxadiazolothiazinone, the Ca2+ ion and the protein. We have supported this hypothesis by means of pharmacophore generation and through the docking of the pharmacophore into a homology model of the protein. We also studied with docking experiments the interaction with a homology model of P-glycoprotein, which is inhibited by this series of molecules, and provided further evidence toward the relevance of this scaffold in biological interactions.

  7. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    International Nuclear Information System (INIS)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang; Zhang, Yi

    2013-01-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients

  8. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  9. Inhibition of GRP78 abrogates radioresistance in oropharyngeal carcinoma cells after EGFR inhibition by cetuximab.

    Directory of Open Access Journals (Sweden)

    Chaonan Sun

    Full Text Available The EGFR-specific mAb cetuximab is one of the most effective treatments for oropharyngeal carcinoma, while patient responses to EGFR inhibitors given alone are modest. Combination treatment with radiation can improve the efficacy of treatment through increasing radiosensitivity, while resistance to radiation after administration of cetuximab limits its efficiency. Radiation and drugs can damage the endoplasmic reticulum (ER homeostatic state and result in ER stress (ERS, subsequently causing resistance to radiation and drugs. Whether the ERS pathway is involved in radioresistance after administration of cetuximab has not been reported. Herein, we show that cetuximab could increase the radiosensitivity of FaDu cells but not Detroit562 cells. In addition, cetuximab inhibited the radiation-induced activation of the ERS signalling pathway IRE1α/ATF6-GRP78 in FaDu cells, while this effect was absent in Detroit562 cells. Silencing GRP78 increased the radiosensitivity of oropharyngeal carcinoma cells and inhibited radiation-induced DNA double-strand-break (DSB repair and autophagy. More interestingly, silencing GRP78 abrogated resistance to cetuximab and radiation in Detroit562 cells and had a synergistic effect with cetuximab in increasing the radiosensitivity of FaDu cells. Immunohistochemistry showed that overexpression of both GRP78 and EGFR was associated with a poor prognosis in oropharyngeal carcinoma patients (P<0.05. Overall, the results of this study show that radioresistance after EGFR inhibition by cetuximab is mediated by the ERS signalling pathway IRE1α/ATF6-GRP78. This suppression was consequently unable to inhibit radiation-induced DSB repair and autophagy in oropharyngeal carcinoma cells, which conferred resistance to radiotherapy and cetuximab. These results suggest that the cooperative effects of radiotherapy and cetuximab could be further improved by inhibiting GRP78 in non-responsive oropharyngeal carcinoma patients.

  10. Cyperus scariosus Chloroform Fraction Inhibits T cell Responses in ...

    African Journals Online (AJOL)

    Erah

    CSC did not significantly (p < 0.01) suppress Th2 (IL-4) system. Conclusion: The findings from this investigation reveal that C. scariosus causes immunosuppression by inhibiting Th1 cytokines. Keywords: Cyperus scariosus; Immunosuppression; Humoral antibody titre; Cell-mediated immune response; CD 4+ T- helper cells ...

  11. MicroRNA-144 inhibits hepatocellular carcinoma cell proliferation

    Indian Academy of Sciences (India)

    MiR-144 was shown to besignificantly down-regulated in HCC tissues and cell lines. Subsequently, overexpression of miR-144 was transfectedinto HCC cell lines so as to investigate its biological function, including MTT, colony formation, and transwell assays.Gain of function assay revealed miR-144 remarkably inhibited ...

  12. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis

    Directory of Open Access Journals (Sweden)

    Li Q

    2013-07-01

    Full Text Available Qingli Li,1,2 Mark J Lambrechts,1 Qiuyang Zhang,1 Sen Liu,1 Dongxia Ge,1 Rutie Yin,2 Mingrong Xi,2 Zongbing You1 1Departments of Structural and Cellular Biology and Orthopaedic Surgery, Tulane Cancer Center and Louisiana Cancer Research Consortium, Tulane Center for Stem Cell Research and Regenerative Medicine, and Tulane Center for Aging, Tulane University Health Sciences Center, New Orleans, LA, USA; 2Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu, People’s Republic of China Abstract: Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA, are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy. Keywords: serine hydroxymethyltransferase, prostate cancer, apoptosis

  13. Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

    International Nuclear Information System (INIS)

    Brooks, J.C.; Brooks, M.

    1985-01-01

    Permeabilized cells treated with the adenosine triphosphate analog, ( 35 S)adenosine-5'-0-3(3-thiotriphosphate) ((γ- 35 S)ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35 S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35 S from the cells over a 20 min period. Treatment of permeabilized cells with ATPγS inhibited secretion and 35 S incorporation into the cells. Pretreatment with ATPγS resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35 S from (γ- 35 S)ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells. 15 references, 1 figure, 3 tables

  14. Thrombomodulin inhibits the activation of eosinophils and mast cells.

    Science.gov (United States)

    Roeen, Ziaurahman; Toda, Masaaki; D'Alessandro-Gabazza, Corina N; Onishi, Masahiro; Kobayashi, Tetsu; Yasuma, Taro; Urawa, Masahito; Taguchi, Osamu; Gabazza, Esteban C

    2015-01-01

    Eosinophils and mast cells play critical roles in the pathogenesis of bronchial asthma. Activation of both cells leads to the release of pro-inflammatory mediators in the airway of asthmatic patients. Recently, we have shown that inhaled thrombomodulin inhibits allergic bronchial asthma in a mouse model. In the present study, we hypothesize that thrombomodulin can inhibit the activation of eosinophils and mast cells. The effect of thrombomodulin on the activation and release of inflammatory mediators from eosinophils and mast cells was evaluated. Thrombomodulin inhibited the eotaxin-induced chemotaxis, upregulation of CD11b and degranulation of eosinophils. Treatment with thrombomodulin also significantly suppressed the degranulation and synthesis of inflammatory cytokines and chemokines in eosinophils and mast cells. Mice treated with a low-dose of inhaled thrombomodulin have decreased number of eosinophils and activated mast cells and Th2 cytokines in the lungs compared to untreated mice. The results of this study suggest that thrombomodulin may modulate allergic responses by inhibiting the activation of both eosinophils and mast cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Quercetin inhibits adipogenesis of muscle progenitor cells in vitro

    Directory of Open Access Journals (Sweden)

    Tomoko Funakoshi

    2018-03-01

    Full Text Available Muscle satellite cells are committed myogenic progenitors capable of contributing to myogenesis to maintain adult muscle mass and function. Several experiments have demonstrated that muscle satellite cells can differentiate into adipocytes in vitro, supporting the mesenchymal differentiation potential of these cells. Moreover, muscle satellite cells may be a source of ectopic muscle adipocytes, explaining the lipid accumulation often observed in aged skeletal muscle (sarcopenia and in muscles of patients` with diabetes. Quercetin, a polyphenol, is one of the most abundant flavonoids distributed in edible plants, such as onions and apples, and possesses antioxidant, anticancer, and anti-inflammatory properties. In this study, we examined whether quercetin inhibited the adipogenesis of muscle satellite cells in vitro with primary cells from rat limbs by culture in the presence of quercetin under adipogenic conditions. Morphological observations, Oil Red-O staining results, triglyceride content analysis, and quantitative reverse transcription polymerase chain reaction revealed that quercetin was capable of inhibiting the adipogenic induction of muscle satellite cells into adipocytes in a dose-dependent manner by suppressing the transcript levels of adipogenic markers, such as peroxisome proliferator-activated receptor-γ and fatty acid binding protein 4. Our results suggested that quercetin inhibited the adipogenesis of muscle satellite cells in vitro by suppressing the transcription of adipogenic markers. Keywords: Quercetin, Muscle satellite cell, Differentiation, Intramuscular lipid

  16. Anandamide inhibits adhesion and migration of breast cancer cells

    International Nuclear Information System (INIS)

    Grimaldi, Claudia; Pisanti, Simona; Laezza, Chiara; Malfitano, Anna Maria; Santoro, Antonietta; Vitale, Mario; Caruso, Maria Gabriella; Notarnicola, Maria; Iacuzzo, Irma; Portella, Giuseppe; Di Marzo, Vincenzo; Bifulco, Maurizio

    2006-01-01

    The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB 1 receptors could induce a non-invasive phenotype in breast mtastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB 1 antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB 1 receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB 1 receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo

  17. Lutein Inhibits the Migration of Retinal Pigment Epithelial Cells via Cytosolic and Mitochondrial Akt Pathways (Lutein Inhibits RPE Cells Migration

    Directory of Open Access Journals (Sweden)

    Ching-Chieh Su

    2014-08-01

    Full Text Available During the course of proliferative vitreoretinopathy (PVR, the retinal pigment epithelium (RPE cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation.

  18. Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization

    Directory of Open Access Journals (Sweden)

    Jia Shen

    2017-07-01

    Full Text Available Background: The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Methods: Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol’s inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol’s efficacy in vivo. Results: Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. Conclusion: These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment.

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

    Science.gov (United States)

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

    2007-07-01

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

  20. Human mesenchymal stem cells inhibit osteoclastogenesis through osteoprotegerin production.

    Science.gov (United States)

    Oshita, Koichi; Yamaoka, Kunihiro; Udagawa, Nobuyuki; Fukuyo, Shunsuke; Sonomoto, Koshiro; Maeshima, Keisuke; Kurihara, Ryuji; Nakano, Kazuhisa; Saito, Kazuyoshi; Okada, Yosuke; Chiba, Kenji; Tanaka, Yoshiya

    2011-06-01

    Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy. Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers. The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium. The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA. Copyright © 2011 by the American College of Rheumatology.

  1. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Svenningsen, Katrine; Almind Knudsen, Lina

    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...... with glucocorticoids. The evidence for the involvement of ABCC2 and ABCG2 in colonic pathophysiology was weak. CONCLUSION: ABCB1, diet, and gut microbes mutually interact in colonic inflammation, a well-known risk factor for CRC. Further insight may be translated into preventive and treatment strategies....... 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...

  2. Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport

    DEFF Research Database (Denmark)

    Syvanen, S.; Lindhe, O.; Palner, M.

    2009-01-01

    preclinical data to humans. Compounds found to be P-gp substrates in rodents are likely to also be substrates in higher species, but sufficient blood-brain barrier permeability may be retained in humans to allow the compound to act at intracerebral targets Udgivelsesdato: 2009/3......Species differences occur in the brain concentrations of drugs, but the reasons for these differences are not yet apparent. This study was designed to compare brain uptake of three radiolabeled P-glycoprotein (P-gp) substrates across species using positron emission tomography. Brain concentrations...... and brain-to-plasma ratios were compared; [(11)C]verapamil in rats, guinea pigs, and monkeys; [(11)C](S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino )-2(S)-phenylpiperidine (GR205171) in rats, guinea pigs, monkeys, and humans; and [(18)F]altanserin in rats, minipigs, and humans...

  3. Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

    Directory of Open Access Journals (Sweden)

    Claudia Scotti

    Full Text Available Helicobacter pylori (H. pylori is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

  4. The inhibition of repair in UV irradiated human cells

    International Nuclear Information System (INIS)

    Collins, A.R.S.; Schor, S.L.; Johnson, R.T.

    1977-01-01

    Three different assay procedures are used to determine the effects of hydroxyurea on excision repair in UV-irradiated HeLa cells. At the cytological level, incubation of UV-irradiated metaphase cells with hydroxyurea caused chromosome decondensation. Using a modified alkaline sucrose gradient sedimentation technique involving minimal lysis before centrifugation, a marked retardation was found in the sedimentation of DNA from UV-irradiated cells incubated for a short period with hydroxyurea. The effect of hydroxyurea on the incorporation of [ 3 H]thymidine by UV-irradiated G1 cells was found to depend on the concentration of thymidine present in the medium. The results point to an inhibition of repair DNA synthesis by hydroxyurea (or deoxyadenosine), at the level of the supply of DNA precursors, i.e. in the same way that these agents inhibit semiconservative DNA synthesis. In the presence of these inhibitors, single-strand gaps accumulate in the DNA

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

    Science.gov (United States)

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

    2007-05-01

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

  6. P-glycoprotein binds to ezrin at amino acid residues 149-242 in the FERM domain and plays a key role in the multidrug resistance of human osteosarcoma.

    Science.gov (United States)

    Brambilla, Daria; Zamboni, Silvia; Federici, Cristina; Lugini, Luana; Lozupone, Francesco; De Milito, Angelo; Cecchetti, Serena; Cianfriglia, Maurizio; Fais, Stefano

    2012-06-15

    Overexpression of the mdr1 gene encoding P-glycoprotein (Pgp) exerts a major role in reducing the effectiveness of cytotoxic therapy in osteosarcoma. The interaction between actin and Pgp has been shown to be instrumental in the establishment of multidrug resistance (MDR) in human tumor cells. The cytoskeleton linker ezrin exerts a pivotal role in maintaining the functional connection between actin and Pgp. We investigated the role of ezrin in a human multidrug-resistant osteosarcoma cell line overexpressing Pgp and compared it to its counterpart that overexpresses an ezrin deletion mutant. The results showed that Pgp binds at amino acid residues 149-242 of the N-terminal domain of ezrin. The interaction between ezrin and Pgp occurs in the plasma membrane of MDR cells, where they also co-localize with the ganglioside G(M1) located in lipid rafts. The overexpression of the ezrin deletion mutant entirely restored drug susceptibility of osteosarcoma cells, consistent with Pgp dislocation to cytoplasmic compartments and abrogation of G(M1) /Pgp co-localization at the plasma membrane. Our study provides evidence that ezrin exerts a key role in MDR of human osteosarcoma cells through a Pgp-ezrin-actin connection that is instrumental for the permanence of Pgp into plasma membrane lipid rafts. We also show for the first time that Pgp-binding site is localized to amino acid residues 149-242 of the ezrin Band 4.1, Ezrin/Radixin/Moesin (FERM) domain, thus proposing a specific target for future molecular therapy aimed at counteracting MDR in osteosarcoma patients. Copyright © 2011 UICC.

  7. Fluoxetine regulates cell growth inhibition of interferon-α.

    Science.gov (United States)

    Lin, Yu-Min; Yu, Bu-Chin; Chiu, Wen-Tai; Sun, Hung-Yu; Chien, Yu-Chieh; Su, Hui-Chen; Yen, Shu-Yang; Lai, Hsin-Wen; Bai, Chyi-Huey; Young, Kung-Chia; Tsao, Chiung-Wen

    2016-10-01

    Fluoxetine, a well-known anti-depression agent, may act as a chemosensitizer to assist and promote cancer therapy. However, how fluoxetine regulates cellular signaling to enhance cellular responses against tumor cell growth remains unclear. In the present study, addition of fluoxetine promoted growth inhibition of interferon-alpha (IFN-α) in human bladder carcinoma cells but not in normal uroepithelial cells through lessening the IFN-α-induced apoptosis but switching to cause G1 arrest, and maintaining the IFN-α-mediated reduction in G2/M phase. Activations and signal transducer and transactivator (STAT)-1 and peroxisome proliferator-activated receptor alpha (PPAR-α) were involved in this process. Chemical inhibitions of STAT-1 or PPAR-α partially rescued bladder carcinoma cells from IFN-α-mediated growth inhibition via blockades of G1 arrest, cyclin D1 reduction, p53 downregulation and p27 upregulation in the presence of fluoxetine. However, the functions of both proteins were not involved in the control of fluoxetine over apoptosis and maintained the declined G2/M phase of IFN-α. These results indicated that activation of PPAR-α and STAT-1 participated, at least in part, in growth inhibition of IFN-α in the presence of fluoxetine.

  8. ATF3 inhibits PPARγ-stimulated transactivation in adipocyte cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Min-Kyung; Jung, Myeong Ho, E-mail: jung0603@pusan.ac.kr

    2015-01-02

    Highlights: • ATF3 inhibits PPARγ-stimulated transcriptional activation. • ATF3 interacts with PPARγ. • ATF3 suppresses p300-mediated transcriptional coactivation. • ATF3 decreases the binding of PPARγ and recruitment of p300 to PPRE. - Abstract: Previously, we reported that activating transcription factor 3 (ATF3) downregulates peroxisome proliferator activated receptor (PPARγ) gene expression and inhibits adipocyte differentiation in 3T3-L1 cells. Here, we investigated another role of ATF3 on the regulation of PPARγ activity. ATF3 inhibited PPARγ-stimulated transactivation of PPARγ responsive element (PPRE)-containing reporter or GAL4/PPARγ chimeric reporter. Thus, ATF3 effectively repressed rosiglitazone-stimulated expression of adipocyte fatty acid binding protein (aP2), PPARγ target gene, in 3T3-L1 cells. Coimmunoprecipitation and GST pulldown assay demonstrated that ATF3 interacted with PPARγ. Accordingly, ATF3 prevented PPARγ from binding to PPRE on the aP2 promoter. Furthermore, ATF3 suppressed p300-mediated transcriptional coactivation of PPRE-containing reporter. Chromatin immunoprecipitation assay showed that overexpression of ATF3 blocked both binding of PPARγ and recruitment of p300 to PPRE on aP2 promoter induced by rosiglitazone treatment in 3T3-L1 cells. Taken together, these results suggest that ATF3 interacts with PPARγ and represses PPARγ-mediated transactivation through suppression of p300-stimulated coactivation in 3T3-L1 cells, which may play a role in inhibition of adipocyte differentiation.

  9. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    International Nuclear Information System (INIS)

    Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-01-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion

  10. Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

    2015-09-08

    B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

  11. Andrographolide inhibits multiple myeloma cells by inhibiting the TLR4/NF-κB signaling pathway.

    Science.gov (United States)

    Gao, Hui; Wang, Jianrong

    2016-02-01

    Andrographolide is an active component from the extract of Andrographis paniculata [(Burm.f) Nees], a medicinal plant from the Acanthaceae family. Pharmacological studies have revealed that andrographolide possesses anti-bacterial, anti-inflammatory, anti-viral, immune regulatory and hepatoprotective properties, and is efficacious in the treatment of cardiovascular diseases, while exhibiting low toxicity and low cost. The present study aimed to determine the inhibitory effects of andrographolide on the growth of multiple myeloma (MM) cells and its possible impact on the Toll-like receptor (TLR)4/nuclear factor (NF)-κB signaling pathway. Cell proliferation was detected using an MTT assay, cellular apoptosis was measured using flow cytometry, and caspase-9/3 activation were assessed using colorimetric assay kits. Furthermore, TLR4 and NF-κB protein expression was determined by western blot analysis. The results revealed that andrographolide reduced the proliferation, while increasing cellular apoptosis and caspase-9/3 activation of MM cells, in addition to downregulating the expression of TLR4 and NF-κB protein. Of note, TLR4- or NF-κB-targeting small-interfering (si)RNA enhanced the andrographolide-induced inhibition of cell proliferation and induction of apoptosis of MM cells. The results of the present study therefore suggested that andrographolide inhibited multiple myeloma cells via the TLR4/NF-κB signaling pathway.

  12. Acrolein in cigarette smoke inhibits T-cell responses.

    Science.gov (United States)

    Lambert, Cherie; McCue, Jesica; Portas, Mary; Ouyang, Yanli; Li, JiMei; Rosano, Thomas G; Lazis, Alexander; Freed, Brian M

    2005-10-01

    Cigarette smoking inhibits T-cell responses in the lungs, but the immunosuppressive compounds have not been fully identified. Cigarette smoke extracts inhibit IL-2, IFN-gamma, and TNF-alpha production in stimulated lymphocytes obtained from peripheral blood, even when the extracts were diluted 100-fold to 1000-fold. The objective of these studies was to identify the immunosuppressive compounds found in cigarette smoke. Gas chromatography/mass spectroscopy and HPLC were used to identify and quantitate volatile compounds found in cigarette smoke extracts. Bioactivity was measured by viability and production of cytokine mRNA and protein levels in treated human lymphocytes. The vapor phase of the cigarette smoke extract inhibited cytokine production, indicating that the immunosuppressive compounds were volatile. Among the volatile compounds identified in cigarette smoke extracts, only the alpha,beta-unsaturated aldehydes, acrolein (inhibitory concentration of 50% [IC50] = 3 micromol/L) and crotonaldehyde (IC50 = 6 micromol/L), exhibited significant inhibition of cytokine production. Although the levels of aldehydes varied 10-fold between high-tar (Camel) and ultralow-tar (Carlton) extracts, even ultralow-tar cigarettes produced sufficient levels of acrolein (34 micromol/L) to suppress cytokine production by >95%. We determined that the cigarette smoke extract inhibited transcription of cytokine genes. The inhibitory effects of acrolein could be blocked with the thiol compound N-acetylcysteine. The vapor phase from cigarette smoke extracts potently suppresses cytokine production. The compound responsible for this inhibition appears to be acrolein.

  13. Phytochemicals radiosensitize cancer cells by inhibiting DNA repair

    International Nuclear Information System (INIS)

    Singh, Rana P.

    2017-01-01

    Solid tumors are mostly treated with radiotherapy. Radiotherapy is toxic to normal tissues and also promote the invasiveness and radioresistance in cancer cells. The resistance against radiotherapy and adverse effects to normal cells reduce the overall therapeutic effects of the treatment. Radiosensitizing agents usually show limited success during clinical trials. Therefore, the search and development of new radiosensitizers showing selective response to only cancer cells is desirable. We analyzed the radiosensitizing effects including cell death effect of silibinin, a phytochemical on prostate cancer cells. Silibinin enhanced gamma radiation (2.5-10 Gy) induced inhibition in colony formation selectively in prostate cancer cells. In cell cycle progression, G2/M phase is the most sensitive phase for radiation-induced damage which was delayed by the compound treatment in radiation exposed cells. The lower concentrations of silibinin substantially enhanced radiation-induced apoptosis. A prolonged reactive oxygen species production was also observed in these treatments EGFR signaling pathway can contribute to radiation-induced pro-survival mechanisms and to the therapeutic resistance. Agent treatment reduced the IR-induced EGFR phosphorylation and consequently reversed the resistance mediating mechanisms within the cancer cell. Thus, inhibiting DNA repair in cancer cells would enhance therapeutic response of radiation in cancer cells. Silibinin affected the localization of EGFR and DNA-dependent protein kinase, the DNA-PK is known to be an important mediator of DSB repair in human cells, and showed increased number of pH2AX (ser139) foci, and thus indicating lower DNA repair in these cancer cells. This was also confirmed in the tumor xenograft study. Our findings suggest that a combination of silibinin with radiation could be an effective treatment of radioresistant human prostate cancer and warrants further investigation. (author)

  14. XIAP antagonist embelin inhibited proliferation of cholangiocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Cody J Wehrkamp

    Full Text Available Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 cells. Assessment of nuclear morphology demonstrated a concentration-dependent increase in nuclear staining. Interestingly, embelin induced nuclear morphology changes as a single agent, independent of the addition of TNF-related apoptosis inducing ligand (TRAIL. However, caspase activity assays revealed that increasing embelin concentrations resulted in slight inhibition of caspase activity, not activation. In addition, the use of a pan-caspase inhibitor did not prevent nuclear morphology changes. Finally, embelin treatment of cholangiocarcinoma cells did not induce DNA fragmentation or PARP cleavage. Apoptosis does not appear to contribute to the effects of embelin on cholangiocarcinoma cells. Instead, embelin caused inhibition of cell proliferation and cell cycle analysis indicated that embelin increased the number of cells in S and G2/M phase. Our results demonstrate that embelin decreased proliferation in cholangiocarcinoma cell lines. Embelin treatment resulted in decreased XIAP protein expression, but did not induce or enhance apoptosis. Thus, in cholangiocarcinoma cells the mechanism of action of embelin may not be dependent on apoptosis.

  15. Elaeocarpusin Inhibits Mast Cell-Mediated Allergic Inflammation

    Directory of Open Access Journals (Sweden)

    Min-Jong Kim

    2018-06-01

    Full Text Available Mast cells are major effector cells for allergic responses that act by releasing inflammatory mediators, such as histamine and pro-inflammatory cytokines. Accordingly, different strategies have been pursued to develop anti-allergic and anti-inflammatory candidates by regulating the function of mast cells. The purpose of this study was to determine the effectiveness of elaeocarpusin (EL on mast cell-mediated allergic inflammation. We isolated EL from Elaeocarpus sylvestris L. (Elaeocarpaceae, which is known to possess anti-inflammatory properties. For this study, various sources of mast cells and mouse anaphylaxis models were used. EL suppressed the induction of markers for mast cell degranulation, such as histamine and β-hexosaminidase, by reducing intracellular calcium levels. Expression of pro-inflammatory cytokines, such as tumor necrosis factor-α and IL-4, was significantly decreased in activated mast cells by EL. This inhibitory effect was related to inhibition of the phosphorylation of Fyn, Lyn, Syk, and Akt, and the nuclear translocation of nuclear factor-κB. To confirm the effect of EL in vivo, immunoglobulin E-mediated passive cutaneous anaphylaxis (PCA and ovalbumin-induced active systemic anaphylaxis (ASA models were induced. EL reduced the PCA reaction in a dose dependent manner. In addition, EL attenuated ASA reactions such as hypothemia, histamine release, and IgE production. Our results suggest that EL is a potential therapeutic candidate for allergic inflammatory diseases that acts via the inhibition of mast cell degranulation and expression of proinflammatory cytokines.

  16. Inhibition of glycolysis by misonidazole in hypoxic cells

    International Nuclear Information System (INIS)

    Ling, L.; Sutherland, R.

    1984-01-01

    Inhibition of glycolysis has been postulated to be a mechanism of misonidazole (MISO) toxicity in hypoxic cells. To investigate the effect of MISO on glycolysis, glucose transport and its consumption and lactate formation were measured. Exponential EMT6 cells (10/sup 6/ cells/ml) were made hypoxix by continuous gassing in 3% CO/sub 2/ in N/sub 2/. They were then treated with 5mM MISO for various times, then washed and analysed for their rates of anaerobic glycolysis. Glucose and lactate content were determined enzymatically. The rates of both glucose consumption and lactate formation decreased after 30 min hypoxic incubation with MISO. After 90 min, the rates were not measurable even though the cells still excluded Trypan Blue. There was, however, a parallel decrease in plating efficiency. These data suggest that the inhibition of glycolysis is an important mechanism of hypoxic toxicity of MISO. To locate the site of inhibition, studies were initiated to look at glucose transport by following the uptake of /sup 14/-C-3-0-methyl-glucose, a nonmetabolised glucose analog. Results obtained so far indicate that up to 90 min of hypoxic incubation with MISO, there was no change in the kinetics of the uptake of his analog. Therefore, the results showed that in hypoxic cells treated with MISO, the glucose transport system was unaffected. However, there was a rapid decrease in anaerobic glycolysis

  17. RNA interference targeting raptor inhibits proliferation of gastric cancer cells

    International Nuclear Information System (INIS)

    Wu, William Ka Kei; Lee, Chung Wa; Cho, Chi Hin; Chan, Francis Ka Leung; Yu, Jun; Sung, Joseph Jao Yiu

    2011-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G 0 /G 1 -phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D 3 and p21 Waf1 , which stabilizes cyclin D/cdk4 complex for G 1 -S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  19. Radiosensitization of colorectal carcinoma cell lines by histone deacetylase inhibition

    International Nuclear Information System (INIS)

    Flatmark, Kjersti; Nome, Ragnhild V; Folkvord, Sigurd; Bratland, Åse; Rasmussen, Heidi; Ellefsen, Mali Strand; Fodstad, Øystein; Ree, Anne Hansen

    2006-01-01

    The tumor response to preoperative radiotherapy of locally advanced rectal cancer varies greatly, warranting the use of experimental models to assay the efficacy of molecular targeting agents in rectal cancer radiosensitization. Histone deacetylase (HDAC) inhibitors, agents that cause hyperacetylation of histone proteins and thereby remodeling of chromatin structure, may override cell cycle checkpoint responses to DNA damage and amplify radiation-induced tumor cell death. Human colorectal carcinoma cell lines were exposed to ionizing radiation and HDAC inhibitors, and cell cycle profiles and regulatory factors, as well as clonogenicity, were analyzed. In addition to G 2 /M phase arrest following irradiation, the cell lines displayed cell cycle responses typical for either intact or defective p53 function (the presence or absence, respectively, of radiation-induced expression of the cell cycle inhibitor p21 and subsequent accumulation of G 1 phase cells). In contrast, histone acetylation was associated with complete depletion of the G 1 population of cells with functional p53 but accumulation of both G 1 and G 2 /M populations of cells with defective p53. The cellular phenotypes upon HDAC inhibition were consistent with the observed repression of Polo-like kinase-1, a regulatory G 2 /M phase kinase. Following pre-treatment with HDAC inhibitors currently undergoing clinical investigation, the inhibitory effect of ionizing radiation on clonogenicity was significantly amplified. In these experimental models, HDAC inhibition sensitized the tumor cells to ionizing radiation, which is in accordance with the concept of increased probability of tumor cell death when chromatin structure is modified

  20. An antitubulin agent BCFMT inhibits proliferation of cancer cells and induces cell death by inhibiting microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Ankit Rai

    Full Text Available Using cell based screening assay, we identified a novel anti-tubulin agent (Z-5-((5-(4-bromo-3-chlorophenylfuran-2-ylmethylene-2-thioxothiazolidin-4-one (BCFMT that inhibited proliferation of human cervical carcinoma (HeLa (IC(50, 7.2 ± 1.8 µM, human breast adenocarcinoma (MCF-7 (IC(50, 10.0 ± 0.5 µM, highly metastatic breast adenocarcinoma (MDA-MB-231 (IC(50, 6.0 ± 1 µM, cisplatin-resistant human ovarian carcinoma (A2780-cis (IC(50, 5.8 ± 0.3 µM and multi-drug resistant mouse mammary tumor (EMT6/AR1 (IC(50, 6.5 ± 1 µM cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 µM, BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably state by 135% and reduced the dynamicity (dimer exchange per unit time of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 ± 1.8 µM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i of 5.2 ± 1.5 µM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2 at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug

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

    Science.gov (United States)

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

    2017-11-01

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

  2. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

    1990-01-01

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  3. Intraglomerular inhibition maintains mitral cell response contrast across input frequencies.

    Science.gov (United States)

    Shao, Zuoyi; Puche, Adam C; Shipley, Michael T

    2013-11-01

    Odor signals are transmitted to the olfactory bulb by olfactory nerve (ON) synapses onto mitral/tufted cells (MTCs) and external tufted cells (ETCs); ETCs provide additional feed-forward excitation to MTCs. Both are strongly regulated by intraglomerular inhibition that can last up to 1 s and, when blocked, dramatically increases ON-evoked MC spiking. Intraglomerular inhibition thus limits the magnitude and duration of MC spike responses to sensory input. In vivo, sensory input is repetitive, dictated by sniffing rates from 1 to 8 Hz, potentially summing intraglomerular inhibition. To investigate this, we recorded MTC responses to 1- to 8-Hz ON stimulation in slices. Inhibitory postsynaptic current area (charge) following each ON stimulation was unchanged from 1 to 5 Hz and modestly paired-pulse attenuated at 8 Hz, suggesting there is no summation and only limited decrement at the highest input frequencies. Next, we investigated frequency independence of intraglomerular inhibition on MC spiking. MCs respond to single ON shocks with an initial spike burst followed by reduced spiking decaying to baseline. Upon repetitive ON stimulation peak spiking is identical across input frequencies but the ratio of peak-to-minimum rate before the stimulus (max-min) diminishes from 30:1 at 1 Hz to 15:1 at 8 Hz. When intraglomerular inhibition is selectively blocked, peak spike rate is unchanged but trough spiking increases markedly decreasing max-min firing ratios from 30:1 at 1 Hz to 2:1 at 8 Hz. Together, these results suggest intraglomerular inhibition is relatively frequency independent and can "sharpen" MC responses to input across the range of frequencies. This suggests that glomerular circuits can maintain "contrast" in MC encoding during sniff-sampled inputs.

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

    Science.gov (United States)

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

    2018-02-01

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

  5. Canine tracheal epithelial cells are more sensitive than rat tracheal epithelial cells to transforming growth factor beta induced growth inhibition

    International Nuclear Information System (INIS)

    Hubbs, A.F.; Hahn, F.F.; Kelly, G.; Thomassen, D.G.

    1988-01-01

    Transforming growth factor beta (TGFβ) markedly inhibited growth of canine tracheal epithelial (CTE) cells. Reduced responsiveness to TGFβ-induced growth inhibition accompanied neoplastic progression of these cells from primary to transformed to neoplastic. This was similar to the relationship between neoplastic progression and increased resistance to TGFβ-induced growth inhibition seen for rat tracheal epithelial (RTE) cells. The canine cells were more sensitive than rat cells to TGFβ-induced growth inhibition at all stages in the neoplastic process. (author)

  6. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    International Nuclear Information System (INIS)

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-01-01

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  7. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  8. Angiopoietin1 inhibits mast cell activation and protects against anaphylaxis.

    Directory of Open Access Journals (Sweden)

    Jun-Hua Yao

    Full Text Available Since morbidity and mortality rates of anaphylaxis diseases have been increasing year by year, how to prevent and manage these diseases effectively has become an important issue. Mast cells play a central regulatory role in allergic diseases. Angiopoietin1 (Ang-1 exhibits anti-inflammatory properties by inhibiting vascular permeability, leukocyte migration and cytokine production. However, Ang-1's function in mast cell activation and anaphylaxis diseases is unknown. The results of our study suggest that Ang-1 decreased lipopolysaccharide (LPS-induced pro-inflammatory cytokines production of mast cells by suppressing IκB phosphorylation and NF-κB nuclear translocation. Ang-1 also strongly inhibited compound 48/80 induced and FcεRI-mediated mast cells degranulation by decreasing intracellular calcium levels in vitro. In vivo lentivirus-mediated delivery of Ang-1 in mice exhibited alleviated leakage in IgE-dependent passive cutaneous anaphylaxis (PCA. Furthermore, exogenous Ang-1 intervention treatment prevented mice from compound 48/80-induced mesentery mast cell degranulation, attenuated increases in pro-inflammatory cytokines, relieved lung injury, and improved survival in anaphylaxis shock. The results of our study reveal, for the first time, the important role of Ang-1 in the activation of mast cells, and identify a therapeutic effect of Ang-1 on anaphylaxis diseases.

  9. Barium inhibits arsenic-mediated apoptotic cell death in human squamous cell carcinoma cells.

    Science.gov (United States)

    Yajima, Ichiro; Uemura, Noriyuki; Nizam, Saika; Khalequzzaman, Md; Thang, Nguyen D; Kumasaka, Mayuko Y; Akhand, Anwarul A; Shekhar, Hossain U; Nakajima, Tamie; Kato, Masashi

    2012-06-01

    Our fieldwork showed more than 1 μM (145.1 μg/L) barium in about 3 μM (210.7 μg/L) arsenic-polluted drinking well water (n = 72) in cancer-prone areas in Bangladesh, while the mean concentrations of nine other elements in the water were less than 3 μg/L. The types of cancer include squamous cell carcinomas (SCC). We hypothesized that barium modulates arsenic-mediated biological effects, and we examined the effect of barium (1 μM) on arsenic (3 μM)-mediated apoptotic cell death of human HSC-5 and A431 SCC cells in vitro. Arsenic promoted SCC apoptosis with increased reactive oxygen species (ROS) production and JNK1/2 and caspase-3 activation (apoptotic pathway). In contrast, arsenic also inhibited SCC apoptosis with increased NF-κB activity and X-linked inhibitor of apoptosis protein (XIAP) expression level and decreased JNK activity (antiapoptotic pathway). These results suggest that arsenic bidirectionally promotes apoptotic and antiapoptotic pathways in SCC cells. Interestingly, barium in the presence of arsenic increased NF-κB activity and XIAP expression and decreased JNK activity without affecting ROS production, resulting in the inhibition of the arsenic-mediated apoptotic pathway. Since the anticancer effect of arsenic is mainly dependent on cancer apoptosis, barium-mediated inhibition of arsenic-induced apoptosis may promote progression of SCC in patients in Bangladesh who keep drinking barium and arsenic-polluted water after the development of cancer. Thus, we newly showed that barium in the presence of arsenic might inhibit arsenic-mediated cancer apoptosis with the modulation of the balance between arsenic-mediated promotive and suppressive apoptotic pathways.

  10. Clonal cell populations unresponsive to radiosensitization induced by telomerase inhibition

    International Nuclear Information System (INIS)

    Ju, Yeun-Jin; Shin, Hyun-Jin; Park, Jeong-Eun; Juhn, Kyoung-Mi; Woo, Seon Rang; Kim, Hee-Young; Han, Young-Hoon; Hwang, Sang-Gu; Hong, Sung-Hee; Kang, Chang-Mo; Yoo, Young-Do; Park, Won-Bong; Cho, Myung-Haing; Park, Gil Hong; Lee, Kee-Ho

    2010-01-01

    Research highlights: → In our present manuscript, we have clearly showed an interesting but problematic obstacle of a radiosensitization strategy based on telomerase inhibition by showing that: Clonal population unresponsive to this radiosensitization occasionally arise. → The telomere length of unsensitized clones was reduced, as was that of most sensitized clones. → The unsensitized clones did not show chromosome end fusion which was noted in all sensitized clones. → P53 status is not associated with the occurrence of unsensitized clone. → Telomere end capping in unsensitized clone is operative even under telomerase deficiency. -- Abstract: A combination of a radiotherapeutic regimen with telomerase inhibition is valuable when tumor cells are to be sensitized to radiation. Here, we describe cell clones unresponsive to radiosensitization after telomere shortening. After extensive division of individual transformed clones of mTERC -/- cells, about 22% of clones were unresponsive to radiosensitization even though telomerase action was inhibited. The telomere lengths of unsensitized mTERC -/- clones were reduced, as were those of most sensitized clones. However, the unsensitized clones did not exhibit chromosomal end-to-end fusion to the extent noted in all sensitized clones. Thus, a defense mechanism preventing telomere erosion is operative even when telomeres become shorter under conditions of telomerase deficiency, and results in unresponsiveness to the radiosensitization generally mediated by telomere shortening.

  11. Gimeracil sensitizes cells to radiation via inhibition of homologous recombination

    International Nuclear Information System (INIS)

    Takagi, Masaru; Sakata, Koh-ichi; Someya, Masanori; Tauchi, Hiroshi; Iijima, Kenta; Matsumoto, Yoshihisa; Torigoe, Toshihiko; Takahashi, Akari; Hareyama, Masato; Fukushima, Masakazu

    2010-01-01

    Background and purpose: 5-Chloro-2,4-dihydroxypyridine (Gimeracil) is a component of an oral fluoropyrimidine derivative S-1. Gimeracil is originally added to S-1 to yield prolonged 5-FU concentrations in tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We found that Gimeracil by itself had the radiosensitizing effect. Methods and materials: We used various cell lines deficient in non-homologous end-joining (NHEJ) or homologous recombination (HR) as well as DLD-1 and HeLa in clonogenic assay. γ-H2AX focus formation and SCneo assay was performed to examine the effects of Gimeracil on DNA double strand break (DSB) repair mechanisms. Results: Results of γ-H2AX focus assay indicated that Gimeracil inhibited DNA DSB repair. It did not sensitize cells deficient in HR but sensitized those deficient in NHEJ. In SCneo assay, Gimeracil reduced the frequency of neo-positive clones. Additionally, it sensitized the cells in S-phase more than in G0/G1. Conclusions: Gimeracil inhibits HR. Because HR plays key roles in the repair of DSBH caused by radiotherapy, Gimeracil may enhance the efficacy of radiotherapy through the suppression of HR-mediated DNA repair pathways.

  12. Caffeine inhibits cell proliferation by G0/G1 phase arrest in JB6 cells.

    Science.gov (United States)

    Hashimoto, Takashi; He, Zhiwei; Ma, Wei-Ya; Schmid, Patricia C; Bode, Ann M; Yang, Chung S; Dong, Zigang

    2004-05-01

    Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.

  13. Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

    Directory of Open Access Journals (Sweden)

    Shuei-Kuen Tseng

    2014-09-01

    Conclusion: Arecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

  14. Inhibiting prenylation augments chemotherapy efficacy in renal cell carcinoma through dual inhibition on mitochondrial respiration and glycolysis.

    Science.gov (United States)

    Huang, Jiangrong; Yang, Xiaoyu; Peng, Xiaochun; Huang, Wei

    2017-11-18

    Prenylation is a posttranslational lipid modification required for the proper functions of a number of proteins involved in cell regulation. Here, we show that prenylation inhibition is important for renal cell carcinoma (RCC) growth, survival and response to chemotherapy, and its underlying mechanism may be contributed to mitochondrial dysfunction. We first demonstrated that a HMG-CoA reductase inhibitor pitavastatin inhibited mevalonate pathway and thereby prenylation in RCC cells. In addition, pitavastatin is effective in inhibiting growth and inducing apoptosis in a panel of RCC cell lines. Combination of pitavastatin and paclitaxel is significantly more effective than pitavastatin or paclitaxel alone as shown by both in vitro cell culture system and in vivo RCC xenograft model. Importantly, pitavastatin treatment inhibits mitochondrial respiration via suppressing mitochondrial complex I and II enzyme activities. Interestingly, different from mitochondrial inhibitor phenformin that inhibits mitochondrial respiration but activates glycolytic rate in RCC cells, pitavastatin significantly decreases glycolytic rate. The dual inhibitory action of pitavastatin on mitochondrial respiration and glycolysis results in remarkable energy depletion and oxidative stress in RCC cells. In addition, inhibition of prenylation by depleting Isoprenylcysteine carboxylmethyltransferase (Icmt) also mimics the inhibitory effects of pitavastatin in RCC cells. Our work demonstrates the previously unappreciated association between prenylation inhibition and energy metabolism in RCC, which can be therapeutically exploited, likely in tumors that largely rely on energy metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    Science.gov (United States)

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. ERβ inhibits proliferation and invasion of breast cancer cells

    Science.gov (United States)

    Lazennec, Gwendal; Bresson, Damien; Lucas, Annick; Chauveau, Corine; Vignon, Françoise

    2001-01-01

    Recent studies indicate that the expression of ERβ in breast cancer is lower than in normal breast, suggesting that ERβ could play an important role in carcinogenesis. To investigate this hypothesis, we engineered estrogen-receptor negative MDA-MB-231 breast cancer cells to reintroduce either ERα or ERβ protein with an adenoviral vector. In these cells, ERβ (as ERα) expression was monitored using RT-PCR and Western blot. ERβ protein was localized in the nucleus (immunocytochemistry) and able to transactivate estrogen-responsive reporter constructs in the presence of estradiol. ERβ and ERα induced the expression of several endogenous genes such as pS2, TGFα or the cyclin kinase inhibitor p21, but in contrast to ERα, ERβ was unable to regulate c-myc proto-oncogene expression. The pure antiestrogen ICI 164, 384 completely blocked ERα and ERβ estrogen-induced activities. ERβ inhibited MDA-MB-231 cell proliferation in a ligand-independent manner, whereas ERα inhibition of proliferation is hormone-dependent. Moreover, ERβ and ERα, decreased cell motility and invasion. Our data bring the first evidence that ERβ is an important modulator of proliferation and invasion of breast cancer cells and support the hypothesis that the loss of ERβ expression could be one of the events leading to the development of breast cancer. PMID:11517191

  17. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Cheng-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan (China); Kuan, Yu-Hsiang [Department of Pharmacology, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Pharmacy, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Ou, Yen-Chuan; Li, Jian-Ri [Division of Urology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Wu, Chih-Cheng [Department of Anesthesiology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Department of Financial and Computational Mathematics, Providence University, Taichung 433, Taiwan (China); Pan, Pin-Ho [Department of Pediatrics, Tungs’ Taichung MetroHarbor Hospital, Taichung 435, Taiwan (China); Chen, Wen-Ying [Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Huang, Hsuan-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Chen, Chun-Jung, E-mail: cjchen@vghtc.gov.tw [Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan (China); Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Center for General Education, Tunghai University, Taichung 407, Taiwan (China); Department of Nursing, HungKuang University, Taichung 433, Taiwan (China)

    2014-09-10

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK.

  18. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

    International Nuclear Information System (INIS)

    Chang, Cheng-Yi; Kuan, Yu-Hsiang; Ou, Yen-Chuan; Li, Jian-Ri; Wu, Chih-Cheng; Pan, Pin-Ho; Chen, Wen-Ying; Huang, Hsuan-Yi; Chen, Chun-Jung

    2014-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK

  19. BET bromodomain inhibition promotes neurogenesis while inhibiting gliogenesis in neural progenitor cells

    Directory of Open Access Journals (Sweden)

    Jingjun Li

    2016-09-01

    Full Text Available Neural stem cells and progenitor cells (NPCs are increasingly appreciated to hold great promise for regenerative medicine to treat CNS injuries and neurodegenerative diseases. However, evidence for effective stimulation of neuronal production from endogenous or transplanted NPCs for neuron replacement with small molecules remains limited. To identify novel chemical entities/targets for neurogenesis, we had established a NPC phenotypic screen assay and validated it using known small-molecule neurogenesis inducers. Through screening small molecule libraries with annotated targets, we identified BET bromodomain inhibition as a novel mechanism for enhancing neurogenesis. BET bromodomain proteins, Brd2, Brd3, and Brd4 were found to be downregulated in NPCs upon differentiation, while their levels remain unaltered in proliferating NPCs. Consistent with the pharmacological study using bromodomain selective inhibitor (+-JQ-1, knockdown of each BET protein resulted in an increase in the number of neurons with simultaneous reduction in both astrocytes and oligodendrocytes. Gene expression profiling analysis demonstrated that BET bromodomain inhibition induced a broad but specific transcription program enhancing directed differentiation of NPCs into neurons while suppressing cell cycle progression and gliogenesis. Together, these results highlight a crucial role of BET proteins as epigenetic regulators in NPC development and suggest a therapeutic potential of BET inhibitors in treating brain injuries and neurodegenerative diseases.

  20. Cold atmospheric plasma treatment inhibits growth in colorectal cancer cells.

    Science.gov (United States)

    Schneider, Christin; Arndt, Stephanie; Zimmermann, Julia L; Li, Yangfang; Karrer, Sigrid; Bosserhoff, Anja-Katrin

    2018-06-01

    Plasma oncology is a relatively new field of research. Recent developments have indicated that cold atmospheric plasma (CAP) technology is an interesting new therapeutic approach to cancer treatment. In this study, p53 wildtype (LoVo) and human p53 mutated (HT29 and SW480) colorectal cancer cells were treated with the miniFlatPlaSter - a device particularly developed for the treatment of tumor cells - that uses the Surface Micro Discharge (SMD) technology for plasma production in air. The present study analyzed the effects of plasma on colorectal cancer cells in vitro and on normal colon tissue ex vivo. Plasma treatment had strong effects on colon cancer cells, such as inhibition of cell proliferation, induction of cell death, and modulation of p21 expression. In contrast, CAP treatment of murine colon tissue ex vivo for up to 2 min did not show any toxic effect on normal colon cells compared to H2O2 positive control. In summary, these results suggest that the miniFlatPlaSter plasma device is able to kill colorectal cancer cells independent of their p53 mutation status. Thus, this device presents a promising new approach in colon cancer therapy.

  1. Blue light inhibits the growth of B16 melanoma cells

    International Nuclear Information System (INIS)

    Ohara, Masayuki; Katoh, Osamu; Watanabe, Hiromitsu

    2002-01-01

    Although a number of studies have been carried out to examine the biological effects of radiation and ultraviolet radiation (UV), little is known concerning the effects of visible light. In the present study, exposure of B16 melanoma cells to blue light (wavelength 470 nm, irradiance 5.7 mW/cm 2 ) from a light-emitting diode (LED) inhibited cell growth in proportion to the period of exposure, with no increase observed in the number of dead cells. The number of B16 melanoma colonies that formed after exposure to blue light for 20 min was only slightly less than that in non-exposed controls, but the colony size as assessed by the area covered by colonies and cell counts per colony were markedly decreased. The percentages of G0/G1 and G2/M phase cells were markedly increased, with a reduction in S phase cells as determined by flow cytometry after exposure to blue light. Furthermore, analysis of the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA also showed a reduction in the percentage of S phase cells after exposure. These results indicate that blue light exerts cytostatic effects, but not a cytocidal action, on B16 melanoma cells. (author)

  2. Blue light inhibits the growth of B16 melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Ohara, Masayuki; Katoh, Osamu; Watanabe, Hiromitsu [Hiroshima Univ. (Japan). Research Inst. for Radiation Biology and Medicine; Kawashima, Yuzo [Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima (Japan)

    2002-05-01

    Although a number of studies have been carried out to examine the biological effects of radiation and ultraviolet radiation (UV), little is known concerning the effects of visible light. In the present study, exposure of B16 melanoma cells to blue light (wavelength 470 nm, irradiance 5.7 mW/cm{sup 2}) from a light-emitting diode (LED) inhibited cell growth in proportion to the period of exposure, with no increase observed in the number of dead cells. The number of B16 melanoma colonies that formed after exposure to blue light for 20 min was only slightly less than that in non-exposed controls, but the colony size as assessed by the area covered by colonies and cell counts per colony were markedly decreased. The percentages of G0/G1 and G2/M phase cells were markedly increased, with a reduction in S phase cells as determined by flow cytometry after exposure to blue light. Furthermore, analysis of the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA also showed a reduction in the percentage of S phase cells after exposure. These results indicate that blue light exerts cytostatic effects, but not a cytocidal action, on B16 melanoma cells. (author)

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  4. Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells

    International Nuclear Information System (INIS)

    Mamo, Tewodros; Mladek, Ann C.; Shogren, Kris L.; Gustafson, Carl; Gupta, Shiv K.; Riester, Scott M.; Maran, Avudaiappan; Galindo, Mario; Wijnen, Andre J. van; Sarkaria, Jann N.; Yaszemski, Michael J.

    2017-01-01

    Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PK CS ), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PK CS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PK CS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PK CS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PK CS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. - Highlights: • DNA-PKcs is consistently expressed in human osteosarcoma tissue and cell lines. • The DNA-PKcs inhibitor, KU60648, effectively radiosensitizes osteosarcoma cells. • Combining KU60648 with radiation increases G2/M accumulation and DNA damage.

  5. Effects of PPARα inhibition in head and neck paraganglioma cells.

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    Rosalba Florio

    Full Text Available Head and neck paragangliomas (HNPGLs are rare tumors that may cause important morbidity, because of their tendency to infiltrate the skull base. At present, surgery is the only therapeutic option, but radical removal may be difficult or impossible. Thus, effective targets and molecules for HNPGL treatment need to be identified. However, the lack of cellular models for this rare tumor hampers this task. PPARα receptor activation was reported in several tumors and this receptor appears to be a promising therapeutic target in different malignancies. Considering that the role of PPARα in HNPGLs was never studied before, we analyzed the potential of modulating PPARα in a unique model of HNPGL cells. We observed an intense immunoreactivity for PPARα in HNPGL tumors, suggesting that this receptor has an important role in HNPGL. A pronounced nuclear expression of PPARα was also confirmed in HNPGL-derived cells. The specific PPARα agonist WY14643 had no effect on HNPGL cell viability, whereas the specific PPARα antagonist GW6471 reduced HNPGL cell viability and growth by inducing cell cycle arrest and caspase-dependent apoptosis. GW6471 treatment was associated with a marked decrease of CDK4, cyclin D3 and cyclin B1 protein expression, along with an increased expression of p21 in HNPGL cells. Moreover, GW6471 drastically impaired clonogenic activity of HNPGL cells, with a less marked effect on cell migration. Notably, the effects of GW6471 on HNPGL cells were associated with the inhibition of the PI3K/GSK3β/β-catenin signaling pathway. In conclusion, the PPARα antagonist GW6471 reduces HNPGL cell viability, interfering with cell cycle and inducing apoptosis. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3β/β-catenin pathway. Therefore, PPARα could represent a novel therapeutic target for HNPGL.

  6. Cellulose synthesis inhibition, cell expansion, and patterns of cell wall deposition in Nitella internodes

    International Nuclear Information System (INIS)

    Richmond, P.A.; Metraux, J.P.

    1984-01-01

    The authors have investigated the pattern of wall deposition and maturation and correlated it with cell expansion and cellulose biosynthesis. The herbicide 2,6-dichlorobenzonitrile (DCB) was found to be a potent inhibitor of cellulose synthesis, but not of cell expansion in Nitella internodal cells. Although cellulose synthesis is inhibited during DCB treatment, matrix substances continue to be synthesized and deposited. The inhibition of cellulose microfibril deposition can be demonstrated by various techniques. These results demonstrate that matrix deposition is by apposition, not by intussusception, and that the previously deposited wall moves progressively outward while stretching and thinning as a result of cell expansion

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

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  8. Multiple Linear Regression Analysis Indicates Association of P-Glycoprotein Substrate or Inhibitor Character with Bitterness Intensity, Measured with a Sensor.

    Science.gov (United States)

    Yano, Kentaro; Mita, Suzune; Morimoto, Kaori; Haraguchi, Tamami; Arakawa, Hiroshi; Yoshida, Miyako; Yamashita, Fumiyoshi; Uchida, Takahiro; Ogihara, Takuo

    2015-09-01

    P-glycoprotein (P-gp) regulates absorption of many drugs in the gastrointestinal tract and their accumulation in tumor tissues, but the basis of substrate recognition by P-gp remains unclear. Bitter-tasting phenylthiocarbamide, which stimulates taste receptor 2 member 38 (T2R38), increases P-gp activity and is a substrate of P-gp. This led us to hypothesize that bitterness intensity might be a predictor of P-gp-inhibitor/substrate status. Here, we measured the bitterness intensity of a panel of P-gp substrates and nonsubstrates with various taste sensors, and used multiple linear regression analysis to examine the relationship between P-gp-inhibitor/substrate status and various physical properties, including intensity of bitter taste measured with the taste sensor. We calculated the first principal component analysis score (PC1) as the representative value of bitterness, as all taste sensor's outputs shared significant correlation. The P-gp substrates showed remarkably greater mean bitterness intensity than non-P-gp substrates. We found that Km value of P-gp substrates were correlated with molecular weight, log P, and PC1 value, and the coefficient of determination (R(2) ) of the linear regression equation was 0.63. This relationship might be useful as an aid to predict P-gp substrate status at an early stage of drug discovery. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

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

    Science.gov (United States)

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

    2013-03-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

  11. A new model of flavonoids affinity towards P-glycoprotein: genetic algorithm-support vector machine with features selected by a modified particle swarm optimization algorithm.

    Science.gov (United States)

    Cui, Ying; Chen, Qinggang; Li, Yaxiao; Tang, Ling

    2017-02-01

    Flavonoids exhibit a high affinity for the purified cytosolic NBD (C-terminal nucleotide-binding domain) of P-glycoprotein (P-gp). To explore the affinity of flavonoids for P-gp, quantitative structure-activity relationship (QSAR) models were developed using support vector machines (SVMs). A novel method coupling a modified particle swarm optimization algorithm with random mutation strategy and a genetic algorithm coupled with SVM was proposed to simultaneously optimize the kernel parameters of SVM and determine the subset of optimized features for the first time. Using DRAGON descriptors to represent compounds for QSAR, three subsets (training, prediction and external validation set) derived from the dataset were employed to investigate QSAR. With excluding of the outlier, the correlation coefficient (R 2 ) of the whole training set (training and prediction) was 0.924, and the R 2 of the external validation set was 0.941. The root-mean-square error (RMSE) of the whole training set was 0.0588; the RMSE of the cross-validation of the external validation set was 0.0443. The mean Q 2 value of leave-many-out cross-validation was 0.824. With more informations from results of randomization analysis and applicability domain, the proposed model is of good predictive ability, stability.

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

    Directory of Open Access Journals (Sweden)

    Daniel G. Gerardi

    2014-01-01

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

  13. Tacrolimus is a class II low-solubility high-permeability drug: the effect of P-glycoprotein efflux on regional permeability of tacrolimus in rats.

    Science.gov (United States)

    Tamura, Shigeki; Ohike, Atsuo; Ibuki, Rinta; Amidon, Gordon L; Yamashita, Shinji

    2002-03-01

    The objective of this study is to investigate the role of P-glycoprotein (P-gp), a membrane efflux pump associated with multidrug resistance (MDR) and a known substrate for tacrolimus, in determining the regional intestinal permeability of tacrolimus in rats. Thus, isolated segments of rat jejunum, ileum, or colon were perfused with tacrolimus solutions containing polyethoxylated hydrogenated castor oil 60 surfactant, and with or without verapamil, a P-gp substrate used to reverse the MDR phenotype. The results indicated that the intrinsic permeability of tacrolimus in the jejunum, calculated on the basis of the concentration of non-micellized free tacrolimus, was quite high ( approximately 1.4 x 10(-4) cm/s). The apparent permeability (P(app)) in the jejunum was unaffected by the presence of verapamil; however, the P(app) in the ileum and the colon increased significantly in the presence of verapamil and were similar to the values observed in the jejunum. The results suggest that systemic absorption of tacrolimus from the gastrointestinal tract could be significantly affected by P-gp efflux mechanisms. It is also possible that differences in P-gp function at various intestinal sites in a subject or at a given intestinal site in various subjects could lead to large intra- and interindividual variability in bioavailability of tacrolimus following oral administration. Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association .

  14. The methoxychlor metabolite, HPTE, inhibits rat luteal cell progesterone production.

    Science.gov (United States)

    Akgul, Yucel; Derk, Raymond C; Meighan, Terence; Rao, K Murali Krishna; Murono, Eisuke P

    2011-07-01

    The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum. Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to progesterone formation and P450scc catalytic activity of hCG- or 8 Br-cAMP-stimulated luteal cells. However, HPTE did not alter mRNA and protein levels of P450scc. Compounds acting as estrogen (17 β-estradiol, bisphenol-A or octylphenol), antiestrogen (ICI) or antiandrogen (monobutyl phthalate, flutamide or M-2) added alone to luteal cells did not mimic the action of HPTE on progesterone and P450scc activity. These results suggest that HPTE directly inhibits P450scc catalytic activity resulting in reduced progesterone formation, and this action was not mediated through estrogen or androgen receptors. Published by Elsevier Inc.

  15. IL-17 inhibits chondrogenic differentiation of human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Kondo

    Full Text Available OBJECTIVE: Mesenchymal stem cells (MSCs can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs. METHODS: Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression. RESULTS: Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA, which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis. CONCLUSIONS: IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation.

  16. Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

    Science.gov (United States)

    Cai, Bin; Li, Wenjun; Mao, XiaoOu; Winters, Ali; Ryou, Myoung-Gwi; Liu, Ran; Greenberg, David A; Wang, Ning; Jin, Kunlin; Yang, Shao-Hua

    2016-03-01

    Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

  17. ITE inhibits growth of human pulmonary artery endothelial cells.

    Science.gov (United States)

    Pang, Ling-Pin; Li, Yan; Zou, Qing-Yun; Zhou, Chi; Lei, Wei; Zheng, Jing; Huang, Shi-An

    2017-10-01

    Pulmonary arterial hypertension (PAH), a deadly disorder is associated with excessive growth of human pulmonary artery endothelial (HPAECs) and smooth muscle (HPASMCs) cells. Current therapies primarily aim at promoting vasodilation, which only ameliorates clinical symptoms without a cure. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous aryl hydrocarbon receptor (AhR) ligand, and mediates many cellular function including cell growth. However, the roles of ITE in human lung endothelial cells remain elusive. Herein, we tested a hypothesis that ITE inhibits growth of human pulmonary artery endothelial cells via AhR. Immunohistochemistry was performed to localize AhR expression in human lung tissues. The crystal violet method and MTT assay were used to determine ITE's effects on growth of HPAECs. The AhR activation in HPAECs was confirmed using Western blotting and RT-qPCR. The role of AhR in ITE-affected proliferation of HPAECs was assessed using siRNA knockdown method followed by the crystal violet method. Immunohistochemistry revealed that AhR was present in human lung tissues, primarily in endothelial and smooth muscle cells of pulmonary veins and arteries, as well as in bronchial and alveolar sac epithelia. We also found that ITE dose- and time-dependently inhibited proliferation of HPAECs with a maximum inhibition of 83% at 20 µM after 6 days of treatment. ITE rapidly decreased AhR protein levels, while it increased mRNA levels of cytochrome P450 (CYP), family 1, member A1 (CYP1A1) and B1 (CYP1B1), indicating activation of the AhR/CYP1A1 and AhR/CYP1B1 pathways in HPAECs. The AhR siRNA significantly suppressed AhR protein expression, whereas it did not significantly alter ITE-inhibited growth of HPAECs. ITE suppresses growth of HPAECs independent of AhR, suggesting that ITE may play an important role in preventing excessive growth of lung endothelial cells.

  18. Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

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    Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T. [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Aftab, Blake T. [Department of Medical Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M.; Wong, John [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Rudin, Charles M. [Department of Medical Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Tran, Phuoc T. [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Department of Medical Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Hales, Russell K., E-mail: rhales1@jhmi.edu [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States)

    2013-05-01

    Purpose: Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials: We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of Kras{sup G12D}-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results: In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions: Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer.

  19. Ginkgo Biloba Extract Kaempferol Inhibits Cell Proliferation and Induces Apoptosis in Pancreatic Cancer Cells

    Science.gov (United States)

    Zhang, Yuqing; Chen, Aaron Y.; Li, Min; Chen, Changyi; Yao, Qizhi

    2010-01-01

    Background Kaempferol is one of the most important constituents in ginkgo flavonoids. Recent studies indicate kaempferol may have anti-tumor activities. The objective in this study was to determine the effect and mechanisms of kaempferol on pancreatic cancer cell proliferation and apoptosis. Materials and Methods Pancreatic cancer cell lines MIA PaCa-2 and Panc-1 were treated with Kampferol, and the inhibitory effects of kaempferol on pancreatic cancer cell proliferation were examined by direct cell counting, 3H-thymidine incorporation and MTS assay. Lactate dehydrogenase (LDH) release from cells was determined as an index of cytotoxicity. Apoptosis was analyzed by TUNEL assay. Results Upon the treatment with 70 μM kaempferol for 4 days, MIA PaCa-2 cell proliferation was significantly inhibited by 79% and 45.7% as determined by direct cell counting and MTS assay, respectively, compared with control cells (Pkaempferol significantly inhibited Panc-1 cell proliferation. Kaempferol treatment also significantly reduced 3H-thymidine incorporation in both MIA PaCa-2 and Panc-1 cells. Combination treatment of low concentrations of kaempferol and 5-fluorouracil (5-FU) showed an additive effect on the inhibition of MIA PaCa-2 cell proliferation. Furthermore, kaempferol had a significantly less cytotoxicity than 5-FU in normal human pancreatic ductal epithelial cells (P=0.029). In both MIA PaCa-2 and Panc-1 cells, apoptotic cell population was increased when treated with kaempferol in a concentration-dependent manner. Conclusions Ginkgo biloba extract kaempferol effectively inhibits pancreatic cancer cell proliferation and induces cancer cell apoptosis, which may sensitize pancreatic tumor cells to chemotherapy. Kaempferol may have clinical applications as adjuvant therapy in the treatment of pancreatic cancer. PMID:18570926

  20. Depletion of Pokemon gene inhibits hepatocellular carcinoma cell growth through inhibition of H-ras.

    Science.gov (United States)

    Zhang, Quan-Le; Tian, De-An; Xu, Xiang-Jiang

    2011-01-01

    Pokemon is a transcription repressor which plays a critical role in cell transformation and malignancy. However, little is known about its effect on the development and progression of hepatocellular carcinoma (HCC). The aim of this study was to investigate the expression of Pokemon in human HCC tissues and the biological behavior of Pokemon in HCC cells in which it is overexpressed. We also explored the expression of potential downstream cofactors of Pokemon. Reverse transcription polymerase chain reaction and Western blot analysis were used to investigate the expression of Pokemon in tissues of 30 HCC patients. We then examined cell proliferation or apoptosis and β-catenin or H-ras expression in Pokemon-depleted HepG(2) cells using DNA vector-based RNA interference technology. Pokemon was markedly expressed in 22/30 (73.3%) HCC tissues, with expression levels higher than in adjacent normal liver tissues (p Pokemon inhibited proliferation of HepG(2) or induced apoptosis. Also, H-ras expression decreased to a large extent. Pokemon exerts its oncogenic activity in the development of HCC by promoting cancer cell growth and reducing apoptosis, and the effect may be mediated by H-ras. Copyright © 2011 S. Karger AG, Basel.

  1. Apoptosis and radiosensitization of Hodgkin cells by proteasome inhibition

    International Nuclear Information System (INIS)

    Pajonk, Frank; Pajonk, Katja; McBride, William H.

    2000-01-01

    Purpose: Malignant cells from Hodgkin's disease have been reported to be defective in regulation of NF-κB activity. Ionizing radiation is known to activate NF-κB, and it has been suggested that this pathway may protect cells from apoptosis following exposure to radiation and other therapeutic agents. Defective NF-κB regulation in Hodgkin cells could therefore dictate the response of this disease to therapy, as well as be responsible for maintaining the malignant phenotype. The purpose of this study was to explore whether NF-κB activity could be modulated in Hodgkin cells and whether it determines the response of these cells to treatment with ionizing radiation and/or dexamethasone. Methods and Materials: Activation of NF-κB in cells is accomplished in large part by degradation of its inhibitor IκB through the 26s proteasome. HD-My-Z Hodgkin cells were treated with the proteasome inhibitor MG-132 or transduced with a dominant negative super-repressor IκBα. Clonogenic survival, apoptosis, proteasome activity, and NF-κB binding activity were monitored in response to ionizing radiation and/or dexamethasone treatment. Results: HD-My-Z Hodgkin cells had modest NF-κB levels but, unlike other cell types, did not decrease their level of constitutively active NF-κB in response to proteasome inhibition with MG-132. In contrast, transduction with a non-phosphorable IκBα construct abolished expression. MG-132 did, however, induce apoptosis in HD-My-Z cells and sensitized them to ionizing radiation. Dexamethasone treatment had no effect on NF-κB activity or clonogenic survival of Hodgkin cells, but protected them from irradiation. Conclusion: We conclude that inhibition of 26s proteasome activity can induce apoptosis in HD-My-Z Hodgkin cells and radiosensitize them, in spite of the fact that their constitutively active NF-κB levels are unaltered. The proteasome may be a promising new therapeutic target for intervention in this disease. In contrast, the use of

  2. Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction.

    Directory of Open Access Journals (Sweden)

    Paul Lin

    Full Text Available Human mesenchymal stem cells (hMSCs can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1-8 µg/mL negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr. Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical.

  3. Improved Performance in Mammalian Cell Perfusion Cultures by Growth Inhibition.

    Science.gov (United States)

    Wolf, Moritz K F; Closet, Aurélie; Bzowska, Monika; Bielser, Jean-Marc; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo

    2018-05-21

    Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0 °C) with respect to control conditions (36.5 °C, no VA). Low temperature significantly reduces cellular growth, thus, resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improves and maintained stable leading to media savings per mass of product. VA shows initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor resulting in a recovery of the cellular growth. Cell cycle and Western blot analyses support the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. INNO-406, a novel BCR-ABL/Lyn dual tyrosine kinase inhibitor, suppresses the growth of Ph+ leukemia cells in the central nervous system, and cyclosporine A augments its in vivo activity.

    Science.gov (United States)

    Yokota, Asumi; Kimura, Shinya; Masuda, Satohiro; Ashihara, Eishi; Kuroda, Junya; Sato, Kiyoshi; Kamitsuji, Yuri; Kawata, Eri; Deguchi, Yasuyuki; Urasaki, Yoshimasa; Terui, Yasuhito; Ruthardt, Martin; Ueda, Takanori; Hatake, Kiyohiko; Inui, Ken-ichi; Maekawa, Taira

    2007-01-01

    Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome-positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate-resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

  5. Migrastatin analogues inhibit canine mammary cancer cell migration and invasion.

    Directory of Open Access Journals (Sweden)

    Kinga Majchrzak

    Full Text Available BACKGROUND: Cancer spread to other organs is the main cause of death of oncological patients. Migration of cancer cells from a primary tumour is the crucial step in the complex process of metastasis, therefore blocking this process is currently the main treatment strategy. Metastasis inhibitors derived from natural products, such as, migrastatin, are very promising anticancer agents. Thus, the aim of our study was to investigate the effect of six migrastatin analogues (MGSTA-1 to 6 on migration and invasion of canine mammary adenocarcinoma cell lines isolated from primary tumours and their metastases to the lungs. Canine mammary tumours constitute a valuable tool for studying multiple aspect of human cancer. RESULTS: OUR RESULTS SHOWED THAT TWO OF SIX FULLY SYNTHETIC ANALOGUES OF MIGRASTATIN: MGSTA-5 and MGSTA-6 were potent inhibitors of canine mammary cancer cells migration and invasion. These data were obtained using the wound healing test, as well as trans-well migration and invasion assays. Furthermore, the treatment of cancer cells with the most effective compound (MGSTA-6 disturbed binding between filamentous F-actin and fascin1. Confocal microscopy analyses revealed that treatment with MGSTA-6 increased the presence of unbound fascin1 and reduced co-localization of F-actin and fascin1 in canine cancer cells. Most likely, actin filaments were not cross-linked by fascin1 and did not generate the typical filopodial architecture of actin filaments in response to the activity of MGSTA-6. Thus, administration of MGSTA-6 results in decreased formation of filopodia protrusions and stress fibres in canine mammary cancer cells, causing inhibition of cancer migration and invasion. CONCLUSION: Two synthetic migrastatin analogues (MGSTA-5 and MGSTA-6 were shown to be promising compounds for inhibition of cancer metastasis. They may have beneficial therapeutic effects in cancer therapy in dogs, especially in combination with other anticancer drugs

  6. Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells

    International Nuclear Information System (INIS)

    Savion, N.; Disatnik, M.H.; Nevo, Z.

    1987-01-01

    Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells attach, invade, and penetrate confluent vascular endothelial cell monolayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [ 35 S]O 4 - -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The degradation of [ 35 S]O 4 - -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10μg/ml), arteparon (10μg/ml), and heparin at a concentration of 3 μg/ml. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage haparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis

  7. Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

    Science.gov (United States)

    Meiler, Eugenia; Nieto-Pelegrín, Elvira; Martinez-Quiles, Narcisa

    2012-01-01

    Background Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading. PMID:22479425

  8. Hydroxyurea inhibits parvovirus B19 replication in erythroid progenitor cells.

    Science.gov (United States)

    Bonvicini, Francesca; Bua, Gloria; Conti, Ilaria; Manaresi, Elisabetta; Gallinella, Giorgio

    2017-07-15

    Parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells (EPCs) of the human bone marrow, leading to transient arrest of erythropoiesis and severe complications mainly in subjects with underlying hematological disorders or with immune system deficits. Currently, there are no specific antiviral drugs for B19V treatment, but identification of compounds inhibiting B19V replication can be pursued by a drug repositioning strategy. In this frame, the present study investigates the activity of hydroxyurea (HU), the only disease-modifying therapy approved for sickle cell disease (SCD), towards B19V replication in the two relevant cellular systems, the UT7/EpoS1 cell line and EPCs. Results demonstrate that HU inhibits B19V replication with EC 50 values of 96.2µM and 147.1µM in UT7/EpoS1 and EPCs, respectively, providing experimental evidence of the antiviral activity of HU towards B19V replication, and confirming the efficacy of a drug discovery process by drug repositioning strategy. The antiviral activity occurs in vitro at concentrations lower than those affecting cellular DNA replication and viability, and at levels measured in plasma samples of SCD patients undergoing HU therapy. HU might determine a dual beneficial effect on SCD patients, not only for the treatment of the disease but also towards a virus responsible for severe complications. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Praziquantel synergistically enhances paclitaxel efficacy to inhibit cancer cell growth.

    Directory of Open Access Journals (Sweden)

    Zhen Hua Wu

    Full Text Available The major challenges we are facing in cancer therapy with paclitaxel (PTX are the drug resistance and severe side effects. Massive efforts have been made to overcome these clinical challenges by combining PTX with other drugs. In this study, we reported the first preclinical data that praziquantel (PZQ, an anti-parasite agent, could greatly enhance the anticancer efficacy of PTX in various cancer cell lines, including PTX-resistant cell lines. Based on the combination index value, we demonstrated that PZQ synergistically enhanced PTX-induced cell growth inhibition. The co-treatment of PZQ and PTX also induced significant mitotic arrest and activated the apoptotic cascade. Moreover, PZQ combined with PTX resulted in a more pronounced inhibition of tumor growth compared with either drug alone in a mouse xenograft model. We tried to investigate the possible mechanisms of this synergistic efficacy induced by PZQ and PTX, and we found that the co-treatment of the two drugs could markedly decrease expression of X-linked inhibitor of apoptosis protein (XIAP, an anti-apoptotic protein. Our data further demonstrated that down-regulation of XIAP was required for the synergistic interaction between PZQ and PTX. Together, this study suggested that the combination of PZQ and PTX may represent a novel and effective anticancer strategy for optimizing PTX therapy.

  10. Inhibition of canonical WNT signaling attenuates human leiomyoma cell growth

    Science.gov (United States)

    Ono, Masanori; Yin, Ping; Navarro, Antonia; Moravek, Molly B.; Coon, John S.; Druschitz, Stacy A.; Gottardi, Cara J.; Bulun, Serdar E.

    2014-01-01

    Objective Dysregulation of WNT signaling plays a central role in tumor cell growth and progression. Our goal was to assess the effect of three WNT/β-catenin pathway inhibitors, Inhibitor of β-Catenin And TCF4 (ICAT), niclosamide, and XAV939 on the proliferation of primary cultures of human uterine leiomyoma cells. Design Prospective study of human leiomyoma cells obtained from myomectomy or hysterectomy. Setting University research laboratory. Patient(s) Women (n=38) aged 27–53 years undergoing surgery. Intervention(s) Adenoviral ICAT overexpression or treatment with varying concentrations of niclosamide or XAV939. Main Outcome Measure(s) Cell proliferation, cell death, WNT/β-catenin target gene expression or reporter gene regulation, β-catenin levels and cellular localization. Result(s) ICAT, niclosamide, or XAV939 inhibit WNT/β-catenin pathway activation and exert anti-proliferative effects in primary cultures of human leiomyoma cells. Conclusion(s) Three WNT/β-catenin pathway inhibitors specifically block human leiomyoma growth and proliferation, suggesting that the canonical WNT pathway may be a potential therapeutic target for the treatment of uterine leiomyoma. Our findings provide rationale for further preclinical and clinical evaluation of ICAT, niclosamide, and XAV939 as candidate anti-tumor agents for uterine leiomyoma. PMID:24534281

  11. Dextran Sulfate Sodium Inhibits Alanine Synthesis in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Carolyn M. Slupsky

    2011-04-01

    Full Text Available To understand and characterize the pathogenic mechanisms of inflammatory bowel disease, dextran sulfate sodium (DSS has been used to induce acute and chronic colitis in animal models by causing intestinal epithelium damage. The mechanism of action of DSS in producing this outcome is not well understood. In an effort to understand how DSS might impact epithelial cell metabolism, we studied the intestinal epithelial cell line Caco-2 incubated with 1% DSS over 56 hours using 1H NMR spectroscopy. We observed no difference in cell viability as compared to control cultures, and an approximately 1.5-fold increase in IL-6 production upon incubation with 1% DSS. The effect on Caco-2 cell metabolism as measured through changes in the concentration of metabolites in the cell supernatant included a three-fold decrease in the concentration of alanine. Given that the concentrations of other amino acids in the cell culture supernatant were not different between treated and control cultures over 56 hours suggest that DSS inhibits alanine synthesis, specifically alanine aminotransferase, without affecting other key metabolic pathways. The importance of alanine aminotransferase in inflammatory bowel disease is discussed.

  12. Resetting cancer stem cell regulatory nodes upon MYC inhibition.

    Science.gov (United States)

    Galardi, Silvia; Savino, Mauro; Scagnoli, Fiorella; Pellegatta, Serena; Pisati, Federica; Zambelli, Federico; Illi, Barbara; Annibali, Daniela; Beji, Sara; Orecchini, Elisa; Alberelli, Maria Adele; Apicella, Clara; Fontanella, Rosaria Anna; Michienzi, Alessandro; Finocchiaro, Gaetano; Farace, Maria Giulia; Pavesi, Giulio; Ciafrè, Silvia Anna; Nasi, Sergio

    2016-12-01

    MYC deregulation is common in human cancer and has a role in sustaining the aggressive cancer stem cell populations. MYC mediates a broad transcriptional response controlling normal biological programmes, but its activity is not clearly understood. We address MYC function in cancer stem cells through the inducible expression of Omomyc-a MYC-derived polypeptide interfering with MYC activity-taking as model the most lethal brain tumour, glioblastoma. Omomyc bridles the key cancer stemlike cell features and affects the tumour microenvironment, inhibiting angiogenesis. This occurs because Omomyc interferes with proper MYC localization and itself associates with the genome, with a preference for sites occupied by MYC This is accompanied by selective repression of master transcription factors for glioblastoma stemlike cell identity such as OLIG2, POU3F2, SOX2, upregulation of effectors of tumour suppression and differentiation such as ID4, MIAT, PTEN, and modulation of the expression of microRNAs that target molecules implicated in glioblastoma growth and invasion such as EGFR and ZEB1. Data support a novel view of MYC as a network stabilizer that strengthens the regulatory nodes of gene expression networks controlling cell phenotype and highlight Omomyc as model molecule for targeting cancer stem cells. © 2016 The Authors.

  13. Antibiotic drug tigecycline inhibited cell proliferation and induced autophagy in gastric cancer cells

    International Nuclear Information System (INIS)

    Tang, Chunling; Yang, Liqun; Jiang, Xiaolan; Xu, Chuan; Wang, Mei; Wang, Qinrui; Zhou, Zhansong; Xiang, Zhonghuai; Cui, Hongjuan

    2014-01-01

    Highlights: • Tigecycline inhibited cell growth and proliferation in human gastric cancer cells. • Tigecycline induced autophagy not apoptosis in human gastric cancer cells. • AMPK/mTOR/p70S6K pathway was activated after tigecycline treatment. • Tigecycline inhibited tumor growth in xenograft model of human gastric cancer cells. - Abstract: Tigecycline acts as a glycylcycline class bacteriostatic agent, and actively resists a series of bacteria, specifically drug fast bacteria. However, accumulating evidence showed that tetracycline and their derivatives such as doxycycline and minocycline have anti-cancer properties, which are out of their broader antimicrobial activity. We found that tigecycline dramatically inhibited gastric cancer cell proliferation and provided an evidence that tigecycline induced autophagy but not apoptosis in human gastric cancer cells. Further experiments demonstrated that AMPK pathway was activated accompanied with the suppression of its downstream targets including mTOR and p70S6K, and ultimately induced cell autophagy and inhibited cell growth. So our data suggested that tigecycline might act as a candidate agent for pre-clinical evaluation in treatment of patients suffering from gastric cancer

  14. Antibiotic drug tigecycline inhibited cell proliferation and induced autophagy in gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Chunling; Yang, Liqun; Jiang, Xiaolan [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China); Xu, Chuan [Division of Scientific Research and Training, General Hospital of PLA Chengdu Military Area Command, Chengdu, Sichuan 610083 (China); Wang, Mei; Wang, Qinrui [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China); Zhou, Zhansong, E-mail: zhouzhans@sina.com [Institute of Urinary Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Xiang, Zhonghuai [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China); Cui, Hongjuan, E-mail: hcui@swu.edu.cn [State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716 (China)

    2014-03-28

    Highlights: • Tigecycline inhibited cell growth and proliferation in human gastric cancer cells. • Tigecycline induced autophagy not apoptosis in human gastric cancer cells. • AMPK/mTOR/p70S6K pathway was activated after tigecycline treatment. • Tigecycline inhibited tumor growth in xenograft model of human gastric cancer cells. - Abstract: Tigecycline acts as a glycylcycline class bacteriostatic agent, and actively resists a series of bacteria, specifically drug fast bacteria. However, accumulating evidence showed that tetracycline and their derivatives such as doxycycline and minocycline have anti-cancer properties, which are out of their broader antimicrobial activity. We found that tigecycline dramatically inhibited gastric cancer cell proliferation and provided an evidence that tigecycline induced autophagy but not apoptosis in human gastric cancer cells. Further experiments demonstrated that AMPK pathway was activated accompanied with the suppression of its downstream targets including mTOR and p70S6K, and ultimately induced cell autophagy and inhibited cell growth. So our data suggested that tigecycline might act as a candidate agent for pre-clinical evaluation in treatment of patients suffering from gastric cancer.

  15. Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin.

    Science.gov (United States)

    Jenssen, Håvard; Sandvik, Kjersti; Andersen, Jeanette H; Hancock, Robert E W; Gutteberg, Tore J

    2008-09-01

    The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.

  16. [Inhibition effects of black rice pericarp extracts on cell proliferation of PC-3 cells].

    Science.gov (United States)

    Jiang, Weiwei; Yu, Xudong; Ren, Guofeng

    2013-05-01

    To observe the inhibitive effects of black rice pericarp extracts on cell proliferation of human prostate cancer cell PC-3 and to explore its effecting mechanism. The black rice pericarp extract was used to treat the PC-3 cells. The inhibitory effect of black rice pericarp extract on cells proliferation of PC-3 was tested by MTT method. Cell apoptosis rates and cell cycle were measured by flow cytometric assay (FCM). Western blot was used to study the protein expression levels of p38, p-p38, JNK, p-JNK. A dose-dependent and time-dependent proliferation inhibition of black rice pericarp extract was demonstrated in PC-3. The most prominent experiment condition was inhibitory concentration with 300microg/ml and treated for 72 h. The experiment result of flow cytometry analysis demonstrates that the apoptosis rate of PC-3 cells increased along with the increasing of black rice pericarp extract concentration, and a G1-S cell cycle arrest was induced in a dose-dependent manner. After PC-3 cell was treated with black rice pericarp extract for 72 h, the expressions of p-p38, p-JNK protein increased. Black rice pericarp extract could inhibit proliferation, change the cell cycle distributions and induce apoptosis in human prostatic cancer cell PC-3. Its inhibitory effect may be through promoting activation of the JNK, p38 signaling pathway. These results suggest that black rice pericarp extract maybe has an inhibitory effect on prostatic cancer.

  17. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Jing [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Liu, Gexiu [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Yan, Guoyao [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); He, Dongmei [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Zhou, Ying [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Chen, Shengting, E-mail: shengtingchen@sina.cn [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China)

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.

  18. Bifenthrin inhibits neurite outgrowth in differentiating PC12 cells.

    Science.gov (United States)

    Tran, Van; Hoffman, Natalie; Mofunanaya, Adaobi; Pryor, Stephen C; Ojugbele, Olutosin; McLaughlin, Ashlea; Gibson, Lydia; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

    2006-02-01

    Bifenthrin is a third generation member of the synthetic pyrethroid family of insecticides. As a new pesticide within a relatively new class of pesticides, bifenthrin is considered relatively safe. Here, we used the PC12 neuronal cell line to examine the effect of bifenthrin on the formation of neurites and the potential developmental neurotoxicity of this pesticide. PC12 cells were exposed to varying concentrations of technical grade bifenthrin or Ortho Home Defense. Cell viability was determined using the AlmarBlue Toxicity Assay. Nontoxic concentrations of these chemicals were concomitantly with nerve growth factor and neurite outgrowth was assessed. Ortho Home Defense preparation reduced PC12 cell viability by approximately 50% and 70% at dilutions that correlate to bifenthrin concentrations of 10(-5) M and 10(-4) M, respectively. In contrast, technical grade bifenthrin, was not toxic to PC12 cells at 10(-3) M, which was the highest concentration tested that was soluble. At "nontoxic" concentrations of 10(-7) M and 10(-6) M, the Ortho Home Defense inhibited nerve growth factor-mediated neurite outgrowth by 30% and 55% respectively. Furthermore the nontoxic concentrations of technical grade bifenthrin of 10(-6) M and 10(-3) M inhibited neurite outgrowth by approximately 35% and 75% respectively. These data suggest that the toxicity of the Ortho Home Defense preparation was due to the "inert" additives in the preparation and not the bifenthrin itself. Further, these data suggest that, even in the absence of overt toxicity, bifenthrin may have deleterious effects to developing nervous system.

  19. Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

    International Nuclear Information System (INIS)

    Yamagishi, Naoko; Teshima-Kondo, Shigetada; Masuda, Kiyoshi; Nishida, Kensei; Kuwano, Yuki; Dang, Duyen T; Dang, Long H; Nikawa, Takeshi; Rokutan, Kazuhito

    2013-01-01

    Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF

  20. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    International Nuclear Information System (INIS)

    Wang, Hongtao; Gao, Peng; Zheng, Jie

    2014-01-01

    Highlights: • As 2 O 3 inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As 2 O 3 than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As 2 O 3 than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As 2 O 3 is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As 2 O 3 ) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As 2 O 3 induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As 2 O 3 on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As 2 O 3 than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As 2 O 3 than HPV 16-positive CaSki and SiHa cells. After As 2 O 3 treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As 2 O 3 is a potential anticancer drug for cervical cancer

  1. Human retinal pigment epithelial cells inhibit proliferation and IL2R expression of activated T cells

    DEFF Research Database (Denmark)

    Kaestel, Charlotte G; Jørgensen, Annette; Nielsen, Mette

    2002-01-01

    -Thymidine incorporation assay, respectively. T cells and RPE cells were cultured directly together or in a transwell system for determination of the effect of cell contact. The importance of cell surface molecules was examined by application of a panel of blocking antibodies (CD2, CD18, CD40, CD40L, CD54, CD58......) in addition to use of TCR negative T cell lines. The expression of IL2R-alpha -beta and -gamma chains of activated T cells was analysed by flow cytometry after incubation of T cells alone or with RPE cells. Human RPE cells were found to inhibit the proliferation of activated T cells by a cell contact......-beta and -gamma chain expression within 24 hr after removal from the coculture. It is concluded that the cultured human adult and foetal RPE cells inhibit the proliferation of activated T cells by a process that does not involve apoptosis. It depends on cell contact but the involved surface molecules were...

  2. Kaempferol inhibits cell proliferation and glycolysis in esophagus squamous cell carcinoma via targeting EGFR signaling pathway.

    Science.gov (United States)

    Yao, Shihua; Wang, Xiaowei; Li, Chunguang; Zhao, Tiejun; Jin, Hai; Fang, Wentao

    2016-08-01

    Antitumor activity of kaempferol has been studied in various tumor types, but its potency in esophagus squamous cell carcinoma is rarely known. Here, we reported the activity of kaempferol against esophagus squamous cell carcinoma as well as its antitumor mechanisms. Results of cell proliferation and colony formation assay showed that kaempferol substantially inhibited tumor cell proliferation and clone formation in vitro. Flow cytometric analysis demonstrated that tumor cells were induced G0/G1 phase arrest after kaempferol treatment, and the expression of protein involved in cell cycle regulation was dramatically changed. Except the potency on cell proliferation, we also discovered that kaempferol had a significant inhibitory effect against tumor glycolysis. With the downregulation of hexokinase-2, glucose uptake and lactate production in tumor cells were dramatically declined. Mechanism studies revealed kaempferol had a direct effect on epidermal growth factor receptor (EGFR) activity, and along with the inhibition of EGFR, its downstream signaling pathways were also markedly suppressed. Further investigations found that exogenous overexpression of EGFR in tumor cells substantially attenuated glycolysis suppression induced by kaempferol, which implied that EGFR also played an important role in kaempferol-mediated glycolysis inhibition. Finally, the antitumor activity of kaempferol was validated in xenograft model and kaempferol prominently restrained tumor growth in vivo. Meanwhile, dramatic decrease of EGFR activity and hexokinase-2 expression were observed in kaempferol-treated tumor tissue, which confirmed these findings in vitro. Briefly, these studies suggested that kaempferol, or its analogues, may serve as effective candidates for esophagus squamous cell carcinoma management.

  3. Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

    International Nuclear Information System (INIS)

    Wu Xiaofeng; Fan Jia; Wang Xiaoying; Zhou Jian; Qiu Shuangjian; Yu Yao; Liu Yinkun; Tang Zhaoyou

    2007-01-01

    CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment

  4. Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction.

    Science.gov (United States)

    Ruan, Banzhan; Zhang, Bo; Chen, Ang; Yuan, Long; Liang, Jianqing; Wang, Manna; Zhang, Zhengrong; Fan, Jie; Yu, Xiaochen; Zhang, Xin; Niu, Zubiao; Zheng, You; Gu, Songzhi; Liu, Xiaoqing; Du, Hongli; Wang, Jufang; Hu, Xianwen; Gao, Lihua; Chen, Zhaolie; Huang, Hongyan; Wang, Xiaoning; Sun, Qiang

    2018-01-01

    Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Cyclosporine, a P-glycoprotein modulator, increases [18F]MPPF uptake in rat brain and peripheral tissues: microPET and ex vivo studies

    International Nuclear Information System (INIS)

    Lacan, Goran; Way, Baldwin M.; Plenevaux, Alain; Defraiteur, Caroline; Lemaire, Christian; Aerts, Joel; Luxen, Andre; Rubins, Daniel J.; Cherry, Simon R.; Melega, William P.

    2008-01-01

    Pretreatment with cyclosporine, a P-glycoprotein (P-gp) modulator increases brain uptake of 4-(2'-methoxyphenyl)-1-[2'-(N-2''-pyridinyl)-p-[ 18 F] fluorobenzamido] ethylpiper azine ([ 18 F]MPPF) for binding to hydroxytryptamine 1A (5-HT 1A ) receptors. Those increases were quantified in rat brain with in vivo microPET and ex vivo tissue studies. Each Sprague-Dawley rat (n=4) received a baseline [ 18 F]MPPF microPET scan followed by second scan 2-3 weeks later that included cyclosporine pretreatment (50 mg/kg, i.p.). Maximum a posteriori reconstructed images and volumetric ROIs were used to generate dynamic radioactivity concentration measurements for hippocampus, striatum, and cerebellum, with simplified reference tissue method (SRTM) analysis. Western blots were used to semiquantify P-gp regional distribution in brain. MicroPET studies showed that hippocampus uptake of [ 18 F]MPPF was increased after cyclosporine; ex vivo studies showed similar increases in hippocampus and frontal cortex at 30 min, and for heart and kidney at 2.5 and 5 min, without concomitant increases in [ 18 F]MPPF plasma concentration. P-gp content in cerebellum was twofold higher than in hippocampus or frontal cortex. These studies confirm and extend prior ex vivo results (J. Passchier, et al., Eur J Pharmacol, 2000) that showed [ 18 F]MPPF as a substrate for P-gp. Our microPET results showed that P-gp modulation of [ 18 F]MPPF binding to 5-HT 1A receptors can be imaged in rat hippocampus. The heterogeneous brain distribution of P-gp appeared to invalidate the use of cerebellum as a nonspecific reference region for SRTM modeling. Regional quantitation of P-gp may be necessary for accurate PET assessment of 5-HT 1A receptor density when based on tracer uptake sensitive to P-gp modulation. (orig.)

  6. Evaluation of the potential effect of Allium sativum, Momordica charantia, Eugenia jambolana, Ocimum sanctum & Psidium guajava on intestinal P-glycoprotein in rats

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    Devendra Kumar

    2017-03-01

    Full Text Available Aims/Background: This study was evaluated synergistic effect of Polyherbal formulation (PHF of Allium sativum L., Eugenia jambolana Lam., Momordica charantia L., Ocimum sanctum Linn and Psidium guajava L on p-glycoprotein of intestine. These five herbs were traditionally used for diabetes. These herbs are commonly present in ayurvedic product as antidiabetics in India. Methodology: PHF was prepared by five indigenous herbs. Different doses (50, 100 and 200 mg/kg/day of was orally administered to Sprague-Dawley rats of different groups for multiple weeks except control groups. Alteration in Pgp expression was evaluated by RT-PCR and western blotting while modulation in activity of Pgp was evaluated using rhodamine 123 as transport substrate by in-situ absorption and everted gut sac method. Results: In PHF pretreated group received 50, 100 and 200 mg/kg/day for seven days, mRNA level decreased by 1.75, 2.45 and 2.37 fold respectively as compared to control. Similarly when PHF at dose of 100 mg/kg/day was given consequently for four weeks maximum decrease in Pgp expression level was observed only after one week and further increase in the treatment duration did not produce significant decrease compared to first week treatment. Pgp mediated transport of rhodamine 123 was significantly decreased with everted gut sac prepared from PHF pretreated rats (one week compared to those prepared from vehicle treated rats. Conclusions: In conclusion, we report that PHF pretreatment down regulated the expression of intestinal Pgp and this down regulated intestinal Pgp would result in decreased functional activity. Additionally this down regulated Pgp expression might affect the bioavailability of antidiabetic Pgp substrate drugs. [J Complement Med Res 2017; 6(1.000: 68-74

  7. E. coli infection modulates the pharmacokinetics of oral enrofloxacin by targeting P-glycoprotein in small intestine and CYP450 3A in liver and kidney of broilers.

    Science.gov (United States)

    Guo, Mengjie; Sun, Yong; Zhang, Yu; Bughio, Shamsuddin; Dai, Xiaohua; Ren, Weilong; Wang, Liping

    2014-01-01

    P-glycoprotein (P-gp) expression determines the absorption, distribution, metabolism and excretion of many drugs in the body. Also, up-regulation of P-gp acts as a defense mechanism against acute inflammation. This study examined expression levels of abcb1 mRNA and localization of P-gp protein in the liver, kidney, duodenum, jejunum and ileum in healthy and E. coli infected broilers by real time RT-PCR and immunohistochemistry. Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The results indicated that E. coli infection up-regulated expression of abcb1 mRNA levels significantly in the kidney, jejunum and ileum (P0.05). However, the expression level of CYP 3A37 mRNA were observed significantly decreased only in liver and kidney of E. coli infected broilers (Penrofloxacin, significantly decreased Cmax (0.34 vs 0.98 µg mL(-1), P = 0.000) and AUC0-12h (4.37 vs 8.88 µg mL(-1) h, P = 0.042) of enrofloxacin, but increased Tmax (8.32 vs 3.28 h, P = 0.040), T1/2a(2.66 vs 1.64 h(-1), P = 0.050) and V/F (26.7 vs 5.2 L, P = 0.040). Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in both healthy and infected broilers. The results suggest that the E. coli infection induces intestine P-gp expression, altering the absorption of orally administered enrofloxacin in broilers.

  8. The role of the polymorphic efflux transporter P-glycoprotein on the brain accumulation of d-methylphenidate and d-amphetamine.

    Science.gov (United States)

    Zhu, Hao-Jie; Wang, Jun-Sheng; DeVane, C Lindsay; Williard, Robin L; Donovan, Jennifer L; Middaugh, Lawrence D; Gibson, Brian B; Patrick, Kennerly S; Markowitz, John S

    2006-07-01

    The psychostimulant medications methylphenidate (MPH) and amphetamine (AMP), available in various ratios or enantiopure formulations of their respective active dextrorotary isomers, constitute the majority of agents used in the treatment of attention-deficit/hyperactivity disorder (ADHD). Substantial interindividual variability occurs in their pharmacokinetics and tolerability. Little is known regarding the potential role of drug transporters such as P-glycoprotein (P-gp) in psychostimulant pharmacokinetics and response. Therefore, experiments were carried out in P-gp knockout (KO) mice versus wild-type (WT) mice after intraperitoneal dosing (2.5 mg/kg) of d-MPH or (3.0 mg/kg) of d-AMP. After the administration of each psychostimulant, locomotor activity was assessed at 30-min intervals for 2 h. Total brain-to-plasma drug concentration ratios were determined at 10-, 30-, and 80-min postdosing time-points. The results showed no statistically supported genotypic difference in d-AMP-induced locomotor activity stimulation or in brain-to-plasma ratio of d-AMP. As for d-MPH, the P-gp KO mice had 33% higher brain concentrations (p brain-to-plasma ratios (p brain concentrations, d-MPH-induced locomotor activity increase was attenuated for P-gp compared with that for WT mice. These data indicate that P-gp has no apparent effect on the pharmacokinetics and pharmacodynamics of d-AMP. In addition, d-MPH is a relatively weak P-gp substrate, and its entry into the brain may be limited by P-gp. Furthermore, the mechanism by which d-MPH-induced locomotor activity was attenuated in P-gp KO mice remains to be elucidated.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Lucy Sanchez-Covarrubias

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

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

    Science.gov (United States)

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

    2011-05-01

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

  13. Relation between expression p-glycoprotein and the findings of the centellography with Tc-99m MIBI in patients with advanced mammary cancer

    International Nuclear Information System (INIS)

    Delgado, L; Alonso, O; Gualco, G; Vargas, C; Ortega, V; Alonso, I; Suarez, L; Nunez, M; Roca, R; Sabini, G; Muse, IM

    2004-01-01

    We have previously shown that pre-treatment tumor uptake of 99m Tc-MIBI, a transport substrate of P-glycoprotein (Pgp), is correlated to clinical response to doxorubicin-based chemotherapy in advanced breast cancer (ABC) patients. The aim of this study was to investigate the possible association between Pgp expression, MIBI uptake and clinical response to chemotherapy in breast cancer lesions. Twenty-seven lesions from 26 patients with ABC were included in the study. Pre-chemotherapy Pgp expression was investigated by immunocytochemistry. MIBI scintigraphy was performed 2-8 days prior chemotherapy. Images were acquired 10 minutes (early phase) and 60 minutes (delayed phase) post injection of 740-1110 MBq of 99m Tc-MIBI. Tumor-to-normal background tissue uptake ratios were calculated in the early (T/Be) and delayed (T/Bd) phase of the study. Both ratios were significantly higher (p< 0.05) in Pgp negative (n=21) than in Pgp positive lesions (n=6). Furthermore, both ratios were higher in responder compared to non-responder lesions (T/Be 2.2 vs 1.4; T/Bd 1.8 vs 1.4; p< 0.05). All lesions with a T/Be ratio higher than 1.5 were classified as responders. No significant association was found between Pgp expression and response to chemotherapy. We concluded that MIBI scintigraphy may predict MDR1 phenotype and response to doxorubicin-based chemotherapy in ABC. Pgp expression would not be useful for predicting chemotherapy response (Au)

  14. Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication

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    Leonie Schnell

    2016-07-01

    Full Text Available Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenylsemicarbazone (EGA has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT. Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria.

  15. Ketamine inhibits 45Ca influx and catecholamine secretion by inhibiting 22Na influx in cultured bovine adrenal medullary cells

    International Nuclear Information System (INIS)

    Takara, Hiroshi; Wada, Akihiko; Arita, Masahide; Izumi, Futoshi; Sumikawa, Koji

    1986-01-01

    The effects of ketamine, an intravenous anesthetic, on 22 Na influx, 45 Ca influx and catecholamine secretion were investigated in cultured bovine adrenal medullary cells. Ketamine inhibited carbachol-induced 45 Ca influx and catecholamine secretion in a concentration-dependent manner with a similar potency. Ketamine also reduced veratridine-induced 45 Ca influx and catecholamine secretion. The influx of 22 Na caused by carbachol or by veratridine was suppressed by ketamine with a concentration-inhibition curve similar to that of 45 Ca influx and catecholamine secretion. Inhibition by ketamine of the carbachol-induced influx of 22 Na, 45 Ca and secretion of catecholamines was not reversed by the increased concentrations of carbachol. These observations indicate that ketamine, at clinical concentrations, can inhibit nicotinic receptor-associated ionic channels and that the inhibition of Na influx via the receptor-associated ionic channels is responsible for the inhibition of carbachol-induced Ca influx and catecholamine secretion. (Auth.)

  16. Interferon lambda inhibits dengue virus replication in epithelial cells.

    Science.gov (United States)

    Palma-Ocampo, Helen K; Flores-Alonso, Juan C; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Flores-Mendoza, Lilian; Herrera-Camacho, Irma; Rosas-Murrieta, Nora H; Santos-López, Gerardo

    2015-09-28

    In viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection. Dengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR. We found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG's peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression. Evidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

  17. COBRA1 inhibits AP-1 transcriptional activity in transfected cells

    International Nuclear Information System (INIS)

    Zhong Hongjun; Zhu Jianhua; Zhang Hao; Ding Lihua; Sun Yan; Huang Cuifen; Ye Qinong

    2004-01-01

    Mutations in the breast cancer susceptibility gene (BRCA1) account for a significant proportion of hereditary breast and ovarian cancers. Cofactor of BRCA1 (COBRA1) was isolated as a BRCA1-interacting protein and exhibited a similar chromatin reorganizing activity to that of BRCA1. However, the biological role of COBRA1 remains largely unexplored. Here, we report that ectopic expression of COBRA1 inhibited activator protein 1 (AP-1) transcriptional activity in transfected cells in a dose-dependent manner, whereas reduction of endogenous COBRA1 with a small interfering RNA significantly enhanced AP-1-mediated transcriptional activation. COBRA1 physically interacted with the AP-1 family members, c-Jun and c-Fos, and the middle region of COBRA1 bound to c-Fos. Lack of c-Fos binding site in the COBRA1 completely abolished the COBRA1 inhibition of AP-1 trans-activation. These findings suggest that COBRA1 may directly modulate AP-1 pathway and, therefore, may play important roles in cell proliferation, differentiation, apoptosis, and oncogenesis

  18. Inhibition of Geranylgeranyl Transferase-I Decreases Cell Viability of HTLV-1-Transformed Cells

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    Cynthia A. Pise-Masison

    2011-10-01

    Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is the etiological agent of adult T-cell leukemia (ATL, an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G2/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.

  19. Multiple kinase pathways involved in the different de novo sensitivity of pancreatic cancer cell lines to 17-AAG.

    Science.gov (United States)

    Liu, Heping; Zhang, Ti; Chen, Rong; McConkey, David J; Ward, John F; Curley, Steven A

    2012-07-01

    17-Allylamino-17-demethoxygeldanamycin (17-AAG) specifically targets heat shock protein (HSP)90 and inhibits its chaperoning functions for multiple kinases involved in cancer cell growth and survival. To select responsive patients, the molecular mechanisms underlying the sensitivity of cancer cells to 17-AAG must be elucidated. We used cytotoxicity assays and Western blotting to explore the effects of 17-AAG and sorafenib on cell survival and expression of multiple kinases in the pancreatic cancer cell lines AsPC-1 and Panc-1. Gene cloning and transfection, siRNA silencing, and immunohistochemistry were used to evaluate the effects of mutant p53 protein on 17-AAG sensitivity. AsPC-1 and Panc-1 responded differently to 17-AAG, with half maximal inhibitory concentration (IC(50)) values of 0.12 and 3.18 μM, respectively. Comparable expression of HSP90, HSP70, and HSP27 was induced by 17-AAG in AsPC-1 and Panc-1 cells. P-glycoprotein and mutant p53 did not affect 17-AAG sensitivity in these cell lines. Multiple kinases are more sensitive to HSP90 inhibition in AsPC-1 than in Panc-1 cells. After 17-AAG treatment, p-Bad (S112) decreased in AsPC-1 cells and increased in Panc-1 cells. Sorafenib markedly increased p-Akt, p-ERK1/2, p-GSK-3β, and p-S6 in both cell lines. Accordingly, 17-AAG and sorafenib acted antagonistically in AsPC-1 and Panc-1 cells, except at high concentrations in AsPC-1 cells. Differential inhibition of multiple kinases is responsible for the different de novo sensitivity of AsPC-1 and Panc-1 cells to HSP90 inhibition. P-glycoprotein and mutant p53 protein did not play a role in the sensitivity of pancreatic cancer cells to 17-AAG. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-05-01

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

  1. Dextromethorphan Inhibits Activations and Functions in Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Der-Yuan Chen

    2013-01-01

    Full Text Available Dendritic cells (DCs play an important role in connecting innate and adaptive immunity. Thus, DCs have been regarded as a major target for the development of immunomodulators. In this study, we examined the effect of dextromethorphan (DXM, a common cough suppressant with a high safety profile, on the activation and function of DCs. In the presence of DXM, the LPS-induced expression of the costimulatory molecules in murine bone marrow-derived dendritic cells (BMDCs was significantly suppressed. In addition, DXM treatment reduced the production of reactive oxygen species (ROS, proinflammatory cytokines, and chemokines in maturing BMDCs that were activated by LPS. Therefore, DXM abrogated the ability of LPS-stimulated DCs to induce Ag-specific T-cell activation, as determined by their decreased proliferation and IFN-γ secretion in mixed leukocyte cultures. Moreover, the inhibition of LPS-induced MAPK activation and NF-κB translocation may contribute to the suppressive effect of DXM on BMDCs. Remarkably, DXM decreased the LPS-induced surface expression of CD80, CD83, and HLA-DR and the secretion of IL-6 and IL-12 in human monocyte-derived dendritic cells (MDDCs. These findings provide a new insight into the impact of DXM treatment on DCs and suggest that DXM has the potential to be used in treating DC-related acute and chronic diseases.

  2. ROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPARα -mediated inhibition of Glioma cell motility in vitro

    Directory of Open Access Journals (Sweden)

    Del Valle Luis

    2010-06-01

    Full Text Available Abstract Background Glioblastomas are characterized by rapid cell growth, aggressive CNS infiltration, and are resistant to all known anticancer regimens. Recent studies indicate that fibrates and statins possess anticancer potential. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα that can switch energy metabolism from glycolysis to fatty acid β-oxidation, and has low systemic toxicity. Fenofibrate also attenuates IGF-I-mediated cellular responses, which could be relevant in the process of glioblastoma cell dispersal. Methods The effects of fenofibrate on Glioma cell motility, IGF-I receptor (IGF-IR signaling, PPARα activity, reactive oxygen species (ROS metabolism, mitochondrial potential, and ATP production were analyzed in human glioma cell lines. Results Fenofibrate treatment attenuated IGF-I signaling responses and repressed cell motility of LN-229 and T98G Glioma cell lines. In the absence of fenofibrate, specific inhibition of the IGF-IR had only modest effects on Glioma cell motility. Further experiments revealed that PPARα-dependent accumulation of ROS is a strong contributing factor in Glioma cell lines responses to fenofibrate. The ROS scavenger, N-acetyl-cysteine (NAC, restored cell motility, improved mitochondrial potential, and increased ATP levels in fenofibrate treated Glioma cell lines. Conclusions Our results indicate that although fenofibrate-mediated inhibition of the IGF-IR may not be sufficient in counteracting Glioma cell dispersal, PPARα-dependent metabolic switch and the resulting ROS accumulation strongly contribute to the inhibition of these devastating brain tumor cells.

  3. A novel small molecular STAT3 inhibitor, LY5, inhibits cell viability, cell migration, and angiogenesis in medulloblastoma cells.

    Science.gov (United States)

    Xiao, Hui; Bid, Hemant Kumar; Jou, David; Wu, Xiaojuan; Yu, Wenying; Li, Chenglong; Houghton, Peter J; Lin, Jiayuh

    2015-02-06

    Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studies have demonstrated that inhibition of the persistent STAT3 signaling pathway results in decreased proliferation and increased apoptosis in human cancer cells, indicating that STAT3 is a viable molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable small molecule, named LY5, to target STAT3 in medulloblastoma cells. LY5 inhibited persistent STAT3 phosphorylation and induced apoptosis in human medulloblastoma cell lines expressing constitutive STAT3 phosphorylation. The inhibition of STAT3 signaling by LY5 was confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, bcl-XL, survivin, and micro-RNA-21. LY5 also inhibited the induction of STAT3 phosphorylation by interleukin-6 (IL-6), insulin-like growth factor (IGF)-1, IGF-2, and leukemia inhibitory factor in medulloblastoma cells, but did not inhibit STAT1 and STAT5 phosphorylation stimulated by interferon-γ (IFN-γ) and EGF, respectively. In addition, LY5 blocked the STAT3 nuclear localization induced by IL-6, but did not block STAT1 and STAT5 nuclear translocation mediated by IFN-γ and EGF, respectively. A combination of LY5 with cisplatin or x-ray radiation also showed more potent effects than single treatment alone in the inhibition of cell viability in human medulloblastoma cells. Furthermore, LY5 demonstrated a potent inhibitory activity on cell migration and angiogenesis. Taken together, these findings indicate LY5 inhibits persistent and inducible STAT3 phosphorylation and suggest that LY5 is a promising therapeutic drug candidate for medulloblastoma by inhibiting persistent STAT3 signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. All-trans retinoic acid inhibits craniopharyngioma cell growth: study on an explant cell model.

    Science.gov (United States)

    Li, Qiang; You, Chao; Zhou, Liangxue; Sima, Xiutian; Liu, Zhiyong; Liu, Hao; Xu, Jianguo

    2013-05-01

    The ratio between FABP5 and CRABPII determines cellular response to physiological level of retinoic acid; tumor cells undergo proliferation with high level of FABP5 and apoptosis with high level of CRABPII. We intended to study FABP5 and CRABPII expression in craniopharyngiomas, to establish craniopharyngioma cell model using explants method, and to study the effect of pharmacological dose of retinoic acid on craniopharyngioma cells. Expression of FABP5 and CRABPII in craniopharyngioma tissue from 20 patients was studied using immunohistochemistry. Primary craniopharyngioma cell cultures were established using tissue explants method. Craniopharyngioma cells were treated using various concentrations of all-trans retinoic acid, and cell growth curve, apoptosis, expression of FABP5, CRABPII and NF-κB were assayed in different groups. FABP5/CRABPII ratio was significantly higher in adamatinomatous group than that in papillary group. Cell cultures were established in 19 cases (95 %). Pharmacological level retinoic acid inhibited cell growth and induced cellular apoptosis in dose dependent manner, and apoptosis rate cells treated with 30 μM retinoic acid for 24 h was 43 %. Also, retinoic acid increased CRABPII, and decreased FABP5 and NF-κB expression in craniopharyngioma cells. High FABP5/CRABPII ratio is observed in adamatinomatous craniopharyngioma. Retinoic acid at pharmacological level induced craniopharyngioma cell apoptosis via increasing FABP5/CRABPII ratio and inhibiting NF-κB signaling pathway. Our study demonstrated that all-trans retinoic acid might be a candidate for craniopharyngioma adjuvant chemotherapy in future.

  5. Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xuesong Liu

    2001-01-01

    Full Text Available The serine/threonine kinases, Akti/PKBα, Akt2/PKBβ, and Akt3/PKBγ, play a critical role in preventing cancer cells from undergoing apoptosis. However, the function of individual Akt isoforms in the tumorigenicity of cancer cells is still not well defined. In the current study, we used an AM antisense oligonucleotide (AS to specifically downregulate Akti protein in both cancer and normal cells. Our data indicate that AM AS treatment inhibits the ability of MiaPaCa-2, H460, HCT-15, and HT1080 cells to grow in soft agar. The treatment also induces apoptosis in these cancer cells as demonstrated by FRCS analysis and a caspase activity assay. Conversely, Akti AS treatment has little effect on the cell growth and survival of normal human cells including normal human fibroblast (NHF, fibroblast from muscle (FBM, and mammary gland epithelial 184135 cells. In addition, AM AS specifically sensitizes cancer cells to typical chemotherapeutic agents. Thus, Akti is indispensable for maintaining the tumorigenicity of cancer cells. Inhibition of AM may provide a powerful sensitization agent for chemotherapy specifically in cancer cells.

  6. Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells

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    Noolu Bindu

    2013-01-01

    Full Text Available Abstract Background Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves, a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Methods Hydro-methanolic extract of curry leaves (CLE was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau’s method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. Results CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Conclusions Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death

  7. Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells.

    Science.gov (United States)

    Noolu, Bindu; Ajumeera, Rajanna; Chauhan, Anitha; Nagalla, Balakrishna; Manchala, Raghunath; Ismail, Ayesha

    2013-01-09

    Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves), a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Hydro-methanolic extract of curry leaves (CLE) was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau's method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death. Therefore, identification of active component(s) from the leaf

  8. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

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    Birgit Piater

    Full Text Available The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF. Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX. CLN64 and a previously described single-stranded DNA (ssDNA aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.

  9. Ginger phytochemicals exhibit synergy to inhibit prostate cancer cell proliferation

    Science.gov (United States)

    Brahmbhatt, Meera; Gundala, Sushma R.; Asif, Ghazia; Shamsi, Shahab A; Aneja, Ritu

    2014-01-01

    Dietary phytochemicals offer non-toxic therapeutic management as well as chemopreventive intervention for slow-growing prostate cancers. However, the limited success of several single-agent clinical trials suggest a paradigm shift that the health benefits of fruits and vegetables are not ascribable due to individual phytochemicals rather may be ascribed to but to synergistic interactions among them. We recently reported growth-inhibiting and apoptosis-inducing properties of ginger extract (GE) in in vitro and in vivo prostate cancer models. Nevertheless, the nature of interactions among the constituent ginger biophenolics, viz. 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogoal, remains elusive. Here we show antiproliferative efficacy of the most-active GE biophenolics as single-agents and in binary combinations, and investigate the nature of their interactions using the Chou-Talalay combination-index (CI) method. Our data demonstrate that binary combinations of ginger phytochemicals synergistically inhibit proliferation of PC-3 cells with CI values ranging from 0.03-0.88. To appreciate synergy among phytochemicals present in GE, the natural abundance of ginger biophenolics was quantitated using LC-UV/MS. Interestingly, combining GE with its constituents (in particular, 6-gingerol) resulted in significant augmentation of GE’s antiproliferative activity. These data generate compelling grounds for further preclinical evaluation of GE alone and in combination with individual ginger biophenols for prostate cancer management. PMID:23441614

  10. Indirubin inhibits cell proliferation, migration, invasion and angiogenesis in tumor-derived endothelial cells

    Directory of Open Access Journals (Sweden)

    Li Z

    2018-05-01

    Full Text Available Zhuohong Li, Chaofu Zhu, Baiping An, Yu Chen, Xiuyun He, Lin Qian, Lan Lan, Shijie Li Department of Oncology, The Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China Purpose: Hepatocellular carcinoma is one of the most predominant malignancies with high fatality rate and its incidence is rising at an alarming rate because of its resistance to radio- and chemotherapy. Indirubin is the major active anti-tumor ingredient of a traditional Chinese herbal medicine. The present study aimed to analyze the effects of indirubin on cell proliferation, migration, invasion, and angiogenesis of tumor-derived endothelial cells (Td-EC. Methods: Td-EC were derived from human umbilical vein endothelial cells (HUVEC by treating HUVEC with the conditioned medium of human liver cancer cell line HepG2. Cell proliferation, migration, invasion, and angiogenesis were assessed by MTT, wound healing, in vitro cell invasion, and in vitro tube formation assay. Results: Td-EC were successfully obtained from HUVEC cultured with 50% culture supernatant from serum-starved HepG2 cells. Indirubin significantly inhibited Td-EC proliferation in a dose- and time-dependent manner. Indirubin also inhibited Td-EC migration, invasion, and angiogenesis. However, indirubin’s effects were weaker on HUVEC than Td-EC. Conclusion: Indirubin significantly inhibited Td-EC proliferation, migration, invasion, and angiogenesis. Keywords: indirubin, Td-EC, proliferation, migration, invasion, angiogenesis

  11. Lichen Secondary Metabolite, Physciosporin, Inhibits Lung Cancer Cell Motility

    Science.gov (United States)

    Yang, Yi; Park, So-Yeon; Nguyen, Thanh Thi; Yu, Young Hyun; Nguyen, Tru Van; Sun, Eun Gene; Udeni, Jayalal; Jeong, Min-Hye; Pereira, Iris; Moon, Cheol; Ha, Hyung-Ho; Kim, Kyung Keun; Hur, Jae-Seoun; Kim, Hangun

    2015-01-01

    Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3’-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action. PMID:26371759

  12. Inhibition of EV71 by curcumin in intestinal epithelial cells.

    Science.gov (United States)

    Huang, Hsing-I; Chio, Chi-Chong; Lin, Jhao-Yin

    2018-01-01

    EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.

  13. Inhibition of EV71 by curcumin in intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hsing-I Huang

    Full Text Available EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6, an active ingredient of turmeric (Curcuma longa Linn with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK signaling pathways is not involved. We found that protein kinase C delta (PKCδ plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.

  14. Inhibition of EV71 by curcumin in intestinal epithelial cells

    Science.gov (United States)

    Chio, Chi-Chong; Lin, Jhao-Yin

    2018-01-01

    EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections. PMID:29370243

  15. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

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    M. Ryan Smith

    2016-08-01

    Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

  16. Lysine demethylase inhibition protects pancreatic β cells from apoptosis and improves β-cell function

    DEFF Research Database (Denmark)

    Backe, Marie Balslev; Andersson, Jan Legaard; Bacos, Karl

    2018-01-01

    ) protects β cells from cytokine-induced apoptosis and reduces type 1 diabetes incidence in animals. We hypothesized that also lysine demethylases (KDMs) regulate β-cell fate in response to inflammatory stress. Expression of the demethylase Kdm6B was upregulated by proinflammatory cytokines suggesting......Transcriptional changes control β-cell survival in response to inflammatory stress. Posttranslational modifications of histone and non-histone transcriptional regulators activate or repress gene transcription, but the link to cell-fate signaling is unclear. Inhibition of lysine deacetylases (KDACs...

  17. Dasatinib inhibits both osteoclast activation and prostate cancer PC-3-cell-induced osteoclast formation.

    Science.gov (United States)

    Araujo, John C; Poblenz, Ann; Corn, Paul; Parikh, Nila U; Starbuck, Michael W; Thompson, Jerry T; Lee, Francis; Logothetis, Christopher J; Darnay, Bryant G

    2009-11-01

    Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC(50) of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts, and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.

  18. Differences in antiproliferative effect of STAT3 inhibition in HCC cells with versus without HBV expression

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Yun; Zhou, Lin; Xie, Haiyang; Wang, Weilin [Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Qingchun Road 79, Hangzhou, Zhejiang 310003 (China); Key Laboratory of Combined Multi-organ Transplantation of Ministry of Public Health, Qingchun Road 79, Hangzhou, Zhejiang 310003 (China); Zheng, Shusen, E-mail: shusenzheng@zju.edu.cn [Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Qingchun Road 79, Hangzhou, Zhejiang 310003 (China); Key Laboratory of Combined Multi-organ Transplantation of Ministry of Public Health, Qingchun Road 79, Hangzhou, Zhejiang 310003 (China)

    2015-06-05

    Chronic infection with hepatitis B virus (HBV) plays an important role in the etiology of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 3 (STAT3) inactivation could inhibit the tumor growth of HCC. In this study, differential antiproliferative effect of STAT3 inhibition was observed with HBV-related HCC cells being more resistant than non-HBV-related HCC cells. Resistance of HBV-related HCC cells to STAT3 inhibition was positively correlated to the expression of HBV. Enhanced ERK activation after STAT3 blockade was detected in HBV-related HCC cells but not in non-HBV-related HCC cells. Combined ERK and STAT3 inhibition eliminates the discrepancy between the two types of HCC cells. Moderate reduced HBV expression was found after STAT3 inhibition. These findings disclose a discrepancy in cellular response to STAT3 inhibition between non-HBV-related and HBV-related HCC cells and underscore the complexity of antiproliferative effect of STAT3 inactivation in HBV-related HCC cells. - Highlights: • HBV endows HCC cells with resistance to STAT3 inactivation on proliferation. • Abnormal ERK activation after STAT3 inhibition in HBV-related HCC cells. • Combined ERK and STAT3 inhibition eliminates the discrepancy. • STAT3 inhibition moderately reduces HBV expression.

  19. Differences in antiproliferative effect of STAT3 inhibition in HCC cells with versus without HBV expression

    International Nuclear Information System (INIS)

    Hong, Yun; Zhou, Lin; Xie, Haiyang; Wang, Weilin; Zheng, Shusen

    2015-01-01

    Chronic infection with hepatitis B virus (HBV) plays an important role in the etiology of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 3 (STAT3) inactivation could inhibit the tumor growth of HCC. In this study, differential antiproliferative effect of STAT3 inhibition was observed with HBV-related HCC cells being more resistant than non-HBV-related HCC cells. Resistance of HBV-related HCC cells to STAT3 inhibition was positively correlated to the expression of HBV. Enhanced ERK activation after STAT3 blockade was detected in HBV-related HCC cells but not in non-HBV-related HCC cells. Combined ERK and STAT3 inhibition eliminates the discrepancy between the two types of HCC cells. Moderate reduced HBV expression was found after STAT3 inhibition. These findings disclose a discrepancy in cellular response to STAT3 inhibition between non-HBV-related and HBV-related HCC cells and underscore the complexity of antiproliferative effect of STAT3 inactivation in HBV-related HCC cells. - Highlights: • HBV endows HCC cells with resistance to STAT3 inactivation on proliferation. • Abnormal ERK activation after STAT3 inhibition in HBV-related HCC cells. • Combined ERK and STAT3 inhibition eliminates the discrepancy. • STAT3 inhibition moderately reduces HBV expression

  20. Evaluation of first-pass cytochrome P4503A (CYP3A) and P-glycoprotein activities using alfentanil and fexofenadine in combination.

    Science.gov (United States)

    Kharasch, Evan D; Walker, Alysa; Hoffer, Christine; Sheffels, Pamela

    2005-01-01

    Cytochrome P4503A (CYP3A) and P-glycoprotein (P-gp) are major determinants of oral bioavailability. Development of in vivo probe(s), for both CYP3A and P-gp, which could be administered in combination, is a current goal. Nevertheless, there is considerable overlap in CYP3A and P-gp substrate selectivities; there are few discrete probes. Alfentanil is a selective CYP3A probe but not a P-gp substrate. Fexofenadine is a P-gp probe but not a CYP3A substrate. This investigation tested the hypothesis that alfentanil and fexofenadine could be administered in combination to probe first-pass CYP3A and P-gp activities in humans. Two 3-way crossover studies were conducted in healthy volunteers. In the first protocol, subjects received oral alfentanil alone, fexofenadine alone, or fexofenadine 1 hour after alfentanil. In the second protocol, subjects abstained from citrus and apple products for 5 days and received fexofenadine alone, fexofenadine 1 hour after alfentanil, or alfentanil 4 hours after fexofenadine. An assay using solid-phase extraction and electrospray liquid chromatography/mass spectrometry was developed for the simultaneous quantification of plasma alfentanil and fexofenadine. In both protocols, alfentanil plasma concentrations and area under the concentration versus time curve (AUC) were unaffected by fexofenadine or meal composition. Fexofenadine given 1 hour after alfentanil and followed 1 hour later by a meal containing orange or apple juice had a somewhat lower AUC compared with fexofenadine alone (geometric mean ratio with and without the interacting drug = 0.73, 90% confidence interval [CI] = 0.59-1.04). Fexofenadine given 1 hour after alfentanil and followed 2 hours later by a meal not containing citrus or apple products had an AUC that was unchanged compared with fexofenadine alone (ratio = 0.91, 90% CI = 0.70-1.35). These results show that alfentanil disposition was not affected by fexofenadine. A dosing regimen was identified in which fexofenadine

  1. Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells

    International Nuclear Information System (INIS)

    Kim, Soon-hee; Park, Hee-Sook; Lee, Myoung-su; Cho, Yong-Jin; Kim, Young-Sup; Hwang, Jin-Taek; Sung, Mi Jeong; Kim, Myung Sunny; Kwon, Dae Young

    2008-01-01

    Inhibition of adipocyte differentiation is one approach among the anti-obesity strategies. This study demonstrates that vitisin A, a resveratrol tetramer, inhibits adipocyte differentiation most effectively of 18 stilbenes tested. Fat accumulation and PPARγ expression were decreased by vitisin A in a dose-dependent manner. Vitisin A significantly inhibited preadipocyte proliferation and consequent differentiation within the first 2 days of treatment, indicating that the anti-adipogenic effect of vitisin A was derived from anti-proliferation. Based on cell cycle analysis, vitisin A blocked the cell cycle at the G1-S phase transition, causing cells to remain in the preadipocyte state. Vitisin A increased p21 expression, while the Rb phosphorylation level was reduced. Therefore, vitisin A seems to induce G1 arrest through p21- and consequent Rb-dependent suppression of transcription. On the other hand, ERK and Akt signaling pathways were not involved in the anti-mitotic regulation by vitisin A. Taken together, these results suggest that vitisin A inhibits adipocyte differentiation through preadipocyte cell cycle arrest

  2. Involvement of P-glycoprotein and multidrug resistance associated protein 1 in the transport of tanshinone IIB, a primary active diterpenoid quinone from the roots of Salvia miltiorrhiza, across the blood-brain barrier.

    Science.gov (United States)

    Zhou, Zhi-Wei; Chen, Xiao; Liang, Jun; Yu, Xi-Yong; Wen, Jing-Yuan; Zhou, Shu-Feng

    2007-08-01

    Tanshinone IIB (TSB) is a major constituent of Salvia miltiorrhiza, which is widely used in treatment of cardiovascular and central nervous system (CNS) diseases such as coronary heart disease and stroke. This study aimed to investigate the role of various drug transporters in the brain penetration of TSB using several in vitro and in vivo mouse and rat models. The uptake and efflux of TSB in rat primary microvascular endothelial cells (RBMVECs) were ATP-dependent and significantly altered in the presence of a P-glycoprotein (P-gp) or multidrug resistance associated protein (Mrp1/2) inhibitor. A polarized transport of TSB was found in RBMVEC monolayers with facilitated efflux from the abluminal to luminal side. Addition of a P-gp inhibitor (e.g. verapamil) in both abluminal and luminal sides attenuated the polarized transport. In an in situ rat brain perfusion model, TSB crossed the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a P-gp or Mrp1/2 inhibitor. The brain levels of TSB were only about 30% of that in the plasma and it could be increased to up to 72% of plasma levels when verapamil, quinidine, or probenecid was co-administered in rats. The entry of TSB to CNS increased by 67-97% in rats subjected to middle cerebral artery occlusion or treatment with the neurotoxin, quinolinic acid, compared to normal rats. Furthermore, The brain levels of TSB in mdr1a(-/-) and mrp1(-/-) mice were 28- to 2.6-fold higher than those in the wild-type mice. TSB has limited brain penetration through the BBB due to the contribution of P-gp and to a lesser extent of Mrp1 in rodents. Further studies are needed to confirm whether these corresponding transporters in humans are involved in limiting the penetration of TSB across the BBB and the clinical relevance.

  3. Nicotine inhibits potassium currents in Aplysia bag cell neurons

    Science.gov (United States)

    White, Sean H.; Sturgeon, Raymond M.

    2016-01-01

    Acetylcholine and the archetypal cholinergic agonist, nicotine, are typically associated with the opening of ionotropic receptors. In the bag cell neurons, which govern the reproductive behavior of the marine snail, Aplysia californica, there are two cholinergic responses: a relatively large acetylcholine-induced current and a relatively small nicotine-induced current. Both currents are readily apparent at resting membrane potential and result from the opening of distinct ionotropic receptors. We now report a separate current response elicited by applying nicotine to cultured bag cell neurons under whole cell voltage-clamp. This current was ostensibly inward, best resolved at depolarized voltages, presented a noncooperative dose-response with a half-maximal concentration near 1.5 mM, and associated with a decrease in membrane conductance. The unique nicotine-evoked response was not altered by intracellular perfusion with the G protein blocker GDPβS or exposure to classical nicotinic antagonists but was occluded by replacing intracellular K+ with Cs+. Consistent with an underlying mechanism of direct inhibition of one or more K+ channels, nicotine was found to rapidly reduce the fast-inactivating A-type K+ current as well as both components of the delayed-rectifier K+ current. Finally, nicotine increased bag cell neuron excitability, which manifested as reduction in spike threshold, greater action potential height and width, and markedly more spiking to continuous depolarizing current injection. In contrast to conventional transient activation of nicotinic ionotropic receptors, block of K+ channels could represent a nonstandard means for nicotine to profoundly alter the electrical properties of neurons over prolonged periods of time. PMID:26864763

  4. Inhibition of Zoledronic Acid on Cell Proliferation and Invasion of Lung Cancer Cell Line 95D

    Directory of Open Access Journals (Sweden)

    Mingming LI

    2009-03-01

    Full Text Available Background and objective Abnormal proliferation and metastasis is the basic characteristic of malignant tumors. The aim of this work is to explore the effects of zoledronic acid on cell proliferation and invasion in lung cancer cell line 95D. Methods The effect of zoledrnic acid (ZOL on proliferation of lung cancer cell line 95D was detected by MTT. The expression of proliferation and invasion-relation genes and proteins were detected by Western blot, RT-PCR and immunofluorescence. Changes of invasion of lung cancer cell numbers were measured by polycarbonates coated with Matrigel. Results ZOL could inhibit the proliferation of lung cancer cell line 95D in vitro in a time-dependant and a dose-dependant manner. With time extending after ZOL treated, the mRNA expresion of VEGF, MMP9, MMP2 and protein expression of VEGF, MMP9, ERK1/ ERK2 were decreased. The results of Tanswell invasion showed the numbers of invasive cells were significantly reduced in 95D cells treated with ZOL 4 d and 6 d later. Conclusion ZOL could inhibit cell proliferation and invasion of lung cancer cell line 95D.

  5. Mesenchymal Stem Cells from Patients with Rheumatoid Arthritis Display Impaired Function in Inhibiting Th17 Cells

    Directory of Open Access Journals (Sweden)

    Yue Sun

    2015-01-01

    Full Text Available Mesenchymal stem cells (MSCs possess multipotent and immunomodulatory properties and are suggested to be involved in the pathogenesis of immune-related diseases. This study explored the function of bone marrow MSCs from rheumatoid arthritis (RA patients, focusing on immunomodulatory effects. RA MSCs showed decreased proliferative activity and aberrant migration capacity. No significant differences were observed in cytokine profiles between RA and control MSCs. The effects of RA MSCs on proliferation of peripheral blood mononuclear cells (PBMCs and distribution of specific CD4+ T cell subtypes (Th17, Treg, and Tfh cells were investigated. RA MSCs appeared to be indistinguishable from controls in suppressing PBMC proliferation, decreasing the proportion of Tfh cells, and inducing the polarization of Treg cells. However, the capacity to inhibit Th17 cell polarization was impaired in RA MSCs, which was related to the low expression of CCL2 in RA MSCs after coculture with CD4+ T cells. These findings indicated that RA MSCs display defects in several important biological activities, especially the capacity to inhibit Th17 cell polarization. These functionally impaired MSCs may contribute to the development of RA disease.

  6. Solena amplexicaulis induces cell cycle arrest, apoptosis and inhibits angiogenesis in hepatocarcinoma cells and HUVECs.

    Science.gov (United States)

    Ren, Jie; Xu, Yuan Yuan; Jiang, He Fei; Yang, Meng; Huang, Qian Hui; Yang, Jie; Hu, Kun; Wei, Kun

    2014-01-01

    Solena amplexicaulis (Lam.) Gandhi (SA) has been used as a traditional medicine for the treatment of dysentery, multiple abscess, gastralgia, urethritis, and eczema in the minority area of China. This study was aimed to examine the cell proliferation inhibitory activity of the SA extract (SACE) and its mechanism of action in human hepatoma cell line (HepG2) and evaluate its anti-angiogenesis activity in human umbilical vein endothelial cell line (HUVEC). SACE could inhibit the growth of HepG2 cells in a dose- and time-dependent manner. FCM analysis showed that SACE could induce G2/M phase arrest, cell apoptosis, the mitochondrial membrane potential loss (ΔΨm) and increase the production of intracellular ROS of HepG2 cells. After treatment with SACE, topical morphological changes of apoptotic body formation, obvious increase of apoptosis-related protein expressions, such as Bax, cytochrome c, caspase-3, PARP-1, and decrease of Bcl-2, procaspase-9 protein expressions were observed at the same time. Moreover, SACE caused the significant inhibition of endothelial cell migration and tube formation in HUVEC cells. The results suggested that SACE could act as an angiogenesis inhibitor and induce cell apoptosis via a caspase-dependent mitochondrial pathway. Therefore, SACE could be a potent candidate for the prevention and treatment of liver cancer.

  7. D-Glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K

    International Nuclear Information System (INIS)

    Oh, Hyun-Ji; Lee, Jason S.; Song, Dae-Kyu; Shin, Dong-Hoon; Jang, Byeong-Churl; Suh, Seong-Il; Park, Jong-Wook; Suh, Min-Ho; Baek, Won-Ki

    2007-01-01

    Although D-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of D-glucosamine is still unclear. Since there are several reports suggesting D-glucosamine inhibits protein synthesis, we examined whether D-glucosamine affects p70S6 K activity, an important signaling molecule involved in protein translation. In the present study, we found D-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. D-Glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that D-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, D-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that D-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K

  8. Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

    International Nuclear Information System (INIS)

    Cheng, Gang; Zielonka, Jacek; McAllister, Donna M; Mackinnon, A Craig Jr; Joseph, Joy; Dwinell, Michael B; Kalyanaraman, Balaraman

    2013-01-01

    Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect

  9. Inhibition of pancreatic tumoral cells by snake venom disintegrins.

    Science.gov (United States)

    Lucena, Sara; Castro, Roberto; Lundin, Courtney; Hofstetter, Amanda; Alaniz, Amber; Suntravat, Montamas; Sánchez, Elda Eliza

    2015-01-01

    Pancreatic cancer often has a poor prognosis, even when diagnosed early. Pancreatic cancer typically spreads rapidly and is rarely detected in its early stages, which is a major reason it is a leading cause of cancer death. Signs and symptoms may not appear until pancreatic cancer is quite advanced, and complete surgical removal is not possible. Furthermore, pancreatic cancer responds poorly to most chemotherapeutic agents. The importance of integrins in several cell types that affect tumor progression has made them an appealing target for cancer therapy. Some of the proteins found in the snake venom present a great potential as anti-tumor agents. In this study, we summarize the activity of two integrins antagonist, recombinant disintegrins mojastin 1 and viridistatin 2, on human pancreatic carcinoma cell line (BXPC-3). Both recombinant disintegrins inhibited some essential aspects of the metastasis process such as proliferation, adhesion, migration, and survival through apoptosis, making these proteins prominent candidates for the development of drugs for the treatment of pancreatic cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    Science.gov (United States)

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hongtao [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China); Gao, Peng [Department of Internal Medicine, University of Iowa, Iowa City, IA 52242 (United States); Zheng, Jie, E-mail: jiezheng54@126.com [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China)

    2014-09-05

    Highlights: • As{sub 2}O{sub 3} inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As{sub 2}O{sub 3} is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As{sub 2}O{sub 3}) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As{sub 2}O{sub 3} induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As{sub 2}O{sub 3} on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As{sub 2}O{sub 3} than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I ce