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Sample records for cells expressing p-glycoprotein

  1. Evaluation of the expression of P-glycoprotein in propoxur-resistant Caco-2 cells.

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    Shabnam Yazdian

    2014-10-01

    Full Text Available There is a great concern about the effect of propoxur, as one of the more common N-methyl carbamate pesticides, on human health due to its extensive use in agricultural and non-agricultural applications. Caco-2 cells became resistant to propoxur, and the resistance was confirmed through MTT assay. Then the cell membrane integrity and P-glycoprotein expression were measured by LDH assay and western blot analysis, respectively and compared to the parent cells.  Contrary to what was expected, the expression of P-glycoprotein in propoxur resistant cells was lower than parent cells.This study indicates that the resistance to propoxur may not be related to P-glycoprotein expression directly, since P-glycoprotein expression has decreased in these cells.

  2. 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.

  3. Expression of P-glycoprotein in adult T-cell leukemia cells

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    Kuwazuru, Y.; Hanada, S.; Furukawa, T.; Yoshimura, A.; Sumizawa, T.; Utsunomiya, A.; Ishibashi, K.; Saito, T.; Uozumi, K.; Maruyama, M. (Kagoshima Univ. (Japan))

    1990-11-15

    We have examined the expression of P-glycoprotein (P-gp) in adult T-cell leukemia (ATL) samples from 25 patients. Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation. All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy. The expression of MDR1 mRNA in P-gp-positive ATL cells was increased at the relapsed stage of one patient. P-gp of this patient was photolabeled with (3H)azidopine and the labeling was inhibited with nimodipine, vinblastine and progesterone. These results suggest that P-gp expressed in ATL cells from patients at relapsed stage has the same binding site(s) for the drugs as that in multidrug resistant cells, and is correlated with the refractory nature of the cells to chemotherapy.

  4. Effects of dietary ingredients on function and expression of P-glycoprotein in human intestinal epithelial cells.

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    Okura, Takashi; Ibe, Michiko; Umegaki, Keizo; Shinozuka, Kazumasa; Yamada, Shizuo

    2010-01-01

    The present study was conducted to investigate the functional and transcriptional modulation of P-glycoprotein (MDR-1) by several dietary ingredients (piperine, capsaicin, daidzein, genistein, sesamin, curcumin, taurine) in vinblastine-resistant colon carcinoma LS-180 cells (LS-180V cells). The amount of rhodamine 123 accumulated in LS-180V cells was significantly increased by capsaicin, piperine and sesamin, whereas it was significantly reduced by daidzein and genistein which stimulated the efflux of rhodamine 123. These results suggest that the P-glycoprotein-mediated efflux is inhibited by piperine, capsaicin and sesamin and stimulated by daidzein and genistein. The concurrent addition of piperine and capsaicin seemed to inhibit synergistically the P-glycoprotein-mediated efflux. Pretreatment with sesamin for 48 h caused a significant increase in MDR1 mRNA expression without a significant effect on the expression of P-glycoprotein or accumulation of rhodamine 123. Similar pretreatment with other ingredients had little effect on the expression of MDR1 mRNA or P-glycoprotein, suggesting that they do not cause transcriptional modulation of P-glycoprotein. Piperine, genistein and curcumin have been suggested to stimulate P-glycoprotein-mediated efflux without increasing P-glycoprotein expression. In LS-180V cells, significant increases in mRNA levels of multi-drug resistance associated protein 1 (MRP1) or MRP3 were observed on pretreatment with capsaicin, daidzein, piperine and sesamin. In conclusion, our results suggest that P-glycoprotein-mediated efflux is significantly affected by dietary ingredients. Also, capsaicin, daidzein, piperine and sesamin increased significantly the mRNA expression of MRP1 or MRP3. Thus, the present study provides further evidence that repeated exposure to dietary ingredients can cause drug-food interactions by affecting the function and mRNA expression of intestinal transporters such as P-glycoprotein.

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

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    Xing Hua Tang

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

  6. [Effect of Siwu decoction on function and expression of P-glycoprotein in Caco-2 cells].

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    Jiang, Yi; Ma, Zeng-chun; Huang, Xian-ju; You, Qing; Tan, Hong-ling; Wang, Yu-guang; Liang, Qian-de; Tang, Xiang-lin; Xiao, Cheng-rong; Gao, Yue

    2015-03-01

    To study the effect of Siwu decoction on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells. The Real-time quantitative poly-merase chain reaction (Q-PCR) was used to analyze the mRNA expression of MDR1 gene in Caco-2 cells. Flow cytometer was used to study the effect of Siwu decoction on the uptake of Rhodamine 123 in Caco-2 cells, in order to evaluate the efflux function of P-gp. Western blotting method was used to detect the effect of Siwu decoction on the P-gp protein expression of Caco-2 cells. Compared with the blank control group, after Caco-2 incubation with Siwu decoction at concentrations of 3.3, 5.0, 10.0 g x L(-1) for 24, 48, 72 h, the mRNA expression of MDR1 was up-regulated, suggesting the effect of Siwu decoction in inducing the expression of MDR1. After the administration with Siwu decoction in Caco-2 cells for 48 h, the uptake of Rhodamine 123 in Caco-2 cells decreased by respectively 16.6%, 22.1% (P Caco-2 cells. After the incubation of Caco-2 cells with Siwu decoction for 48 h, the P-gp protein expression on Caco-2 cell emebranes, demonstrating the effect of Siwu decoction in inducing the protein expression of P-gp.

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

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    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

  8. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression.

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    Perazzoli, Gloria; Prados, Jose; Ortiz, Raul; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2'-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.

  9. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression.

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    Gloria Perazzoli

    Full Text Available The use of temozolomide (TMZ has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated.Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2'-deoxycytidine was used to demethylate the MGMT promoter and O(6-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed.Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229 and high (SF268 and SK-N-SH basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines.These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.

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

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

    2010-08-27

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

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

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    Wei Xu

    2010-08-01

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

  12. Analysis of the impact of extracellular acidity on the expression and activity of P-glycoprotein and on the P-glycoprotein-mediated cytotoxicity of daunorubicin in cancer cell by microfluidic chip technology.

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    Li, Yuan; Xiang, Jiao; Zhang, Sha-sha; Liu, Bei-zhong; Gong, Fang; Peng, Ming-qing

    2015-02-01

    To explore the impact of extracellular acidic environment on the expression and activity of P-glycoprotein (P-gp) and on the P-gp-mediated cytotoxicity of daunomycin in cancer cells by using microfluidic chip technology. The A549 cells cultured on a microfluidic chip were divided into experiment group and control group. The experiment group was exposed to an acidic cell culture medium (pH 6.6), while the control group was treated with a neutral cell culture medium (pH 7.4). The expression of P-gp was detected by cell immunofluorescense analysis and the activity of P-gp was evaluated by Rhodamine 123 efflux experiment. Meanwhile, the cytotoxicity of daunomycin was analyzed by cell live/dead fluorescence staining method. Microfluidic chip designed in this study could provide a suitable microenvironment for the growth of A549 cells and the A549 cells reached the confluence of 90% after inoculation for 72 h. Treatment of the acidic cell culture media on A549 cells did not make a significant difference on the expression level of P-gp. However, the activity of P-gp was significantly enhancement and peaked at 6 h after treatment with acidic cell culture media. Meanwhile, the cytotoxicity of daunomycin reduced significantly after treatment with acidic cell culture medium for 6 h,and a reversal effect was obtained when synergy with verapamil. Microfluidic chip technology can shorten the analysis time and reduce the reagent consumption. It can be used as a new technology platform for understanding the mechanisms of multi-drug resistance and for screening highly efficient multi-drug resistance reversal agents.

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

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    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...

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

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    Dong, YePing; Pan, QiongXi; Jiang, Li; Chen, Zhen; Zhang, FangFang; Liu, YanJun; Xing, Hui; Shi, Mei; Li, Jiao; Li, XiYuan; Zhu, YaoDan; Chen, Yun; Bruce, Iain C.; Jin, Jian, E-mail: jinjian31@126.com; Ma, Xin, E-mail: maxin@jiangnan.edu.cn

    2014-03-28

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

  15. Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells.

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    McInerney, Mitchell P; Pan, Yijun; Short, Jennifer L; Nicolazzo, Joseph A

    2017-09-01

    An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  16. Effects of nonsteroidal anti-inflammatory drugs on the expression and function of P-glycoprotein/MDR1 in Caco-2 cells.

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    Takara, Kohji; Hayashi, Ryuhei; Kokufu, Misato; Yamamoto, Kazuhiro; Kitada, Noriaki; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2009-01-01

    The aim of this study was to examine the effects of 16 kinds of nonsteroidal anti-inflammatory drugs (NSAIDs) on P-glycoprotein/MDR1 in Caco-2 cells as an intestinal epithelial cell model. Cells were treated with NSAIDs for 24 hours, and then, the expression of MDR1 mRNA was evaluated by reverse-transcriptase polymerase chain reaction. The function of MDR1 in cells pretreated with NSAIDs for 48 hours was evaluated by measuring the cellular amount of rhodamine123, which is a substrate of MDR1. The expression of MDR1 mRNA was increased by diclofenac, fenbufen, indomethacin, and nimesulide and the tended to be increased by meloxicam, mepirizole, and sulindac. However, pretreatment for 48 hours with diclofenac, indomethacin, or nimesulide, but not fenbufen, resulted in a significant increase in the amount of rhodamine123 accumulated. Although NSAIDs without effects on the expression of MDR1 mRNA altered the accumulation of rhodamine123 significantly, the efflux of rhodamine123 from cells was unchanged. In conclusion, the expression of MDR1 mRNA in Caco-2 cells was demonstrated to be increased by treatment with some NSAIDs, although the transport function of MDR1 was unchanged. These findings imply that the NSAIDs did not cause the drug interaction via MDR1 induction.

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

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    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)

  18. Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid resistant pro-inflammatory CD8+T cells.

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    Hodge, Greg; Hodge, Sandra; Nguyen, Phan Tien; Yeo, Aeneas; Sarkar, Parama; Badiei, Arash; Holmes-Liew, C L; Reynolds, Paul N; Holmes, Mark

    2018-01-20

    Immunosuppressive therapy fails to suppress the production of pro-inflammatory cytokines, particularly by CD8+T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and IFNγ/TNFα pro-inflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (± prednisolone ± Pgp inhibitor, cyclosporin A ± GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFNγ/TNFα pro-inflammatory cytokines. Pgp expression was increased in CD8+Pgp+T cells and correlated with IFNγ/TNFα expression and BOS grade. Reduced GCR was observed in CD8+Pgp-T, NKT-like and NK cells from stable patients compared with controls, and further reduced in CD8+Pgp-T cells in BOS. Addition of 2.5ng/ml cyclosporine A and 1µM prednisolone significantly inhibit IFNγ/TNFα production by CD8+Pgp+ T cells from BOS patients. Addition of 10µM Compound A and 1µM prednisolone significantly inhibit IFNγ/TNFα production by CD8+Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid resistant pro-inflammatory CD8+T cells. Treatments that inhibit Pgp and upregulate GCR in CD8+T cells may improve graft survival. This article is protected by copyright. All rights reserved. © 2018 British Society for Immunology.

  19. Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

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    Kwatra, Deep; Budda, Balasubramanyam; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K

    2012-07-02

    The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drug of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 μM naringin and 3 μM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and Western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The Vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be 10- and 3-fold lower in MMC as compared to MDCK-WT and MDCK-MDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT, indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined activities of CYP3A4 and P-gp. Transport of cortisol increased 5-fold in the presence of naringin in MMC and doubled in MDCK-MDR1. Cortisol transport in MMC was significantly lower than that in MDCK-WT in the presence of naringin. The permeability increased 3-fold in the presence of morphine, which is a weaker inhibitor of CYP3A4. Formation of 6β-hydroxy cortisol was found to decrease in the presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes toward drug-drug interactions.

  20. 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.

  1. Mechanism Underlying the Reversal of Drug Resistance in P-Glycoprotein-Expressing Leukemia Cells by Pinoresinol and the Study of a Derivative

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    María C. Carpinella

    2017-04-01

    Full Text Available P-glycoprotein (P-gp is a membrane protein associated with multidrug resistance (MDR due to its key role in mediating the traffic of chemotherapeutic drugs outside cancer cells, leading to a cellular response that hinders efforts toward successful therapy. With the aim of finding agents that circumvent the MDR phenotype mediated by P-gp, 15 compounds isolated from native and naturalized plants of Argentina were screened. Among these, the non-cytotoxic lignan (± pinoresinol successfully restored sensitivity to doxorubicin from 7 μM in the P-gp overexpressed human myelogenous leukemia cells, Lucena 1. This resistance-reversing effect was confirmed by competitively increasing the intracellular doxorubicin accumulation and by significantly inhibiting the efflux of doxorubicin and, to a lesser extent, that of rhodamine 123. The activity obtained was similar to that observed with verapamil. No such results were observed in the sensitive parental K562 cell line. To gain deeper insight into the mode of action of pinoresinol, its effect on P-gp function and expression was examined. The docking simulations indicated that the lignan bound to P-gp at the apex of the V-shaped transmembrane cavity, involving transmembrane helices 4, 5, and 6, and partially overlapped the binding region of tariquidar, which was used as a positive control. These results would shed some light on the nature of its interaction with P-gp at molecular level and merit further mechanistic and kinetic studies. In addition, it showed a maximum 29% activation of ATP hydrolysis and antagonized verapamil-stimulated ATPase activity with an IC50 of 20.9 μM. On the other hand, pinoresinol decreased the presence of P-gp in the cell surface. Derivatives of pinoresinol with improved activity were identified by docking studies. The most promising one, the non-cytotoxic 1-acetoxypinoresinol, caused a reversion of doxorubicin resistance from 0.11 μM and thus higher activity than the lead

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

    National Research Council Canada - National Science Library

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

    2015-01-01

    .... 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...

  3. Involvement of the multidrug resistance P-glycoprotein in acetaminophen-induced toxicity in hepatoma-derived HepG2 and Hep3B cells.

    Science.gov (United States)

    Manov, Irena; Bashenko, Yulia; Hirsh, Mark; Iancu, Theodore C

    2006-09-01

    Acetaminophen overdose causes severe hepatic failure. Although the mechanisms of acetaminophen hepatotoxicity have been well investigated, little is known about the involvement of the P-glycoprotein in acetaminophen transport and toxicity. P-Glycoprotein is a membrane efflux pump, playing a significant role in regulating absorption, excretion, and tissue distribution of many drugs. To evaluate the contribution of P-glycoprotein transporter in the course of acetaminophen-induced toxicity, HepG2 and Hep3B cells with different P-glycoprotein expression and activity, were treated by acetaminophen (1-10 mM) for different time periods, with or without the P-glycoprotein inhibitor verapamil. P-Glycoprotein activity was determined by rhodamine 123 efflux assay and western blot analysis. To assess the acetaminophen-induced toxicity and effect of verapamil, we investigated cellular redox status, phosphatidylserine externalization, nuclear fragmentation and ultrastructural changes. Verapamil markedly enhanced acetaminophen-induced oxidative damage and cell death. Moreover, verapamil revealed acetaminophen toxicity even at subtoxic levels. High acetaminophen concentrations increased P-glycoprotein activity and content in both HepG2 and Hep3B cells. These observations suggest the involvement of P-glycoprotein in acetaminophen transport. Notwithstanding the differences of the investigated hepatoma cell lines in P-glycoprotein function, acetaminophen-induced toxicity was similar, possibly due to different functions of drug-metabolizing systems. We conclude that acetaminophen is a P-glycoprotein substrate and P-glycoprotein is involved in acetaminophen transport and toxicity in HepG2 and Hep3B cells. This study establishes the fact that acetaminophen can modulate P-glycoprotein in tumour cells, suggesting that its routine use in cancer patients in combination with anticancer drugs, may influence the result of chemotherapy.

  4. Clinical relevance of P-glycoprotein expression in haematological malignancies

    NARCIS (Netherlands)

    K. Nooter (Kees); P. Sonneveld (Pieter)

    1994-01-01

    textabstractAlthough, generally speaking, haematological malignancies are chemotherapy-responsive tumours and high remission induction rates are obtained, disease-related death is the rule rather than the exception. The appearance of cell populations, resistant to multidrug-based chemotherapy,

  5. Interrogation of multidrug resistance (MDR1) P-glycoprotein (ABCB1) expression in human pancreatic carcinoma cells: correlation of 99mTc-Sestamibi uptake with western blot analysis.

    Science.gov (United States)

    Harpstrite, Scott E; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

    2014-10-01

    Histopathological studies indicate that ∼63% of pancreatic tumors express multidrug resistance (MDR1) P-glycoprotein (Pgp) and its polymorphic variants. However, Pgp expression detected at the mRNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations and the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate the status of the functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using Tc-Sestamibi were performed and correlated with western blot analysis. Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug-sensitive KB-3-1 cells, and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug-resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Tc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp inhibitor, and correlate with western blot analysis. These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from the pancreatic duct and Tc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas.

  6. 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

  7. β-elemene reverses the drug resistance of A549/DDP lung cancer cells by activating intracellular redox system, decreasing mitochondrial membrane potential and P-glycoprotein expression, and inducing apoptosis.

    Science.gov (United States)

    Yao, Chengcai; Jiang, Jie; Tu, Yuanrong; Ye, Shefang; Du, Haoxin; Zhang, Yi

    2014-07-01

    β-elemene (β-ELE) injection is a new anticancer drug extracted from Curcuma zedoaria Roscoe that has been widely used to treat malignant tumors. Recent studies show that β-ELE reverses the drug resistance of tumor cells. To explore the possible mechanisms of β-ELE, we investigated its effects on cisplatin (DDP)-resistant human lung adenocarcinoma A549/DDP cells. The effects of β-ELE on the growth of A549/DDP cells in vitro were determined by MTT assay. Apoptosis was assessed by fluorescence microscopy with Hoechst 33258 staining, flow cytometry with Annexin V-FITC/propium iodide double staining; mitochondrial membrane potential using JC-1 fluorescence probe and laser confocal scanning microscopy, and intracellular reactive oxygen species levels were measured by 2',7'-dichlorfluorescein-diacetate staining and flow cytometry; and contents of cytosolic glutathione were determined by glutathione assay kits. Intracellular Rhodamine-123 fluorescence intensity was detected by flow cytometry, and the expression of P-glycoprotein (P-gp) was detected by Western blotting. β-ELE inhibited the proliferation of A549/DDP cells in a time- and dose-dependent manner. Furthermore, β-ELE enhanced the sensitivity of A549/DDP cells to cisplatin and reversed the drug resistance of A549/DDP cells. Consistent with a role in activating apoptosis, β-ELE decreased mitochondrial membrane potential, increased intracellular reactive oxygen species concentration and intracellular accumulation of Rhodamine-123, decreased the cytoplasmic glutathione levels and the expression of P-gp in a time- and dose-dependent manner. These results define a pathway of β-ELE function that involves decreased mitochondrial membrane potential and P-gp expression activated intracellular redox system, and induced apoptosis leading to reverse drug resistance.

  8. Characterization of P-glycoprotein and multidrug resistance proteins in rat kidney and intestinal cell lines.

    NARCIS (Netherlands)

    Water, F.M. van de; Boleij, J.M.; Peters, J.G.P.; Russel, F.G.M.; Masereeuw, R.

    2007-01-01

    The activity of P-glycoprotein (Pgp/MDR1/ABCB1) and multidrug resistance proteins (MRP/ABCC) influence the pharmacokinetics and bioavailability of many drugs. Few suitable cell lines for the study of drug transport exist. Additional non-human cell lines may help clarify species differences and

  9. Increased Expression of P-Glycoprotein Is Associated With Chlorpyrifos Resistance in the German Cockroach (Blattodea: Blattellidae).

    Science.gov (United States)

    Hou, Weiyuan; Jiang, Chu; Zhou, Xiaojie; Qian, Kun; Wang, Lei; Shen, Yanhui; Zhao, Yan

    2016-12-01

    A principal method for control of the German cockroach, Blattella germanica (L.), is the broad-spectrum organophosphorus insecticide, chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate); however, extensive and repeated application has resulted in the development of resistance to chlorpyrifos in this insect. Evidence suggests that ATP-binding cassette protein transporters, including P-glycoprotein, are involved in insecticide resistance. However, little is known of the role of P-glycoprotein in insecticide resistance in the German cockroach. Here, we developed a chlorpyrifos-resistant strain of German cockroach and investigated the relationship between P-glycoprotein and chlorpyrifos resistance using toxicity assays; inhibition studies with two P-glycoprotein inhibitors, verapamil and quinine; P-glycoprotein-ATPase activity assays; and western blotting analysis. After 23 generations of selection from susceptible strain cockroaches, we obtained animals with high resistance to chlorpyrifos. When P-glycoprotein-ATPase activity was inhibited by verapamil and quinine, we observed enhanced susceptibility to chlorpyrifos in both control and chlorpyrifos-resistant cockroaches. No significant alterations of P-glycoprotein expression or ATPase activity were observed in cockroaches acutely exposed to LD50 doses of chlorpyrifos for 24 h, while P-glycoprotein expression and ATPase activity were clearly elevated in the chlorpyrifos-resistant cockroach strain. Thus, we conclude that P-glycoprotein is associated with chlorpyrifos resistance in the German cockroach and that elevated levels of P-glycoprotein expression and ATPase activity may be an important mechanism of chlorpyrifos resistance in the German cockroach. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Resistance to paclitaxel in a cisplatin-resistant ovarian cancer cell line is mediated by P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Britta Stordal

    Full Text Available The IGROVCDDP cisplatin-resistant ovarian cancer cell line is also resistant to paclitaxel and models the resistance phenotype of relapsed ovarian cancer patients after first-line platinum/taxane chemotherapy. A TaqMan low-density array (TLDA was used to characterise the expression of 380 genes associated with chemotherapy resistance in IGROVCDDP cells. Paclitaxel resistance in IGROVCDDP is mediated by gene and protein overexpression of P-glycoprotein and the protein is functionally active. Cisplatin resistance was not reversed by elacridar, confirming that cisplatin is not a P-glycoprotein substrate. Cisplatin resistance in IGROVCDDP is multifactorial and is mediated in part by the glutathione pathway and decreased accumulation of drug. Total cellular glutathione was not increased. However, the enzyme activity of GSR and GGT1 were up-regulated. The cellular localisation of copper transporter CTR1 changed from membrane associated in IGROV-1 to cytoplasmic in IGROVCDDP. This may mediate the previously reported accumulation defect. There was decreased expression of the sodium potassium pump (ATP1A, MRP1 and FBP which all have been previously associated with platinum accumulation defects in platinum-resistant cell lines. Cellular localisation of MRP1 was also altered in IGROVCDDP shifting basolaterally, compared to IGROV-1. BRCA1 was also up-regulated at the gene and protein level. The overexpression of P-glycoprotein in a resistant model developed with cisplatin is unusual. This demonstrates that P-glycoprotein can be up-regulated as a generalised stress response rather than as a specific response to a substrate. Mechanisms characterised in IGROVCDDP cells may be applicable to relapsed ovarian cancer patients treated with frontline platinum/taxane chemotherapy.

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

    Science.gov (United States)

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

    2015-02-11

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

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Structure and expression of the human MDR (P-glycoprotein) gene family.

    OpenAIRE

    Chin, J E; Soffir, R; Noonan, K E; Choi, K.; Roninson, I B

    1989-01-01

    The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragmen...

  14. P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway

    Science.gov (United States)

    Galski, Hanan; Oved-Gelber, Tamar; Simanovsky, Masha; Lazarovici, Philip; Gottesman, Michael M.; Nagler, Arnon

    2014-01-01

    The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. PMID:23774624

  15. P-Glycoprotein Induction Ameliorates Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells

    Science.gov (United States)

    Lee, Sun-hyo; Kim, Jin-sun; Ravichandran, Kameswaran; Gil, Hyo-Wook; Song, Ho-yeon; Hong, Sae-yong

    2015-01-01

    The pathogenesis of colistin induced nephrotoxicity is poorly understood. Currently there are no effective therapeutic or prophylactic agents available. This study was aimed to determine the mechanism of colistin induced nephrotoxicity and to determine whether P-glycoprotein (P-gp) induction could prevent colistin induced nephrotoxicity. Colistin induced cell toxicity in cultured human proximal tubular cells in both dose and time dependent manner. Colistin provoked ROS in a dose dependent manner as measured by DCF-DA. To investigate apoptosis, caspase 3/7 activity was determined. Caspase 3/7 activity was increased dose dependently (25, 50, 100 μg/ml) at 6 h. Autophagosome formation was assessed by measuring LC3- II/LC3-I ratio. The ratio of LC3-II to LC3- I was increased at 2 h (25 μg/ml). Suppression of autophagosome formation increased colistin induced nephrotoxicity. The expression of P-gp and the cell toxicity was determined in colistin with or without dexamethasone (P-gp inducer) and verapamil (selective P-gp inhibitor). Colistin itself suppressed the expression of P-gp. P-gp expression and activity decreased colistin induced nephrotoxicity with dexamethasone treatment. In addition induced P-gp transporter was shown to improve the efflux effect on colistin treated HK2 cell line, which was demonstrated by calcein-AM fluorescence accumulation assay. The increased activity could be blocked by N-acetylcysteine. In conclusion, colistin induces nephrotoxicity by suppressing P-gp. Induction of P-gp could ameliorate colistin induced nephrotoxicity by decreasing apoptosis. PMID:26287374

  16. P-Glycoprotein Induction Ameliorates Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells.

    Directory of Open Access Journals (Sweden)

    Sun-hyo Lee

    Full Text Available The pathogenesis of colistin induced nephrotoxicity is poorly understood. Currently there are no effective therapeutic or prophylactic agents available. This study was aimed to determine the mechanism of colistin induced nephrotoxicity and to determine whether P-glycoprotein (P-gp induction could prevent colistin induced nephrotoxicity. Colistin induced cell toxicity in cultured human proximal tubular cells in both dose and time dependent manner. Colistin provoked ROS in a dose dependent manner as measured by DCF-DA. To investigate apoptosis, caspase 3/7 activity was determined. Caspase 3/7 activity was increased dose dependently (25, 50, 100 μg/ml at 6 h. Autophagosome formation was assessed by measuring LC3- II/LC3-I ratio. The ratio of LC3-II to LC3- I was increased at 2 h (25 μg/ml. Suppression of autophagosome formation increased colistin induced nephrotoxicity. The expression of P-gp and the cell toxicity was determined in colistin with or without dexamethasone (P-gp inducer and verapamil (selective P-gp inhibitor. Colistin itself suppressed the expression of P-gp. P-gp expression and activity decreased colistin induced nephrotoxicity with dexamethasone treatment. In addition induced P-gp transporter was shown to improve the efflux effect on colistin treated HK2 cell line, which was demonstrated by calcein-AM fluorescence accumulation assay. The increased activity could be blocked by N-acetylcysteine. In conclusion, colistin induces nephrotoxicity by suppressing P-gp. Induction of P-gp could ameliorate colistin induced nephrotoxicity by decreasing apoptosis.

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

    Directory of Open Access Journals (Sweden)

    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. Targeted inhibitors of P-glycoprotein increase chemotherapeutic-induced mortality of multidrug resistant tumor cells.

    Science.gov (United States)

    Nanayakkara, Amila K; Follit, Courtney A; Chen, Gang; Williams, Noelle S; Vogel, Pia D; Wise, John G

    2018-01-17

    Overexpression of ATP-binding cassette (ABC) transporters is often linked to multidrug resistance (MDR) in cancer chemotherapies. P-glycoprotein (P-gp) is one of the best studied drug transporters associated with MDR. There are currently no approved drugs available for clinical use in cancer chemotherapies to reverse MDR by inhibiting P-glycoprotein. Using computational studies, we previously identified several compounds that inhibit P-gp by targeting its nucleotide binding domain and avoiding its drug binding domains. Several of these compounds showed successful MDR reversal when tested on a drug resistant prostate cancer cell line. Using conventional two-dimensional cell culture of MDR ovarian and prostate cancer cells and three dimensional prostate cancer microtumor spheroids, we demonstrated here that co-administration with chemotherapeutics significantly decreased cell viability and survival as well as cell motility. The P-gp inhibitors were not observed to be toxic on their own. The inhibitors increased cellular retention of chemotherapeutics and reporter compounds known to be transport substrates of P-gp. We also showed that these compounds are not transport substrates of P-gp and that two of the three inhibit P-gp, but not the closely related ABC transporter, ABCG2/BCRP. The results presented suggest that these P-gp inhibitors may be promising leads for future drug development.

  19. Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

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    Guillet Catherine

    2002-07-01

    Full Text Available Abstract Background The P-glycoprotein (P-gp, an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. Results Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF. As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. Conclusions These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

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

    Science.gov (United States)

    Zhang, Yue-Hong; Li, Juan; Yang, Wei-Zhong; Xian, Zhuan-Hua; Feng, Qi-Ting; Ruan, Xiang-Cai

    2017-01-01

    To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein (P-gp) in mitochondria of D407 cells. 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. 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. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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

    2017-07-11

    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.

  3. Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif.

    Science.gov (United States)

    Ye, Siying; MacEachran, Daniel P; Hamilton, Joshua W; O'Toole, George A; Stanton, Bruce A

    2008-09-01

    P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

  4. Gastrointestinal Hormone Cholecystokinin Increases P-Glycoprotein Membrane Localization and Transport Activity in Caco-2 Cells.

    Science.gov (United States)

    Yano, Kentaro; Shimizu, Saori; Tomono, Takumi; Ogihara, Takuo

    2017-09-01

    It was reported that stimulation of taste receptor type 2 member 38 by a bitter substance, phenylthiocarbamide (PTC), increased P-glycoprotein (P-gp) mRNA level and transport activity via release of the gastrointestinal hormone cholecystokinin-8 (CCK-8) at 9 h. Therefore, we hypothesized that CCK-8 and PTC might also regulate P-gp activity more rapidly via a different mechanism. As a result, we found that the pretreatment of human colon adenocarcinoma (Caco-2) cells with 10-mM PTC significantly decreased the intracellular accumulation of P-gp substrate rhodamine 123 (Rho123) compared with the control after 90-min incubation. Moreover, CCK-8 treatments significantly reduced the accumulation of Rho123 within 30 min, compared with the control. On the other hand, when Caco-2 cells were pretreated with PTC, the efflux ratio of Rho123 was significantly increased compared with control. The efflux ratio of Rho123 in CCK-8 treatment cells was also significantly increased compared with control. Furthermore, CCK-8 increased the phosphorylation of the scaffold proteins ezrin, radixin, and moesin, which regulate translocation of P-gp to the plasma membrane. Therefore, our results indicate that PTC induced release of CCK-8, which in turn induced the phosphorylation of ezrin, radixin, and moesin proteins, leading to upregulation of P-gp transport activity via increased membrane localization of P-gp. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. 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

  6. Intestinal permeability and P-glycoprotein-mediated efflux transport of ticagrelor in Caco-2 monolayer cells.

    Science.gov (United States)

    Marsousi, Niloufar; Doffey-Lazeyras, Fabienne; Rudaz, Serge; Desmeules, Jules A; Daali, Youssef

    2016-12-01

    Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P-glycoprotein (P-gp)-mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco-2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P-gp inhibitor valspodar. Ticagrelor presented an apical-basolateral apparent permeability coefficient (Papp ) of 6.0 × 10(-6) cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P-gp in its oral disposition. Co-incubation of the P-gp inhibitor decreased the efflux Papp of ticagrelor from 1.60 × 10(-5) to 1.13 × 10(-5) cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P-gp across the Caco-2 cell monolayer. The co-administration of ticagrelor with a P-gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients. © 2016 Société Française de Pharmacologie et de Thérapeutique.

  7. Guggulsterone of Commiphora mukul resin reverses drug resistance in imatinib-resistant leukemic cells by inhibiting cyclooxygenase-2 and P-glycoprotein.

    Science.gov (United States)

    Xu, Hong-Bin; Xu, Lu-Zhong; Mao, Xia-Ping; Fu, Jun

    2014-06-15

    The purpose of this study was to investigate the effects of guggulsterone on cyclooxygenase-2 and P-glycoprotein mediated drug resistance in imatinib-resistant K562 cells (K562/IMA). MTT cytotoxicity assay, flow cytometry, western blot analysis, and ELISA were performed to investigate the anti-proliferative effect, the reversal action of drug resistance, and the inhibitory effect on cyclooxygenase-2, P-glycoprotein, BCR/ABL kinase, and PGE2 release in K562/IMA cells by guggulsterone. The results showed that co-administration of guggulsterone resulted in a significant increase in chemo-sensitivity of K562/IMA cells to imatinib, compared with imatinib treatment alone. Rhodamine123 accumulation in K562/IMA cells was significantly enhanced after incubation with guggulsterone (60, 120 μM), compared with untreated K562/IMA cells (p<0.05). When imatinib (1 μM) was combined with guggulsterone (60, 120 μM), the mean apoptotic population of K562/IMA cells was 15.47% and 24.91%. It was increased by 3.82 and 6.79 times, compared with imatinib (1 μM) treatment alone. Furthermore, guggulsterone had significantly inhibitory effects on the levels of cyclooxygenase-2, P-glycoprotein and prostaglandin E2. However, guggulsterone had little inhibitory effect on the activity of BCR/ABL kinase. The present study indicates guggulsterone induces apoptosis by inhibiting cyclooxygenase-2 and down-regulating P-glycoprotein expression in K562/IMA cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

  8. Definition of the domain boundaries is critical to the expression of the nucleotide-binding domains of P-glycoprotein.

    Science.gov (United States)

    Kerr, Ian D; Berridge, Georgina; Linton, Kenneth J; Higgins, Christopher F; Callaghan, Richard

    2003-11-01

    Heterologous expression of domains of eukaryotic proteins is frequently associated with formation of inclusion bodies, consisting of aggregated mis-folded protein. This phenomenon has proved a significant barrier to the characterization of domains of eukaryotic ATP binding cassette (ABC) transporters. We hypothesized that the solubility of heterologously expressed nucleotide binding domains (NBDs) of ABC transporters is dependent on the definition of the domain boundaries. In this paper we have defined a core NBD, and tested the effect of extensions to and deletions of this core domain on protein expression. Of 10 NBDs constructed, only one was expressed as a soluble protein in Escherichia coli, with expression of the remaining NBDs being associated with inclusion body formation. The soluble NBD protein we have obtained corresponds to residues 386-632 of P-glycoprotein and represents an optimally defined domain. The NBD has been isolated and purified to 95% homogeneity by a two-step purification protocol, involving affinity chromatography and gel filtration. Although showing no detectable ATP hydrolysis, the protein retains specific ATP binding and has a secondary structure compatible with X-ray crystallographic data on bacterial NBDs. We have interpreted our results in terms of homology models, which suggest that the N-terminal NBD of P-glycoprotein can be produced as a stable, correctly folded, isolate domain with judicious design of the expression construct.

  9. Balaglitazone reverses P-glycoprotein-mediated multidrug resistance via upregulation of PTEN in a PPARγ-dependent manner in leukemia cells.

    Science.gov (United States)

    Yousefi, Bahman; Azimi, Ako; Majidinia, Maryam; Shafiei-Irannejad, Vahid; Badalzadeh, Reza; Baradaran, Behzad; Zarghami, Nosratollah; Samadi, Nasser

    2017-10-01

    Multidrug resistance in tumor cells is still a big challenge in cancer treatment. Therefore, identification ofsafe and effective multidrug resistance-reversing compounds with minimal side effects is an important approach in cancer treatment. Here, we investigated the role and potential mechanisms of peroxisome proliferator-activated receptor γ in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. The effect of doxorubicin on cell viability following treatment with balaglitazone, a peroxisome proliferator-activated receptor γ agonist, was evaluated using trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Rhodamine123 assay was used to determine the activity of two common drug efflux membrane transporters P-glycoprotein and multidrug resistance protein-1. P-glycoprotein, multidrug resistance protein-1, and phosphatase and tensin homolog deleted on chromosome 10 messenger RNA/protein expression levels were measured by quantitative reverse transcription polymerase chain reaction and western blot analyses. Annexin V/fluorescein isothiocyanate assay was also employed to investigate apoptosis. We showed that balaglitazone considerably enhanced the cytotoxicity of doxorubicin. Balaglitazone also significantly downregulated P-glycoprotein expression and activity in K562/DOX cells and reduced multidrug resistance through elevation of intracellular doxorubicin in cells. Furthermore, upon balaglitazone treatment, phosphatase and tensin homolog deleted on chromosome 10 expression could be restored in K562/DOX cells in a peroxisome proliferator-activated receptor γ-dependent manner. We concluded that peroxisome proliferator-activated receptor γ agonist, balaglitazone, could reverse multidrug resistance by inducing phosphatase and tensin homolog deleted on chromosome 10 and peroxisome proliferator-activated receptor/ phosphatase and tensin homolog deleted on chromosome 10 signaling pathway. These findings suggest that

  10. EM-E-11-4 increases paclitaxel uptake by inhibiting P-glycoprotein-mediated transport in Caco-2 cells.

    Science.gov (United States)

    Liu, Qian; Sun, Hua; Chen, Xiao-Guang; Li, Yan; Chen, Hui; You, Feng; Bao, Xiu-Qi; Zhang, Dan; Shi, Jian-Gong

    2015-01-01

    P-glycoprotein (P-gp) overexpression is the main mechanism involved in chemotherapy drug resistance such as paclitaxel resistance and therapy failure. The most widely studied P-gp inhibitors still have limited ability to reverse resistance in the clinic. In this study, EM-E-11-4, a lathyrane-type diterpenoid isolated from Euphorbia micractina, was found to significantly increase paclitaxel uptake in Caco-2 cells, which functionally overexpressed P-gp. In vitro transport experiments, carried out in the Caco-2 monolayer model, indicated that EM-E-11-4 significantly reduced the efflux ratio of paclitaxel transport by inhibiting P-gp function without affecting P-gp expression. We also found that EM-E-11-4 enhanced the intracellular accumulation of paclitaxel in a dose-dependent manner by LC-MS/MS and EM-E-11-4 showed low cytotoxicity. Hence, EM-E-11-4 is an effective potential agent to reverse P-gp-mediated paclitaxel resistance by inhibiting P-gp transport function and increasing the intracellular concentration of paclitaxel.

  11. 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.

  12. 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.

  13. 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 side....... Using microarray data analysis from 145 neurologically sound adults, this study investigated the association between body mass index (BMI) and ABCB1 expression in the frontal cortex. Increasing BMI values were associated with a statistically significantly reduced expression of ABCB1. Investigation...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Increased intestinal P-glycoprotein expression and activity with progression of diabetes and its modulation by epigallocatechin-3-gallate: Evidence from pharmacokinetic studies.

    Science.gov (United States)

    Dash, Ranjeet Prasad; Ellendula, Bhanuchander; Agarwal, Milee; Nivsarkar, Manish

    2015-11-15

    The aim of this study was to evaluate the change in the expression and the activity of intestinal P-glycoprotein (efflux transporter) with progression of diabetes in rats. Diabetes was induced in Wistar rats using a combination of low dose streptozotocin along with high fat diet. The expression of intestinal P-glycoprotein significantly increased (P≤0.05) with the progression of diabetes which was inferred from the mRNA analysis of mdr1a and mdr1b genes in the ileum segment of rat intestine. Furthermore, a significant increase (P≤0.05) in Na(+)-K(+) ATPase activity was observed in the ileum segment of rat intestine with the progression of diabetes. As a result of this, a significant decrease in the intestinal uptake and peroral bioavailability of the P-glycoprotein substrates (verapamil and atorvastatin) was observed along with the progression of diabetes as compared to normal animals. To address this problem of impaired drug uptake and bioavailability, a reported P-glycoprotein inhibitor, epigallocatechin-3-gallate, was experimentally evaluated. The treatment with epigallocatechin-3-gallate resulted in significant reduction in the expression and activity of P-glycoprotein and subsequent improvement in the intestinal uptake and peroral bioavailability of both verapamil and atorvastatin in normal as well as in diabetic animals. The findings of this study rationalised the use and established the mechanism of action of epigallocatechin-3-gallate to overcome P-glycoprotein mediated drug efflux and will also be helpful in therapeutic drug monitoring in diabetes. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Aliskiren toxicity in juvenile rats is determined by ontogenic regulation of intestinal P-glycoprotein expression

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, Peter, E-mail: peterk.hoffmann@novartis.com [Preclinical Safety, Novartis Institutes for BioMedical Research, East Hanover, NJ (United States); Beckman, David; McLean, Lee Anne [Preclinical Safety, Novartis Institutes for BioMedical Research, East Hanover, NJ (United States); Yan, Jing-He [Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, NJ (United States)

    2014-02-15

    Juvenile rat toxicity studies with the direct renin inhibitor aliskiren were initiated to support treatment in the pediatric population. In Study 1, aliskiren was administered orally to juvenile rats at doses of 0, 30, 100 or 300 mg/kg/day with repeated dosing from postpartum day (PPD) 8 to PPD 35/36. In-life, clinical pathology, anatomic pathology, and toxicokinetics evaluations were performed. In Study 2, single oral doses of aliskiren (0, 100 or 300 mg/kg) were given to 14-, 21-, 24-, 28-, 31- or 36-day-old rats; in-life data and toxicokinetics were evaluated. Study 3 was a single dose (3 mg/kg i.v.) pharmacokinetic study in juvenile rats on PPD 8, 14, 21 and 28. In Study 4, naïve rats were used to investigate ontogenic changes of the multidrug-resistant protein 1 (MDR1) and the organic anion transporting polypeptide (OATP) mRNA in several organs. Oral administration of aliskiren at 100 and 300 mg/kg caused unexpected mortality and severe morbidity in 8-day-old rats. Aliskiren plasma and tissue concentrations were increased in rats aged 21 days and younger. Expression of MDR1 and OATP mRNA in the intestine, liver and brain was significantly lower in very young rats. In conclusion, severe toxicity and increased exposure in very young rats after oral administration of aliskiren are considered to be the result of immature drug transporter systems. Immaturity of MDR1 in enterocytes appears to be the most important mechanism responsible for the high exposure. - Highlights: • Aliskiren was orally administered to juvenile rats. • Unexpected severe toxicity and acute mortality occurred in rats aged 8 days. • Toxicity was associated with increased aliskiren plasma and tissue exposure. • Developmental changes of exposure correlated with ontogeny of transporters. • Immaturity of MDR1 in enterocytes causes increased exposure in very young rats.

  17. Genetic variants and increased expression of Parascaris equorum P-glycoprotein-11 in populations with decreased ivermectin susceptibility.

    Directory of Open Access Journals (Sweden)

    I Jana I Janssen

    Full Text Available Macrocyclic lactones (MLs represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum. Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and identified as orthologs of Caenorhabditis elegans Ppg-11 and an unnamed Caenorhabditis briggsae Pgp designated as Pgp-16 using phylogenetic analysis. Quantitative real-time PCR was used to compare expression between tissues. Significantly higher PeqPgp-11 expression was found in the gut for both genders, whereas for PeqPgp-16 the body wall was identified as predominant expression site. Furthermore, Pgps were analyzed regarding their participation in resistance development. Using SeqDoC analyses, Pgp-sequences of P. equorum populations with different ML susceptibility were compared. This approach revealed three single nucleotide polymorphisms (SNPs causing missense mutations in the PeqPgp-11 sequence which correlated with decreased ML susceptibility. However, no resistance associated differences in mRNA expression levels were detected between embryonated eggs of these populations. In contrast, comparison of two pre-adult groups with different ivermectin (IVM susceptibility revealed the presence of the three SNPs and in addition statistically significant PeqPgp-11 overexpression in the group of worms with reduced susceptibility. These results indicate that Pgp-11 might be involved in IVM resistance in P. equorum as it shows increased expression in an IVM exposed life-cycle stage of an IVM resistant population as well as occurrence of putatively resistance

  18. Interaction of anthelmintic drugs with P-glycoprotein in recombinant LLC-PK1-mdr1a cells.

    Science.gov (United States)

    Dupuy, Jacques; Alvinerie, Michel; Ménez, Cecile; Lespine, Anne

    2010-08-05

    Given the widespread use of formulations combining anthelmintics which are possible P-glycoprotein interfering agents, the understanding of drug interactions with efflux ABC transporters is of concern for improving anthelmintic control. We determined the ability of 14 anthelmintics from different classes to interact with abcb1a (mdr1a, P-glycoprotein, Pgp) by following the intracellular accumulation of rhodamine 123 (Rho 123), a fluorescent Pgp substrate, in LLC-PK1 cells overexpressing Pgp. The cytotoxicity of the compounds that are able to interfere with Pgp activity was evaluated in cells overexpressing Pgp and compared with parental cells using the MTS viability assay. Among all the anthelmintics used, ivermectin (IVM), triclabendazole (TCZ), triclabendazole sulfoxide (TCZ-SO), closantel (CLOS) and rafoxanide (RAF) increased the intracellular Rho 123 in Pgp overexpressing cells, while triclabendazole sulfone, albendazole, mebendazole, oxfendazole, thiabendazole, nitroxynil, levamisole, praziquantel and clorsulon failed to have any effect. The concentration needed to reach the maximal Rho 123 accumulation (E(max)) was obtained with 10 microM for IVM, 80 microM for CLOS, 40 microM for TCZ and TCZ-SO, and 80 microM for RAF. We showed that for these five drugs parental cell line was more sensitive to drug toxicity compared with Pgp recombinant cell line. Such in vitro approach constitutes a powerful tool to predict Pgp-drug interactions when formulations combining several anthelmintics are administered and may contribute to the required optimization of efficacy of anthelmintics. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  19. P-glycoprotein expression and function in patients with temporal lobe epilepsy: a case-control study.

    Science.gov (United States)

    Feldmann, Maria; Asselin, Marie-Claude; Liu, Joan; Wang, Shaonan; McMahon, Adam; Anton-Rodriguez, José; Walker, Matthew; Symms, Mark; Brown, Gavin; Hinz, Rainer; Matthews, Julian; Bauer, Martin; Langer, Oliver; Thom, Maria; Jones, Terry; Vollmar, Christian; Duncan, John S; Sisodiya, Sanjay M; Koepp, Matthias J

    2013-08-01

    Studies in rodent models of epilepsy suggest that multidrug efflux transporters at the blood-brain barrier, such as P-glycoprotein, might contribute to pharmacoresistance by reducing target-site concentrations of antiepileptic drugs. We assessed P-glycoprotein activity in vivo in patients with temporal lobe epilepsy. We selected 16 patients with pharmacoresistant temporal lobe epilepsy who had seizures despite treatment with at least two antiepileptic drugs, eight patients who had been seizure-free on antiepileptic drugs for at least a year after 3 or more years of active temporal lobe epilepsy, and 17 healthy controls. All participants had a baseline PET scan with the P-glycoprotein substrate (R)-[(11)C]verapamil. Pharmacoresistant patients and healthy controls then received a 30-min infusion of the P-glycoprotein-inhibitor tariquidar followed by another (R)-[(11)C]verapamil PET scan 60 min later. Seizure-free patients had a second scan on the same day, but without tariquidar infusion. Voxel-by-voxel, we calculated the (R)-[(11)C]verapamil plasma-to-brain transport rate constant, K1 (mL/min/cm(3)). Low baseline K1 and attenuated K1 increases after tariquidar correspond to high P-glycoprotein activity. Between October, 2008, and November, 2011, we completed (R)-[(11)C]verapamil PET studies in 14 pharmacoresistant patients, eight seizure-free patients, and 13 healthy controls. Voxel-based analysis revealed that pharmacoresistant patients had lower baseline K1, corresponding to higher baseline P-glycoprotein activity, than seizure-free patients in ipsilateral amygdala (0·031 vs 0·036 mL/min/cm(3); p=0·014), bilateral parahippocampus (0·032 vs 0·037; pEURIPIDES number 201380). Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Genetic transfer of non-P-glycoprotein-mediated multidrug resistance (MDR) in somatic cell fusion : Dissection of a compound MDR phenotype

    NARCIS (Netherlands)

    EIJDEMS, EWHM; BORST, P; JONGSMA, APM; de Jong, Steven; DEVRIES, EGE; VANGROENIGEN, M; VERSANTVOORT, CHM; NIEUWINT, AWM; BAAS, F

    1992-01-01

    A non-P-glycoprotein-mediated mechanism of multidrug resistance (non-Pgp MDR) bas been identified in doxorubicin-selected sublines of the human non-small cell lung carcinoma cell lines SW-1573. These sublines are cross-resistant to daunorubicin, VP16-213, Vinca alkaloids, colchicine, gramicidin D,

  2. Caco-2 cell methodology and inhibition of the P-glycoprotein transport of digoxin by Aloe vera juice.

    Science.gov (United States)

    Djuv, Ane; Nilsen, Odd Georg

    2008-12-01

    The aims of this study were to carry out a thorough quality control setup for essential Caco-2 cell characteristics in P-glycoprotein (P-gp) inhibition studies and to explore if Aloe vera juice (AVJ) inhibits the bidirectional transport of the P-gp substrate digoxin (30 nm). Seven AVJ concentrations (0.00001-1.0 mg/mL), anticipated to cover a clinically relevant range, were tested and digoxin apparent permeability coefficients (Papp), net Papp values (Papp(Net)) and net flux values (J(Net)) were calculated. Relevant validation parameters for P-gp inhibition studies in Caco-2 cells are suggested to include, as a minimum, an assay linearity test with and without a known P-gp inhibitor, cell cytotoxicity testing (MTT-test) for substrates and inhibitors, and cell integrity testing by TEER and mannitol transport measurements. The question is also raised whether a minimum effect of a reference P-gp inhibitor as verapamil should be demanded. Cell cytotoxicity was seen for digoxin at concentrations >or=3 microM and for AVJ at 10 mg/mL. AVJ did not inhibit the P-gp transport of digoxin in any of the concentrations tested. This indicates that AVJ is no inhibitor of the P-gp mediated transport of digoxin in vitro if AVJ is present in clinically relevant concentrations. (c) 2008 John Wiley & Sons, Ltd.

  3. Expression of multidrug resistance protein P-glycoprotein in correlation with markers of hypoxia (HIF-1α, EPO, EPO-R) in invasive breast cancer with metastasis to lymph nodes.

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    Badowska-Kozakiewicz, Anna M; Sobol, Maria; Patera, Janusz

    2017-10-01

    Overexpression of the mdr-1 gene is the earliest discovered mechanism of multidrug resistance, which is associated with P-glycoprotein (P-gp) - a cell membrane protein responsible for the efflux of drugs of various structures out of cancer cells. Although the expression of P-glycoprotein has been demonstrated in many cancer types, its relation to markers of hypoxia such as HIF-1α, EPO-R or EPO in invasive breast cancer is not well established. The aim of this research was to analyze the co-expression of P-glycoprotein and the markers of tissue hypoxia HIF-1α, EPO, and EPO-R by immunohistochemistry in invasive breast cancer classified according to the presence of steroid receptors and the HER2 receptors. Tissue samples were collected from 58 patients with the diagnosis of invasive breast cancer with lymph node metastases. The expression of P-gp, HIF-1α, EPO-R and EPO was determined by immunohistochemistry. Of all the invasive breast cancers with lymph node metastases, 15.5% expressed P-gp in cell membrane and tumor blood vessels. In our research, there was a significant positive correlation between HER2-positive tumors that did not express steroid receptors (ER-/PR-/HER2+), and P-gp expression (p = 0.049, r = 0.105). Moreover, there was a significant positive correlation between EPO expression and P-gp (p breast cancer with lymph node metastases. An important result of our study is the demonstration of a correlation between P-gp expression and patients with HER2-positive breast tumors that do not express steroid receptors.

  4. The effects of the human MDR1 genotype on the expression of duodenal P-glycoprotein and disposition of the probe drug talinolol.

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    Siegmund, Werner; Ludwig, Karen; Giessmann, Thomas; Dazert, Peter; Schroeder, Eike; Sperker, Bernhard; Warzok, Rolf; Kroemer, Heyo K; Cascorbi, Ingolf

    2002-11-01

    A single-nucleotide polymorphism (SNP) of the human multidrug-resistance gene in wobble position of exon 26 reportedly predicts expression and function of P-glycoprotein in human enterocytes and lymphocytes. Several other allelic variants of MDR1 have been identified, some of which lead to amino acid exchange with as yet unknown functional relevance. In healthy white volunteers, we investigated the influence of the hereditary variants C3435T in exon 26 and G2677T/A (Ala893Ser/Thr) in exon 21 and the influence of 7 frequent or putative functional SNPs on duodenal MDR1 messenger ribonucleic acid (n = 32) and immunoreactive P-glycoprotein (n = 37) expression. Moreover, the disposition of the probe drug talinolol was evaluated in 55 subjects after oral administration (100 mg) and in 23 subjects after intravenous administration(30 mg). Duodenal MDR1 messenger ribonucleic acid and P-glycoprotein, as assessed by real-time polymerase chain reaction (TaqMan) and immunostaining, were not influenced by any MDR1 polymorphism studied. Talinolol disposition was not affected by the exon 26 mutation C3435T. In carriers of the TT/TA variants of G2677T/A, the area under the serum concentration-time curve values of oral talinolol were slightly but significantly elevated compared with those in carriers of at least 1 wild-type allele (P disposition. We did not identify any influence of MDR1 genotypes on duodenal expression of P-glycoprotein and disposition of talinolol in humans.

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

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    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.

  6. Regulation of multidrug resistance 1 (MDR1)/P-glycoprotein gene expression and activity by heat-shock transcription factor 1 (HSF1).

    Science.gov (United States)

    Vilaboa, N E; Galán, A; Troyano, A; de Blas, E; Aller, P

    2000-08-11

    Infection of HeLa cells with adenovirus-carrying HSF1(+) cDNA, which encodes a mutated form of HSF1 with constitutive transactivation capacity, increased multidrug resistance 1 (MDR1) mRNA level and P-glycoprotein (P-gp) cell surface content and stimulated rhodamine 123 accumulation and vinblastine efflux activity. On the other hand, infection with adenovirus-carrying HSP70 and HSP27 cDNAs did not increase MDR1/P-gp expression. HSF1 regulates MDR1/P-gp expression at the transcriptional level, since HSF1(+) bound the heat-shock consensus elements (HSEs) in the MDR1 gene promoter and also activated the expression of an MDR1 promoter-driven reporter plasmid (pMDR1(-1202)). In addition, heat-shock increased pMDR1(-1202) promoter activity but not the activity of a similar reporter plasmid with point mutations at specific HSEs, and the heat-induced increase was totally inhibited by co-transfection with an expression plasmid carrying HSF1(-), a dominant negative mutant of HSF1. The stress inducers arsenite, butyrate, and etoposide also increased pMDR1(-1202) promoter activity, but the increase was not inhibited (in the case of butyrate) or was only partially inhibited (in the case of arsenite and etoposide) by HSF1(-). These results demonstrate that HSF1 regulates MDR1 expression, and that the HSEs present in the -315 to -285 region mediate the heat-induced activation of the MDR1 promoter. However, other factors may also participate in MDR1 induction by stressing agents.

  7. 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.

  8. Determination of P-glycoprotein surface expression and functional ability after in vitro treatment with darunavir or raltegravir in lymphocytes of healthy donors.

    Science.gov (United States)

    Tempestilli, Massimo; Gentilotti, Elisa; Tommasi, Chiara; Nicastri, Emanuele; Martini, Federico; De Nardo, Pasquale; Narciso, Pasquale; Pucillo, Leopoldo P

    2013-08-01

    It has been shown that P-glycoprotein (P-gp) can greatly affect the cell uptake of antiretroviral drugs, thus hampering their access to HIV-1 replication sites. Lymphocytes are important sites of replication of HIV and target of other drugs, modification on these cells of P-gp could have an effect on pharmacokinetic of antiretrovirals and drug substrates. Blood samples from 16 healthy volunteers were used to determine the expression of P-gp on total, T and T helper lymphocytes after exposure to darunavir, a second generation protease inhibitor, and raltegravir, the first approved integrase inhibitor. Moreover, the effect of the drugs on P-gp functional activity was also studied by the rhodamine-123 efflux test. Darunavir, but not raltegravir, exposure caused a moderate, dose-dependent increment in P-gp expression in total, T and T helper lymphocytes, as demonstrated by the relative frequency of P-gp+ cells and by the amount of P-gp molecules present on cell surface. Functionally, incubation with darunavir led to a marked inhibition of P-gp activity measured by the efflux of rhodamine-123 similar to that observed by verapamil, a specific P-gp inhibitor. Raltegravir was not able to modify the efflux of rhodamine-123 level. Data show that darunavir, unlike raltegravir, may modify the expression and functionality of P-gp on human lymphocytes, thus leading to potential changes in intracellular concentrations of darunavir in patients treated with other drugs substrate of P-gp and vice versa. Our study highlights the need for studies on drug interactions via the P-gp modulation mechanism, especially with the current multi-drug regimens. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Detection of Glycomic Alterations Induced by Overexpression of P-Glycoprotein on the Surfaces of L1210 Cells Using Sialic Acid Binding Lectins

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    Albert Breier

    2012-11-01

    Full Text Available P-glycoprotein (P-gp overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells by selection with vincristine (R cells or transfection with the gene encoding human P-gp (T cells. Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA, wheat germ agglutinin (WGA and Maackia amurensis agglutinin (MAA. Sialic acid is predominantly linked to the surface of S, R and T cells via α-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via α-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase.

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

    Science.gov (United States)

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

    2016-01-01

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

  11. 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 < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  12. 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.

  13. P-glycoprotein mediated efflux in Caco-2 cell monolayers: the influence of herbals on digoxin transport.

    Science.gov (United States)

    Oga, Enoche F; Sekine, Shuichi; Shitara, Yoshihisa; Horie, Toshiharu

    2012-12-18

    Several herbal medicines are concomitantly used with conventional medicines with a resultant increase in the recognition of herb-drug interactions. The phytomedicines Vernonia amygdalina Delile (VA), family Asteraceae; Azadiractha indica A. Juss (NL), family Meliaceae; Morinda lucida Benth (MLB), family Rubiaceae; Cymbopogon citratus Stapf (LG), family Poaceae; Curcuma longa L. (CUR), family Zingiberaceae; Carica papaya L. (CP), family Caricaceae and Tapinanthus sessilifolius Blume (ML), family Loranthaceae are used in African traditional medicine for the treatment of malaria. They are also used in several regions world over in managing other ailments like cancer and diabetes. This study investigated their interaction with digoxin (DIG) with a view to predict the potential of P-glycoprotein (p-gp) mediated drug-herb interactions occurring with p-gp substrate drugs. To assess p-gp mediated transport and inhibition, bidirectional transport studies were carried out on Caco-2 cell monolayers using digoxin (DIG) as a model p-gp substrate. Cell functionality was demonstrated using the determinations of transepithelial electric resistance (TEER), cell cytotoxicity testing utilizing the MTT assay as well as the inclusion of inhibition controls. Under the conditions of this study, extracts of ML, VA and CP showed significant inhibition to (3)H-Digoxin basolateral-to-apical (B-A) transport at 0.02-20mg/mL; the concentrations examined. Their apical-to-basolateral (A-B) transport was further investigated. Increases in the mean A-B transport and significant decreases in the B-A transport and efflux ratio values were observed. The apparent permeability coefficient and efflux ratio were computed providing an estimate of drug absorption. The findings show that extracts of ML, VA and CP significantly inhibit p-gp in vitro and interactions with conventional p-gp substrate drugs are likely to occur on co-administration which may result in altered therapeutic outcomes. Copyright

  14. P-glycoprotein attenuates DNA repair activity in multidrug-resistant cells by acting through the Cbp-Csk-Src cascade.

    Science.gov (United States)

    Lin, Li-Fang; Wu, Ming-Hsi; Pidugu, Vijaya Kumar; Ho, I-Ching; Su, Tsann-Long; Lee, Te-Chang

    2017-07-11

    Recent studies have demonstrated that P-glycoprotein (P-gp) expression impairs DNA interstrand cross-linking agent-induced DNA repair efficiency in multidrug-resistant (MDR) cells. To date, the detailed molecular mechanisms underlying how P-gp interferes with Src activation and subsequent DNA repair activity remain unclear. In this study, we determined that the C-terminal Src kinase-binding protein (Cbp) signaling pathway involved in the negative control of Src activation is enhanced in MDR cells. We also demonstrated that cells that ectopically express P-gp exhibit reduced activation of DNA damage response regulators, such as ATM, Chk2, Braca1 and Nbs1 and hence attenuated DNA double-strand break repair capacity and become more susceptible than vector control cells to DNA interstrand cross-linking (ICL) agents. Moreover, we demonstrated that P-gp can not only interact with Cbp and Src but also enhance the formation of inhibitory C-terminal Src kinase (Csk)-Cbp complexes that reduce phosphorylation of the Src activation residue Y416 and increase phosphorylation of the Src negative regulatory residue Y527. Notably, suppression of Cbp expression in MDR cells restores cisplatin-induced Src activation, improves DNA repair capacity, and increases resistance to ICL agents. Ectopic expression of Cbp attenuates cisplatin-induced Src activation and increases the susceptibility of cells to ICL agents. Together, the current results indicate that P-gp inhibits DNA repair activity by modulating Src activation via Cbp-Csk-Src cascade. These results suggest that DNA ICL agents are likely to have therapeutic potential against MDR cells with P-gp-overexpression.

  15. P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

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    Di Pietro A.

    1999-01-01

    Full Text Available Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp, a plasma membrane ATP-binding cassette (ABC transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR. In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

  16. CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

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    Leyla Norouzi-Barough

    2018-02-01

    Full Text Available Objective(s: Multidrug resistance (MDR is a major obstacle in the successful chemotherapy of ovarian cancer. Inhibition of P-glycoprotein (P-gp, a member of ATP-binding cassette (ABC transporters, is a well-known strategy to overcome MDR in cancer. The aim of this study was to investigate the efficiency and ability of CRISPR/Cas9 genome editing technology to knockdown ABCB1 gene expression in adriamycin resistant (A2780/ADR ovarian cancer cell line and evaluate the sensitivity changes to doxorubicin. Materials and Methods: Three single-guide RNAs (sgRNAs targeting the fourth and fifth exons of human ABCB1 gene were designed in this study. Expression level of ABCB1 was detected using quantitative real time PCR (qRT-PCR after co-transfection of all three sgRNAs into A2780/ADR cell line and subsequent antibiotic selection. Drug sensitivity to doxorubicin was determined by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Results: The results showed that CRISPR/Cas9 system could significantly reduce the expression of P-gp. The dramatic decline in ABCB1 gene expression was associated with increased sensitivity of cells transfected with sgRNAs to doxorubicin. Conclusion: Based on the results of this study, it is concluded that the CRISPR-based systems, used in the present study, effectively down-regulated the target gene and acted as an ideal and cost-effective tool for gene editing of A2780/ADR cell line resulting in restoration of nonmalignant phenotype.

  17. Cyclohexylpiperazine derivative PB28, a sigma2 agonist and sigma1 antagonist receptor, inhibits cell growth, modulates P-glycoprotein, and synergizes with anthracyclines in breast cancer.

    Science.gov (United States)

    Azzariti, Amalia; Colabufo, Nicola A; Berardi, Francesco; Porcelli, Letizia; Niso, Mauro; Simone, Grazia M; Perrone, Roberto; Paradiso, Angelo

    2006-07-01

    sigma Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-independent apoptosis, and to down-regulate P-glycoprotein (P-gp) mRNA levels in some mouse and human models. In this study, we verified whether a mixed sigma(2) agonist/sigma(1) antagonist, PB28, was able to have antitumor activity and to enhance anthracycline efficacy in two human breast cancer cell lines, MCF7 and MCF7 ADR, both characterized by significant sigma(2) receptor expression, by high and low sigma(1) receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high sigma(2) receptor affinity and low sigma(1) receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC(50) in nanomolar range), a consistent time exposure-independent increase of G(0)-G(1)-phase fraction (of approximately 20% of both cell lines) and caspase-independent apoptosis (15% increased after 1-day drug exposure). PB28 also reduced P-gp expression in a concentration- and time-dependent manner ( approximately 60% in MCF7 and 90% in MCF7 ADR). We showed also a strong synergism between PB28 and doxorubicin by adopting either simultaneous or sequential schedules of the two drugs. We suggest that this synergism could depend on PB28-induced increase of intracellular accumulation of doxorubicin ( approximately 50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the sigma(2) agonist PB28 could be an interesting antitumor agent either in monotherapy or in combination with conventional drugs.

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

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    Chen, Yan; Huang, Xian-Jing; Yu, Nian; Xie, Yuan; Zhang, Kang; Wen, Fang; Liu, Hao; Di, Qing

    2015-01-01

    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. PMID:26485677

  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. Uptake characteristics of pinocembrin and its effect on p-glycoprotein at the blood-brain barrier in in vitro cell experiments.

    Science.gov (United States)

    Yang, Zhi-Hong; Sun, Xiao; Qi, Yun; Mei, Chao; Sun, Xiao-Bo; Du, Guan-Hua

    2012-01-01

    One purpose of the present study was to investigate the uptake characteristics of pinocembrin (PCB) and its effect on p-glycoprotein (P-gp) at the blood-brain barrier (BBB). Cultured rat brain microvascular endothelial cells (rBMECs) were used as an in vitro BBB model. Experiments were conducted to examine time-, concentration-, and temperature-dependent elements of PCB uptake, and the effect of classical P-gp inhibitors, cyclosporin A (CsA) and verapamil (Ver), on the steady-state uptake of PCB. Uptake of rhodamine 123 (Rho123), the typical P-gp substance, was measured with or without PCB. Meanwhile, the protein level of P-gp after incubation with PCB was detected by Western blot assay. The results demonstrated that PCB uptake by rBMECs was in a time- and concentration-dependent manner. CsA and Ver slightly increased PCB steady-state uptake by less than 10% (p>0.05). Similar results were observed in Rho123 uptake by co-administration of PCB. Further results were obtained by Western blot assay. PCB might not affect P-gp expression in rBMECs. Overall, the findings demonstrate that the passive transport process may be the main process for PCB to pass through the BBB, and P-gp is likely to have a little effect on the PCB transport process. Furthermore, PCB may not affect the functional activity and the protein expression of the P-gp transporter at the BBB.

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

    Science.gov (United States)

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

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. The effect of maternal obesity on the expression and functionality of placental P-glycoprotein: Implications in the individualized transplacental digoxin treatment for fetal heart failure.

    Science.gov (United States)

    Wang, Chuan; Li, Huaying; Luo, Chunyan; Li, Yifei; Zhang, Yi; Yun, Ding; Mu, Dezhi; Zhou, Kaiyu; Hua, Yimin

    2015-10-01

    Placental P-glycoprotein (P-gp) plays a significant role in controlling digoxin transplacental rate. Investigations on P-gp regulation in placenta of women with different pregnant pathology are of great significance to the individualized transplacental digoxin treatment for fetal heart failure (FHF). This study aimed to explore the effect of maternal obesity on the expression and functionality of placental P-gp both in human and in mice. Placenta tissues from obese and lean women were collected. Female C57BL mice were fed with either a normal chow diet or a high-fat diet for 12 weeks before mating and throughout pregnancy. Maternal plasma glucose, HDL-C, LDL-C, TC, TGs, insulin, IL-1β, IL-6 and TNF-α concentrations was detected. Placental ABCB1/Abcb1a/Abcb1b/IL-1β/IL-6/TNF-α mRNA and P-gp/IL-1β/IL-6/TNF-α protein expression were determined by real-time quantitative PCR and western-blot, respectively. Maternal plasma and fetal-unit digoxin concentrations were detected by a commercial kit assay. Both ABCB1 gene mRNA and protein expression of obesity group was significantly lower than that of control group in human. The high-fat dietary intervention resulted in an overweight phenotype, a significant increased Lee's index, higher levels of plasma glucose, HDL-C, LDL-C, insulin and TGs, increased peri-renal and peri-reproductive gland adipose tissue weight, and larger size of adipose cell. Compared with control group at the same gestational day (E12.5, E15.5, E17.5), placental Abcb1a mRNA and P-gp expression of obese group were significantly decreased in mice, while digoxin transplacental rates were significantly increased. Higher maternal plasma IL-1β/TNF-α concentrations and placental IL-1β/TNF-α expression were observed in obesity groups in comparison with control group at the same gestational age. Maternal obesity could inhibit placental P-gp expression and its functionality both in human and in mice, which might be resulted from a heightened inflammatory

  3. 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.

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

    Science.gov (United States)

    Sivak, Ladislav; Subr, Vladimir; Tomala, Jakub; Rihova, Blanka; Strohalm, Jiri; Etrych, Tomas; Kovar, Marek

    2017-01-01

    Multidrug resistance (MDR) is a common cause of failure in chemotherapy for malignant diseases. MDR is either acquired as a result of previous repeated exposure to cytostatic drugs (P388/MDR cells) or naturally, as some tumors are congenitally resistant to chemotherapy (CT26 cells). One of the most common mechanisms of MDR is upregulation of P-glycoprotein (P-gp) expression. Here, we used HPMA copolymer conjugates, whereby the cytostatic drug doxorubicin (Dox) or the derivative of the P-gp inhibitor reversin 121 (R121) or both were covalently bound through a degradable pH-sensitive hydrazone bond. We proved that R121, when bound to a polymeric carrier, is capable of inhibiting P-gp in P388/MDR cells and sensitizing them in relation to the cytostatic activity of Dox. Conjugate bearing both Dox and R121 was found to be far more potent in P388/MDR cells than conjugate bearing Dox alone or a mixture of conjugates bearing either Dox or R121 when cytostatic activity in vitro, cell cycle arrest, accumulation of Dox in cells and induction of apoptosis were determined. Importantly, conjugate bearing R121 is also effective in vivo as it inhibits P-gp in P388/MDR tumors after intraperitoneal administration, while both the conjugate bearing Dox and R121 induces apoptosis in P388/MDR tumors more effectively than conjugate bearing Dox alone. Only conjugate bearing Dox and R121 significantly inhibited P388/MDR tumor growth and led to the prolonged survival of treated mice. However, the most dramatic antitumor activity of this conjugate was found in the CT26 tumor model where it completely cured six out of eight experimental mice, while conjugate bearing Dox alone cured no mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  6. 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

  7. Astrocyte-mediated regulation of multidrug resistance p-glycoprotein in fetal and neonatal brain endothelial cells: age-dependent effects.

    Science.gov (United States)

    Baello, Stephanie; Iqbal, Majid; Gibb, William; Matthews, Stephen G

    2016-08-01

    Brain endothelial cells (BECs) form a major component of the blood-brain barrier (BBB). In late gestation, these cells express high levels of the multidrug transporter p-glycoprotein (P-gp; encoded by Abcb1), which prevents the passage of an array of endogenous factors and xenobiotics into the fetal brain. P-gp levels in the BECs increase dramatically in late gestation, coincident with astrocyte differentiation. However, the role of astrocytes in modulating P-gp in the developing BBB is unknown. We hypothesized that factors produced by astrocytes positively regulate P-gp in BECs. Astrocytes and BECs were isolated from fetal and postnatal guinea pigs. Levels of Abcb1 mRNA and P-gp were increased in BECs co-cultured with astrocytes compared to BECs in monoculture. Moreover, postnatal astrocytes enhanced P-gp function in fetal BECs but fetal astrocytes had no effect on postnatal BECs. These effects were dependent on secreted proteins with a molecular weight in the range of 3-100 kDa. LC/MS-MS revealed significant differences in proteins secreted by fetal and postnatal astrocytes. We propose that astrocytes are critical modulators of P-gp at the developing BBB. As such, aberrations in astrocyte maturation, observed in neurodevelopmental disorders, will likely decrease P-gp at the BBB. This would allow increased transfer of P-gp endogenous and exogenous substrates into the brain, many of which have neurodevelopmental consequences. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  8. 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.

  9. 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.

  10. 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...

  11. Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy.

    Science.gov (United States)

    Gervasoni, J E; Fields, S Z; Krishna, S; Baker, M A; Rosado, M; Thuraisamy, K; Hindenburg, A A; Taub, R N

    1991-09-15

    Four well defined multidrug-resistant cell lines and their drug-sensitive counterparts were examined for intracellular distribution of daunorubicin (DNR) by laser-assisted confocal fluorescence microscopy: P-glycoprotein-negative HL-60/AR cells, and P-glycoprotein-positive P388/ADR, KBV-1, and MCF-7/ADR cells. Both drug sensitive cell lines (HL-60/S, P388/S, KB3-1, and MCF-7/S) and drug-resistant cell lines (HL-60/AR, P388/ADR, KBV-1, and MCF-7/ADR) exposed to DNR showed a similar rapid distribution of drug from the plasma membrane to the perinuclear region within the first 2 min. From 2-10 min, the drug sensitive HL-60/S, P388/S, and MCF-7/S cells redistributed drug to the nucleus and to the cytoplasm in a diffuse pattern. In contrast, drug-resistant HL-60/AR, P388/ADR, and MCF-7/ADR redistributed DNR from the perinuclear region into vesicles distinct from nuclear structures, thereby assuming a "punctate" pattern. This latter redistribution could be inhibited by glucose deprivation (indicating energy dependence), or by lowering the temperature of the medium below 18 degrees C. The differences in distribution between sensitive and resistant cells did not appear to be a function of intracellular DNR content, nor the result of drug cytotoxicity. Drug-sensitive KB3-1 and -resistant KBV-1 cells did not fully follow this pattern in that they demonstrated an intracellular DNR distribution intermediate between HL-60/S and HL-60/AR cells with both "punctate" and nuclear/cytoplasmic uptake sometimes in the same cell. These data indicate that the intracellular distribution of DNR is an important determinant of drug resistance regardless of the overexpression of P-glycoprotein. The intracellular movement of drug requires the presence of glucose and a temperature above 18 degrees C, implicating energy-dependent processes and vesicle fusion in the distribution process. This intracellular transport of DNR away from the nucleus in multidrug-resistant cells may protect putative

  12. 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....

  13. 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.

  14. Absorption properties and effects of caffeic acid phenethyl ester and its p-nitro-derivative on P-glycoprotein in Caco-2 cells and rats.

    Science.gov (United States)

    Gou, Jing; Yao, Xiaofang; Tang, Hao; Zou, Kaili; Liu, Yujia; Zuo, Hua; Zhao, Xiaoyan; Li, Zhubo

    2016-12-01

    Caffeic acid phenethyl ester (CAPE), isolated from honeybee propolis, has pharmacological applications. A synthesized CAPE derivative, p-nitro-caffeic acid phenethyl ester (CAPE-NO2), showed similar activities with CAPE. The pharmacological activities of CAPE and CAPE-NO2 are related to their absorption properties. To understand the pharmacokinetic profiles of CAPE and CAPE-NO2 in rats and investigate the absorption mechanisms and effects on P-glycoprotein in Caco-2 cells. The pharmacokinetic profiles of CAPE and CAPE-NO2 were obtained after oral administration (10 mg/kg) to rats. Transport studies of CAPE and CAPE-NO2 (5, 10, 20 μM) were performed in Caco-2 cell model. P-gp activities were assayed by rhodamine 123 cellular retention. Expression of P-gp was determined after the cells were administrated with CAPE and CAPE-NO2 (5, 20 μM) for 48 and 72 h. The AUC(0-t) of CAPE-NO2 (3239.9 ± 352 ng × h/mL) was two-time greater than CAPE (1659.6 ± 152 ng × h/mL) in rats. The Papp values of CAPE and CAPE-NO2 were (4.86 ± 0.90) × 10(-6 )cm/s and (12.34 ± 1.6) × 10(-6 )cm/s, respectively. The accumulation of rhodamine 123 was increased by 1.3- to 1.9-fold and 1.4- to 2.3-fold in CAPE and CAPE-NO2 groups after 1 h administration, respectively. However, CAPE and CAPE-NO2 increased the P-gp levels by 2.1- and 1.7-fold, respectively. The absorption of CAPE-NO2 can be enhanced in rats and Caco-2 cells compared with CAPE. The two compounds are potential inhibitors of P-gp. The increased P-gp levels generated by CAPE and CAPE-NO2 played a role as a defense mechanism by limiting intracellular xenobiotic levels.

  15. Modulation of P-glycoprotein by Stemona alkaloids in human multidrug resistance leukemic cells and structural relationships.

    Science.gov (United States)

    Umsumarng, Sonthaya; Pitchakarn, Pornsiri; Yodkeeree, Supachai; Punfa, Wanisa; Mapoung, Sariya; Ramli, Rosdayati Alino; Pyne, Stephen G; Limtrakul, Pornngarm

    2017-10-15

    Multidrug resistance (MDR) is a major reason for the failure of chemotherapy in the treatment of cancer patients. P-gp over-expression in MDR cancer cells is a multifactorial phenomenon with biochemical resistance mechanisms. Stemofoline (STF), isolated from Stemona bukillii, has been reported to be an MDR reversing compound. This study investigated whether other Stemona alkaloids that had been purified from Stemonaceae plants exerted MDR modulation activity. MTT assay was performed to determine the MDR reversing property of the alkaloids. Modulation of P-gp function by these compounds was investigated using cell cycle analysis and P-gp fluorescent substrate accumulation assays. P-gp expression was determined by Western blot analysis. We preliminarily examined the safety of these compounds in normal human fibroblasts and human peripheral blood mononuclear cells (PBMCs) using the MTT assay, and in red blood cells (human and rat) through in vitro hemolysis assays. Three of the eight alkaloids tested, isostemofoline (ISTF), 11Z -didehydrostemofoline (11Z-DSTF) and 11E-didehydrostemofoline (11E-DSTF), enhanced the chemotherapeutic sensitivity of MDR leukemic K562/Adr cells, which overexpressed P-gp. The P-gp functional studies showed that these three alkaloids increased the accumulation of P-gp substrates, calcein-AM (C-AM) and rhodamine123 (Rho 123) in K562/Adr cells, while this effect was not seen in drug sensitive parental K562 cells. Whereas, the alkaloids did not alter P-gp expression as was determined by Western blotting analysis. The alkaloids reversed MDR via the inhibition of P-gp function. For pharmaceutical safety testing, the alkaloids were found to be not toxic to normal human fibroblasts and PBMCs. Moreover, the effective compounds did not induce hemolysis in either human or rat erythrocytes. These compounds may be introduced as potential candidate molecules for treating cancers exhibiting P-gp-mediated MDR. Copyright © 2017 Elsevier GmbH. All rights

  16. Comparative tissue pharmacokinetics and efficacy of moxidectin, abamectin and ivermectin in lambs infected with resistant nematodes: Impact of drug treatments on parasite P-glycoprotein expression.

    Science.gov (United States)

    Lloberas, Mercedes; Alvarez, Luis; Entrocasso, Carlos; Virkel, Guillermo; Ballent, Mariana; Mate, Laura; Lanusse, Carlos; Lifschitz, Adrian

    2013-12-01

    The high level of resistance to the macrocyclic lactones has encouraged the search for strategies to optimize their potential as antiparasitic agents. There is a need for pharmaco-parasitological studies addressing the kinetic-dynamic differences between various macrocyclic lactones under standardized in vivo conditions. The current work evaluated the relationship among systemic drug exposure, target tissue availabilities and the pattern of drug accumulation within resistant Haemonchus contortus for moxidectin, abamectin and ivermectin. Drug concentrations in plasma, target tissues and parasites were measured by high performance liquid chromatography. Additionally, the efficacy of the three molecules was evaluated in lambs infected with resistant nematodes by classical parasitological methods. Furthermore, the comparative determination of the level of expression of P-glycoprotein (P-gp2) in H. contortus recovered from lambs treated with each drug was performed by real time PCR. A longer persistence of moxidectin (P abamectin and ivermectin at day 2 post-treatment. However, the efficacy against H. contortus was 20.1% (ivermectin), 39.7% (abamectin) and 89.6% (moxidectin). Only the ivermectin treatment induced an enhancement on the expression of P-gp2 in the recovered adult H. contortus, reaching higher values at 12 and 24 h post-administration compared to control (untreated) worms. This comparative pharmacological evaluation of three of the most used macrocyclic lactones compounds provides new insights into the action of these drugs.

  17. HMGB1 regulates P-glycoprotein expression in status epilepticus rat brains via the RAGE/NF-κB signaling pathway

    Science.gov (United States)

    Xie, Yuan; Yu, Nian; Chen, Yan; Zhang, Kang; Ma, Hai-Yan; Di, Qing

    2017-01-01

    Overexpression of P-glycoprotein (P-gp) in the brain is an important mechanism involved in drug-resistant epilepsy (DRE). High-mobility group box 1 (HMGB1), an inflammatory cytokine, significantly increases following seizures and may be involved in upregulation of P-gp. However, the underlying mechanisms remain elusive. The aim of the present study was to evaluate the role of HMGB1 and its downstream signaling components, receptor for advanced glycation end-product (RAGE) and nuclear factor-κB (NF-κB), on P-gp expression in rat brains during status epilepticus (SE). Small interfering RNA (siRNA) was administered to rats prior to induction of SE by pilocarpine, to block transcription of the genes encoding HMGB1 and RAGE, respectively. An inhibitor of NF-κB, pyrrolidinedithiocarbamic acid (PDTC), was utilized to inhibit activation of NF-κB. The expression levels of HMGB1, RAGE, phosphorylated-NF-κB p65 (p-p65) and P-gp were detected by western blotting. The relative mRNA expression levels of the genes encoding these proteins were measured using reverse transcription-quantitative polymerase chain reaction and the cellular localization of the proteins was determined by immunofluorescence. Pre-treatment with HMGB1 siRNA reduced the expression levels of RAGE, p-p65 and P-gp. PDTC reduced the expression levels of P-gp. These findings suggested that overexpression of P-gp during seizures may be regulated by HMGB1 via the RAGE/NF-κB signaling pathway, and may be a novel target for treating DRE. PMID:28627626

  18. P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods

    Directory of Open Access Journals (Sweden)

    Jennifer Pasquier

    2013-01-01

    Full Text Available Background. P-gp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. Fluorescence techniques have been widely applied to measure the P-gp activity. In this paper, there is a comparison between the advantages of two fluorescence approaches of commonly available and affordable instruments: the microplate reader (MPR and the flow cytometer to detect the P-gp efflux activity using calcein-AM. Results. The selectivity, sensibility, and reproducibility of the two methods have been defined. Our results showed that the MPR is more powerful for the detection of small inhibition, whereas the flow cytometry method is more reliable at higher concentrations of the inhibitors. We showed that to determine precisely the inhibition efficacy the flow cytometry is better; hence, to get the correct Emax and EC50 values, we cannot only rely on the MPR. Conclusion. Both techniques can potentially be used extensively in the pharmaceutical industry for high-throughput drug screening and in biology laboratories for academic research, monitoring the P-gp efflux in specific assays.

  19. Pregnane X Receptor Not Nuclear Factor-kappa B Up-regulates P-glycoprotein Expression in the Brain of Chronic Epileptic Rats Induced by Kainic Acid.

    Science.gov (United States)

    Yu, Nian; Zhang, Yan-Fang; Zhang, Kang; Cheng, Yong-Fei; Ma, Hai-Yan; Di, Qing

    2017-08-01

    Drug-resistance epilepsy (DRE) is attributed to the brain P-glycoprotein (P-gp) overexpression. We previously reported that nuclear factor-kappa B (NF-κB) played a critical role in regulating P-gp expression at the brain of the acute seizure rats. This study was extended further to investigate the interaction effect of NF-κB and pregnane X receptor (PXR) on P-gp expression at the brain of chronic epileptic rats treated with carbamazepine (CBZ). The chronic epileptic models were induced by the micro-injection of kainic acid (KA) into rats' hippocampus. Subsequently, the successful models were treated with different intervention agents of CBZ; PMA(a non-specific PXR activity inhibitor) or PDTC(a specific NF-κB activity inhibitor) respectively. The expression levels of P-gp and its encoded gene mdr1a/b were significantly up-regulated on the brain of KA-induced chronic epilepsy rats or the epilepsy rats treated with CBZ for 1 week, meanwhile with a high expression of PXR. The treatment of PMA dramatically reduced both PXR and P-gp expressions at the protein and mRNA levels in the chronic epilepsy brain. By compared to the epilepsy model group, the P-gp expression was not markedly attenuated by the inhibition of NF-κB activity with PDTC treatment, nevertheless with a decrease of NF-κB expression in this intervention group. Higher levels of proinflammatory cytokines(IL-1β, IL-6, TNF-α) were found both in the brain tissue and the serum in the epilepsy rats of each group. There was a declined trend of the pro-inflammatory cytokines expression of the PDTC treatment group but with no statistical significance. This study demonstrates for the first time that P-gp up-regulation is due to increase PXR expression in the chronic phase of epilepsy, differently from that NF-κB signaling may induce the P-gp expression in the acute seizure phase. Our results offer insights into the mechanism underlying the development of DRE using or not using CBZ treatment.

  20. Effect of P-glycoprotein on flavopiridol sensitivity

    OpenAIRE

    Boerner, S. A.; Tourne, M E; Kaufmann, S H; Bible, K C

    2001-01-01

    Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC 50 s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CHRC5 (higher Pgp)...

  1. 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.

  2. TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3β pathway and activation of caspases

    Science.gov (United States)

    2010-01-01

    Background The development of new modulator possessing high efficacy, low toxicity and high selectivity is a pivotal approach to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in cancer treatment. In this study, we suggest a new molecular mechanism that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) down-regulates P-glycoprotein (P-gp) through inhibition of DNA-PKcs/Akt/GSK-3β pathway and activation of caspases and thereby sensitize MDR cells to MDR-related drugs. Results MDR variants, CEM/VLB10-2, CEM/VLB55-8 and CEM/VLB100 cells, with gradually increased levels of P-gp derived from human lymphoblastic leukemia CEM cells, were gradually more susceptible to TRAIL-induced apoptosis and cytotoxicity than parental CEM cells. The P-gp level of MDR variants was positively correlated with the levels of DNA-PKcs, pAkt, pGSK-3β and c-Myc as well as DR5 and negatively correlated with the level of c-FLIPs. Hypersensitivity of CEM/VLB100 cells to TRAIL was accompanied by the activation of mitochondrial apoptotic pathway as well as the activation of initiator caspases. In addition, TRAIL-induced down-regulation of DNA-PKcs/Akt/GSK-3β pathway and c-FLIP and up-regulation of cell surface expression of death receptors were associated with the increased susceptibility to TRAIL of MDR cells. Moreover, TRAIL inhibited P-gp efflux function via caspase-3-dependent degradation of P-gp as well as DNA-PKcs and subsequently sensitized MDR cells to MDR-related drugs such as vinblastine and doxorubicin. We also found that suppression of DNA-PKcs by siRNA enhanced the susceptibility of MDR cells to vincristine as well as TRAIL via down-regulation of c-FLIP and P-gp expression and up-regulation of DR5. Conclusion This study showed for the first time that the MDR variant of CEM cells was hypersensitive to TRAIL due to up-regulation of DR5 and concomitant down-regulation of c-FLIP, and degradation of P-gp and DNA-PKcs by activation of caspase-3 might be

  3. Microvilli Morphology Can Affect Efflux Active P-Glycoprotein in Confluent MDCKII -hMDR1-NKI and Caco-2 Cell Monolayers.

    Science.gov (United States)

    Meng, Zhou; Le Marchand, Sylvain; Agnani, Deep; Szapacs, Matthew; Ellens, Harma; Bentz, Joe

    2017-02-01

    From fits of drug transport kinetics across confluent MDCKII-hMDR1-NKI and Caco-2 cell monolayers we estimated the levels of efflux active P-glycoprotein (P-gp) in these two cell lines (companion paper). In the present work, we compared the efflux active P-gp number to the total P-gp level, using liquid chromatography-tandem mass spectrometry, and showed that in Caco-2 cells total P-gp is about 10-fold greater than efflux active P-gp, whereas in MDCKII-hMDR1-NKI cells these values are within twofold. We further visualized the microvilli in MDCKII-hMDR1-NKI and Caco-2 cells using three-dimensional structured illumination super-resolution microscopy and found that the microvilli in Caco-2 cells are taller and more densely packed than those in MDCK-hMDR1-NKI cells. We hypothesized over 10 years ago that only P-gp at the tips of the microvilli contribute significantly to efflux activity, whereas the remaining P-gp are involved in a futile cycle of efflux of amphipathic drugs from the microvillus membrane, followed by their reabsorption into the same or nearby microvillous membranes. The difference between the levels of total and efflux active P-gp in Caco-2 cells can be explained by the more densely packed microvilli in Caco-2 cells, which would lead to a substantial fraction of P-gp not contributing to final release of drug into the apical chamber. Our results suggest that the effect of microvilli morphology differences between in vitro and in vivo systems must be considered when scaling transporter activity for efflux transporters of amphipathic compounds, for example, P-gp. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  4. 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.

  5. Modulation of p-glycoprotein transport function at the blood-brain barrier.

    Science.gov (United States)

    Bauer, Björn; Hartz, Anika M S; Fricker, Gert; Miller, David S

    2005-02-01

    The central nervous system (CNS) effects of many therapeutic drugs are blunted because of restricted entry into the brain. The basis for this poor permeability is the brain capillary endothelium, which comprises the blood-brain barrier. This tissue exhibits very low paracellular (tight-junctional) permeability and expresses potent, multispecific, drug export pumps. Together, these combine to limit use of pharmacotherapy to treat CNS disorders such as brain cancer and bacterial or viral infections. Of all the xenobiotic efflux pumps highly expressed in brain capillary endothelial cells, p-glycoprotein handles the largest fraction of commonly prescribed drugs and thus is an obvious target for manipulation. Here we review recent studies focused on understanding the mechanisms by which p-glycoprotein activity in the blood-brain barrier can be modulated. These include (i) direct inhibition by specific competitors, (ii) functional modulation, and (iii) transcriptional modulation. Each has the potential to specifically reduce p-glycoprotein function and thus selectively increase brain permeability of p-glycoprotein substrates.

  6. 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...

  7. Spinosad is a potent inhibitor of canine P-glycoprotein

    NARCIS (Netherlands)

    Schrickx, Johannes A|info:eu-repo/dai/nl/30483114X

    Inhibition of the drug transporter P-glycoprotein (P-gp) by the oral flea preventative spinosad has been suggested as the underlying cause of the drug-drug interaction with ivermectin. In this study, an in vitro model consisting of canine cells was validated to describe the inhibitory effect of

  8. Grape seed procyanidin reversal of p-glycoprotein associated multi-drug resistance via down-regulation of NF-κB and MAPK/ERK mediated YB-1 activity in A2780/T cells.

    Directory of Open Access Journals (Sweden)

    Bo-xin Zhao

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

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

    Directory of Open Access Journals (Sweden)

    Andrea Picchianti-Diamanti

    2014-03-01

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

  10. Nuclear Factor-Kappa B Activity Regulates Brain Expression of P-Glycoprotein in the Kainic Acid-Induced Seizure Rats

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    Nian Yu

    2011-01-01

    Full Text Available This study was aimed to investigate the effect of NF-κB activity on the seizure susceptibility, brain damage, and P-gp expression in kainic acid- (KA- induced seizure rats. Male SD rats were divided into saline control group (NS group, KA induced epilepsy group (EP group, and epilepsy group intervened with NF-κB inhibitor-pyrrolidine dithiocarbamate salt (PDTC group or with dexamethasone (DEX group. No seizures were observed in the rats of NS group. Compared with NS group, increased P-gp expression and NF-κB activation in the rat brain of the EP group were observed after KA micro-injection. Both PDTC and DEX pre-treatment significantly increased the latency to grade III or V seizure onset compared to EP group but failed to show neuron-protective effect as the number of survival neurons didn't significantly differ from that in EP group. Furthermore, PDTC pre-treatment significantly decreased P-gp expression along with NF-κB activation in the hippocampus CA3 area and amygdala complex of rats compared with the EP group, implying that NF-κB activation involved in the seizure susceptibility and seizure induced brain P-gp over-expression. Additionally, DEX pre-treatment only decreased P-gp expression level without inhibition of NF-κB activation, suggesting NF-κB independent pathway may also participate in regulating seizure induced P-gp over-expression.

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

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    Yubang Wang

    2015-12-01

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

  12. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. (R-[11C]Verapamil PET studies to assess changes in P-glycoprotein expression and functionality in rat blood-brain barrier after exposure to kainate-induced status epilepticus

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    Lammertsma Adriaan A

    2011-01-01

    Full Text Available Abstract Background Increased functionality of efflux transporters at the blood-brain barrier may contribute to decreased drug concentrations at the target site in CNS diseases like epilepsy. In the rat, pharmacoresistant epilepsy can be mimicked by inducing status epilepticus by intraperitoneal injection of kainate, which leads to development of spontaneous seizures after 3 weeks to 3 months. The aim of this study was to investigate potential changes in P-glycoprotein (P-gp expression and functionality at an early stage after induction of status epilepticus by kainate. Methods (R-[11C]verapamil, which is currently the most frequently used positron emission tomography (PET ligand for determining P-gp functionality at the blood-brain barrier, was used in kainate and saline (control treated rats, at 7 days after treatment. To investigate the effect of P-gp on (R-[11C]verapamil brain distribution, both groups were studied without or with co-administration of the P-gp inhibitor tariquidar. P-gp expression was determined using immunohistochemistry in post mortem brains. (R-[11C]verapamil kinetics were analyzed with approaches common in PET research (Logan analysis, and compartmental modelling of individual profiles as well as by population mixed effects modelling (NONMEM. Results All data analysis approaches indicated only modest differences in brain distribution of (R-[11C]verapamil between saline and kainate treated rats, while tariquidar treatment in both groups resulted in a more than 10-fold increase. NONMEM provided most precise parameter estimates. P-gp expression was found to be similar for kainate and saline treated rats. Conclusions P-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate.

  14. The importance of P-glycoprotein multidrug transporter activity measurement in patients with Helicobacter pylori infection.

    Science.gov (United States)

    Babić, Zarko; Kucisec-Tepes, Nastja; Troskot, Rosana; Dorosulić, Zdravko; Svoboda-Beusan, Ivna

    2009-12-01

    P-glycoprotein is important in local antibiotic resistance. Aim was to evaluate the role of P-glycoprotein in local antibiotic resistance in patients with antral gastritis during antibiotic therapy to Helicobacter pylori infection. In the group of 53 patients with pathohistologically verified gastritis and microbiologically confirmed H. pylori infection (no signs of antimicrobial resistance) we have determined P-glycoprotein activity in gastric mucosa biopsy specimens, and compared them with the P-glycoprotein activity in 12 control subjects with normal endoscopic findings. The H. pylori positive patients were treated according to Maastricht protocol with short-term 7-day therapy consisting of two antibiotics (amoxicillin and azithromycin/metronidazole and clarithromycin) and a proton pump inhibitor P-glycoprotein activity was determined in rhodamine dye efflux test and quantified by ratio of the mean fluorescence (RMF) in flow cytometry analysis. H. pylori was successfully eradicated in the first cycle in 20 patients, whereas therapy was continued in 33 patients. The mean pre-treatment RMF values were higher in patients with H. pylori infection then in control subjects (p RMF was also higher in patients with multiple therapeutic failure than in those with successful H. pylori eradication (p RMF increased significantly during the antibiotic therapy (p < 0.05). P-glycoprotein might be one of the causes of therapy failure in patients with H. pylori. Our study confirms the importance of quantitative evaluation of P-glycoprotein expression during antibiotic treatment response.

  15. Automated analysis of polyethylene glycol-induced inhibition of P-glycoprotein activity in vitro.

    Science.gov (United States)

    Hugger, Erin D; Cole, Clayton J; Raub, Thomas J; Burton, Philip S; Borchardt, Ronald T

    2003-01-01

    Previous studies in our laboratories have shown that commonly used polyethoxylated pharmaceutical excipients inhibit P-glycoprotein activity in cell culture models of the intestinal mucosa. The results presented in this technical note show that the TECAN Genesis robotic workstation can be utilized to automate cellular transport studies for evaluating excipient effects on P-glycoprotein activity in vitro and for estimating the permeation of drug-like molecules across cell monolayers. Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

  16. A plasma membrane 'vacuum cleaner' for daunorubicin in non-P-glycoprotein multidrug-resistant SW-1573 human non-small cell lung carcinoma cells. A study using fluorescence resonance energy transfer.

    Science.gov (United States)

    Mülder, H S; van Grondelle, R; Westerhoff, H V; Lankelma, J

    1993-12-15

    A multidrug resistant (MDR) human non-small cell lung carcinoma cell line, SW-1573/2R120 (2R120), not containing the drug-efflux pump P-glycoprotein (PgP), has been studied for the transport of daunorubicin (DN) across the cellular plasma membrane. Earlier, reduced initial DN-uptake rates and lower cellular DN steady-state concentrations were found for this cell line, when it was compared to the SW-1573 wild-type cell line. This finding was an indication for the presence of another cellular drug-efflux pump. However, we found similar DN-efflux rates in drug-free medium for the two cell lines, while for Pgp-containing MDR SW-1573/2R160 (2R160) cells the efflux rate was increased compared to wild-type cells. In order to elucidate differences in DN transport across the cellular plasma membrane, the association of DN with plasma membranes of intact cells was investigated, using fluorescence-resonance-energy transfer. For this purpose, the plasma-membrane probe 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was chosen since, because of the overlap between the emission spectrum of TMA-DPH and the excitation spectrum of DN, transfer of energy can be achieved from TMA-DPH to DN. Cells were loaded with TMA-DPH and, after addition of 10 microM DN, the TMA-DPH fluorescence was quenched. Rapid initial quenching proved to be similar in the MDR 2R160 (Pgp-containing) cells and in the SW-1573 wild-type cells (21 +/- 1% and 20 +/- 2%, respectively), but was less in the MDR 2R120 cells not containing Pgp (14 +/- 1%). This finding correlated with a lowered amount of DN dissolved in the plasma membrane of 2R120 cells. We interpret these data to be the result of a 'vacuum-cleaner' pumping system other than Pgp which removes DN from a plasma membrane compartment and equilibrates relatively slowly with the interior of the cell.

  17. Targeting prostaglandin E2 EP1 receptors prevents seizure-associated P-glycoprotein up-regulation

    NARCIS (Netherlands)

    Pekcec, A.; Unkrüer, B.; Schlichtiger, J.; Soerensen, J.; Hartz, A.M.S.; Bauer, B.; van Vliet, E.A.; Gorter, J.A.; Potschka, H.

    2009-01-01

    Up-regulation of the blood-brain barrier efflux transporter P-glycoprotein in central nervous system disorders results in restricted brain access and limited efficacy of therapeutic drugs. In epilepsies, seizure activity strongly triggers expression of P-glycoprotein. Here, we identified the

  18. High-dose acetaminophen inhibits the lethal effect of doxorubicin in HepG2 cells: the role of P-glycoprotein and mitogen-activated protein kinase p44/42 pathway.

    Science.gov (United States)

    Manov, Irena; Bashenko, Yulia; Eliaz-Wolkowicz, Anat; Mizrahi, Meital; Liran, Oded; Iancu, Theodore C

    2007-09-01

    Doxorubicin (DOX) is a widely used chemotherapeutic drug for human hepatocellular carcinoma (HCC). A major limitation to its effectiveness is the development of multidrug resistance of cancer cells. In clinical trials, patients with advanced HCC were treated with high-dose acetaminophen (HAAP) in an effort to improve the antitumor activity of chemotherapeutics. In this study, we investigated the effect of concomitant treatment of DOX and HAAP on hepatoma-derived HepG2 cells. Viability, cell cycle distribution, and ultrastructure were examined. Unexpectedly, HAAP, when added to DOX-exposed cells, increased cell viability, released cell cycle arrest, and decreased apoptosis. To elucidate the mechanisms by which HAAP reduces the DOX lethal effect to HepG2 cells, we investigated the multidrug resistance P-glycoprotein (P-gp) and p44/42-mitogen-activated protein kinase (MAPK) pathways. The P-gp function was enhanced by DOX and HAAP, and it was further stimulated during combined treatment, leading to decreased DOX retention. Verapamil (VRP), when added to DOX + HAAP exposure, increased DOX accumulation and restored DOX-induced toxicity. The increased phospho-p44/42-MAPK level in DOX-exposed cells was inhibited by HAAP. In addition, suppression of p44/42 activation by the p44/42-MAPK inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked DOX-induced apoptosis. These findings suggest that the antagonistic effect of concomitant DOX + HAAP treatment occurs as a result of interactive stimulation of P-gp, generating decreased intracellular drug concentrations. Furthermore, inhibition of the p44/42-MAPK phosphorylation by HAAP could abolish the DOX-induced cell death pathway. Thus, combined treatment by DOX + HAAP, intended to improve chemotherapeutic efficacy, could have an opposite effect facilitating cancer cell survival.

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

    Science.gov (United States)

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

    2015-01-01

    In this study, a novel pH-responsive cholesterol-PEG adduct-coated solid lipid nanoparticles (C-PEG-SLNs) carrying doxorubicin (DOX) capable of overcoming multidrug resistance (MDR) breast cancer cells is presented. The DOX-loaded SLNs have a mean hydrodynamic diameter of ~100 nm and a low polydispersity index (under 0.20) with a high drug-loading efficiency ranging from 80.8% to 90.6%. The in vitro drug release profiles show that the DOX-loaded SLNs exhibit a pH-controlled drug release behavior with the maximum and minimum unloading percentages of 63.4% at pH 4.7 and 25.2% at pH 7.4, respectively. The DOX-loaded C-PEG-SLNs displayed a superior ability in inhibiting the proliferation of MCF-7/MDR cells. At a DOX concentration of 80 μM, the cell viabilities treated with C-PEG-SLNs were approximately one-third of the group treated with free DOX. The inhibition activity of C-PEG-SLNs could be attributed to the transport of C-PEG to cell membrane, leading to the change of the composition of the cell membrane and thus the inhibition of permeability glycoprotein activity. This hypothesis is supported by the confocal images showing the accumulation of DOX in the nuclei of cancer cells and the localization of C-PEG on the cell membranes. The results of in vivo study further demonstrated that the DOX delivered by the SLNs accumulates predominantly in tumor via enhanced permeability and retention effect, the enhanced passive tumor accumulation due to the loose intercellular junctions of endothelial cells lining inside blood vessels at tumor site, and the lack of lymphatic drainage. The growth of MCF-7/MDR xenografted tumor on Balb/c nude mice was inhibited to ~400 mm(3) in volume as compared with the free DOX treatment group, 1,140 mm(3), and the group treated with 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] solid lipid nanoparticles, 820 mm(3). Analysis of the body weight of nude mice and the histology of organs and tumor after the

  20. MAPK1 of Leishmania donovani modulates antimony susceptibility by downregulating P-glycoprotein efflux pumps.

    Science.gov (United States)

    Garg, Mansi; Goyal, Neena

    2015-07-01

    Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance in Leishmania donovani. The L. donovani MAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Highly Eribulin-resistant KBV20C Oral Cancer Cells Can Be Sensitized by Co-treatment with the Third-generation P-Glycoprotein Inhibitor, Elacridar, at a Low Dose.

    Science.gov (United States)

    Park, Yujin; Son, Ji-Yeon; Lee, Byung-Mu; Kim, Hyung Sik; Yoon, Sungpil

    2017-08-01

    Eribulin mesylate, also called Halaven® (HAL), was recently developed as a microtubule-targeting drug and is used in the clinic for resistant or metastatic cancer. Previously, we showed that P-glycoprotein (P-gp)-overexpressing KBV20C oral cancer cells are highly resistant to HAL compared to sensitive KB cells. This qualitative study was designed to identify specific P-gp inhibitors that increase the sensitivity of highly resistant cancer cells to HAL. In order to identify functional P-gp inhibitors, HAL-treated KBV20C cells were co-treated with P-gp inhibitors, verapamil, elacridar, cyclosporine A, mitotane, piperine, fumagillin, curcumin, indomethacin, probenecid, sulindac, tesmilifene, and C-4. We then evaluated which P-gp inhibitors required a low dose to sensitize KBV20C cells to HAL. We also determined whether a low dose of a P-gp inhibitor could inhibit P-gp efflux pumping. We found that cyclosporine A sensitized HAL-treated KBV20C cells at a low dose, whereas verapamil, another first-generation P-gp inhibitor, required a dose that was nearly 10-fold higher. We also found that the natural products, piperine and mitotane, sensitized KBV20C cells to HAL co-treatment. Interestingly, we found that elacridar, a third-generation P-gp inhibitor, sensitized HAL-treated cells at a low dose. Elacridar required approximately a 500-fold lower dose than that of verapamil to exert a similar effect. All inhibitors showed P-gp inhibitory activity that correlated with sensitivity to HAL. These results suggest that highly HAL-resistant cancer cells can be sensitized with cyclosporine A or elacridar, specific P-gp inhibitors that exert their effects at a low dose. These findings provide important information regarding the sensitization of highly HAL-resistant cells with selective P-gp inhibitors and indicate that elacridar may be used to treat such highly HAL-resistant cancer cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios

  2. 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

  3. Deficiency of either P-glycoprotein or breast cancer resistance protein protect against acute kidney injury.

    NARCIS (Netherlands)

    Huls, M.; Schoeber, J.P.H.; Verfaillie, C.M.; Luttun, A.; Ulloa-Montoya, F.; Menke, A.L.; Bolderen, L.R. van; Woestenenk, R.M.; Merkx, G.F.M.; Wetzels, J.F.M.; Russel, F.G.M.; Masereeuw, R.

    2010-01-01

    The kidney has a high capacity to regenerate after ischemic injury via several mechanisms, one of which involves bone marrow-derived (stem) cells. The ATP binding cassette transporters, P-glycoprotein and breast cancer resistance protein, are determinants for the enriched stem and progenitor cell

  4. Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation.

    Science.gov (United States)

    Zhang, J T; Ling, V

    1991-09-25

    Increased expression of P-glycoprotein (Pgp) has been demonstrated to cause multidrug resistance (MDR) in vitro, and it may be responsible for chemotherapy failure in a number of human cancers. Pgp is a plasma membrane protein thought to function as an energy-dependent drug transporter. From its deduced protein sequence the topology of Pgp was proposed to contain 12 transmembrane domains with six extracellular loops and two cytoplasmic ATP-binding sites. To investigate further the membrane orientation of Pgp, we have expressed a full length cDNA of mouse mdr1, as well as its truncated forms, in a cell-free system supplemented with dog pancreatic microsomal membranes (RM). We determined which domains of the in vitro-synthesized Pgp had transversed the RM membranes by analyzing their resistance to protease digestion and their glycosylation state. To our surprise, this system revealed that a significant portion of in vitro-synthesized Pgp molecules has an additional glycosylated domain in the C-terminal half. Previously, only the first predicted extracellular loop near the N terminus had been thought to be glycosylated. Furthermore, we discovered that Pgp has at least two functional signal recognition particle/docking protein dependent signal sequences, one at the N-terminal half and the other at the C-terminal half. These findings suggest a new topological model for in vitro synthesized P-glycoprotein which may be relevant to its in vivo topology.

  5. 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.

  6. P-glycoprotein acts as an immunomodulator during neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Gijs Kooij

    Full Text Available BACKGROUND: Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system in which autoreactive myelin-specific T cells cause extensive tissue damage, resulting in neurological deficits. In the disease process, T cells are primed in the periphery by antigen presenting dendritic cells (DCs. DCs are considered to be crucial regulators of specific immune responses and molecules or proteins that regulate DC function are therefore under extensive investigation. We here investigated the potential immunomodulatory capacity of the ATP binding cassette transporter P-glycoprotein (P-gp. P-gp generally drives cellular efflux of a variety of compounds and is thought to be involved in excretion of inflammatory agents from immune cells, like DCs. So far, the immunomodulatory role of these ABC transporters is unknown. METHODS AND FINDINGS: Here we demonstrate that P-gp acts as a key modulator of adaptive immunity during an in vivo model for neuroinflammation. The function of the DC is severely impaired in P-gp knockout mice (Mdr1a/1b-/-, since both DC maturation and T cell stimulatory capacity is significantly decreased. Consequently, Mdr1a/1b -/- mice develop decreased clinical signs of experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis. Reduced clinical signs coincided with impaired T cell responses and T cell-specific brain inflammation. We here describe the underlying molecular mechanism and demonstrate that P-gp is crucial for the secretion of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Importantly, the defect in DC function can be restored by exogenous addition of these cytokines. CONCLUSIONS: Our data demonstrate that P-gp downmodulates DC function through the regulation of pro-inflammatory cytokine secretion, resulting in an impaired immune response. Taken together, our work highlights a new physiological role for P-gp as an immunomodulatory molecule and reveals a possible

  7. P-glycoprotein and Its Role in Treatment Resistance

    Directory of Open Access Journals (Sweden)

    Isil Gogcegoz Gul

    2016-03-01

    Full Text Available Polypharmacy which has often used to increase efficacy of treatment and to prevent resistance in psychiatry may lead to pharmacokinetic and pharmacodynamic drug interactions. One of the inten-sively studied topic in recent years to clarify the mechanism of drug interactions, in the pharmacoki-netic area is p-glycoprotein related drug-drug and drug-food interactions. The interactions of some drugs with p-glycoprotein which is a carrier protein, can lead to a decrease in the bioavailability of these drugs and reduction in passage through the blood-brain barrier. In this review, the role of p-glycoprotein on drug pharmacokinetics and bioavailability of psychiatric drugs are discussed. [Psikiyatride Guncel Yaklasimlar - Current Approaches in Psychiatry 2016; 8(1: 19-31

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

    Science.gov (United States)

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

    2016-01-01

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

  9. 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

  10. Induction and activation of P-glycoprotein by dihydroxylated xanthones protect against the cytotoxicity of the P-glycoprotein substrate paraquat.

    Science.gov (United States)

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

    2014-04-01

    Xanthones are a family of compounds with several known biological activities and therapeutic potential for which information on their interaction with membrane transporters is lacking. Knowing that P-glycoprotein (P-gp) acts as a cellular defense mechanism by effluxing its toxic substrates, the aim of this study was to investigate the potential of five dihydroxylated xanthones as inducers of P-gp expression and/or activity and to evaluate whether they could protect Caco-2 cells against the cytotoxicity induced by the toxic P-gp substrate paraquat (PQ). After 24 h of incubation, all tested xanthones 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, after a short 45-min incubation, all the tested xanthones induced a rapid increase in P-gp activity, indicating direct pump activation without increased P-gp protein expression. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, demonstrating to be P-gp substrates. Moreover, when simultaneously incubated with PQ, all xanthones significantly reduced the cytotoxicity of the herbicide, and these protective effects were completely reversed upon incubation with a specific P-gp inhibitor. In silico studies evaluating the interactions between xanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. A quantitative structure-activity relationship model was developed and validated, and the maximal partial charge for an oxygen atom was the descriptor predicted as being implicated in P-gp activation by the dihydroxylated xanthones. These results disclose new perspectives in preventing PQ- and other P-gp substrates-induced poisonings.

  11. P-Glycoprotein in skin contributes to transdermal absorption of topical corticosteroids.

    Science.gov (United States)

    Hashimoto, Naoto; Nakamichi, Noritaka; Yamazaki, Erina; Oikawa, Masashi; Masuo, Yusuke; Schinkel, Alfred H; Kato, Yukio

    2017-04-15

    ATP binding cassette transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are expressed in skin, but their involvement in transdermal absorption of clinically used drugs remains unknown. Here, we examined their role in transdermal absorption of corticosteroids. Skin and plasma concentrations of dexamethasone after dermal application were reduced in P-gp and BCRP triple-knockout (Mdr1a/1b/Bcrp(-/-)) mice. The skin concentration in Mdr1a/1b/Bcrp(-/-) mice was reduced in the dermis, but not in the epidermis, indicating that functional expression of these transporters in skin is compartmentalized. Involvement of these transporters in dermal transport of dexamethasone was also supported by the observation of a higher epidermal concentration in Mdr1a/1b/Bcrp(-/-) than wild-type mice during intravenous infusion. Transdermal absorption after dermal application of prednisolone, but not methylprednisolone or ethinyl estradiol, was also lower in Mdr1a/1b/Bcrp(-/-) than in wild-type mice. Transport studies in epithelial cell lines transfected with P-gp or BCRP showed that dexamethasone and prednisolone are substrates of P-gp, but are minimally transported by BCRP. Thus, our findings suggest that P-gp is involved in transdermal absorption of at least some corticosteroids in vivo. P-gp might be available as a target for inhibition in order to deliver topically applied drugs and cosmetics in a manner that minimizes systemic exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Safa, A.R. (Univ. of Chicago Medical Centers, IL (USA))

    1988-10-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-09-01

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

  15. 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

  16. 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

  17. Marine Natural Products with P-Glycoprotein Inhibitor Properties

    Directory of Open Access Journals (Sweden)

    Dioxelis Lopez

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  19. Expression of multidrug resistance proteins P-glycoprotein, multidrug resistance protein 1, breast cancer resistance protein and lung resistance related protein in locally advanced bladder cancer treated with neoadjuvant chemotherapy: biological and clinical implications.

    NARCIS (Netherlands)

    Diestra, JE; Condom, E; Muro, XG Del; Scheffer, G.L.; Perez, J; Zurita, AJ; Munoz-Segui, J; Vigues, F; Scheper, R.J.; Capella, G; Germa-Lluch, JR; Izquierdo, M.A.

    2003-01-01

    PURPOSE: Resistance to chemotherapy is a major obstacle to overcome in the conservative treatment of patients with locally advanced bladder cancer (LABC). We investigated the predictive value of the response to neoadjuvant chemotherapy (NACT) and prognosis of the expression of multidrug resistance

  20. Marked impact of P-glycoprotein on the absorption of TAK-427 in rats.

    Science.gov (United States)

    Takeuchi, Toshiyuki; Nonaka, Masami; Yoshitomi, Sumie; Higuchi, Tomoaki; Ebihara, Takuya; Maeshiba, Yoshihiro; Kawase, Masahiro; Asahi, Satoru

    2008-09-01

    The role of P-glycoprotein (P-gp, ABCB1) on the absorption process was investigated by drug-drug interaction studies of TAK-427 with P-gp inhibitors (erythromycin, ketoconazole or quinidine) in rats and by transport studies using rat multidrug resistance (MDR1) stably expressing cells and rat small intestine mounted in a Ussing-type chamber. TAK-427 showed high efflux activity with low permeability in rat MDR1a and MDR1b stably expressing cells and was revealed to be a typical substrate for P-gps. Although TAK-427 was mainly absorbed from the small intestine in rats, a large part of the dosed compound remained in the gastrointestinal tract. Orally co-administered P-gp inhibitors (50 mg/kg) increased the AUC of TAK-427 after a 5 mg/kg oral dose 5.4- to 18.3-fold, whereas orally administered P-gp inhibitors had a minor effect on the increase in the AUC of TAK-427 (1.3- to 2.2-fold) after a 0.5 mg/kg intravenous dose. Thus, the bioavailability of TAK-427 after oral administration in rats (7.3%) markedly increased when co-administered with P-gp inhibitors (28.6-57.6%). Moreover, the transport of TAK-427 was predominantly secretory throughout the rat small intestine and was inhibited by P-gp inhibitors. In conclusion, P-gp can markedly reduce the absorption of a typical P-gp substrate by its efflux activity throughout the absorption site.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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....... As in vertebrates, the locust brain–barrier function is morphologically confined to one specific cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model...

  3. Functional Impact of ABCB1 Variants on Interactions between P-Glycoprotein and Methadone

    Science.gov (United States)

    Hung, Chin-Chuan; Chiou, Mu-Han; Teng, Yu-Ning; Hsieh, Yow-Wen; Huang, Chieh-Liang; Lane, Hsien-Yuan

    2013-01-01

    Methadone is a widely used substitution therapy for opioid addiction. Large inter-individual variability has been observed in methadone maintenance dosages and P-glycoprotein (P-gp) was considered to be one of the major contributors. To investigate the mechanism of P-gp’s interaction with methadone, as well as the effect of genetic variants on the interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established in the present study. The RNA and protein expression levels of human P-gp were confirmed by real-time quantitative RT-PCR and western blot, respectively. Utilizing rhodamine 123 efflux assay and calcein-AM uptake study, methadone was demonstrated to be an inhibitor of wild-type human P-gp via non-competitive kinetic (IC50 = 2.17±0.10 µM), while the variant-type human P-gp, P-gp with 1236T-2677T-3435T genotype and P-gp with 1236T-2677A-3435T genotype, showed less inhibition potency (IC50 = 2.97±0.09 µM and 4.43±1.10 µM, respectively) via uncompetitive kinetics. Methadone also stimulated P-gp ATPase and inhibited verapamil-stimulated P-gp ATPase activity under therapeutic concentrations. These results may provide a possible explanation for higher methadone dosage requirements in patients carrying variant-type of P-gp and revealed the possible drug-drug interactions in patients who receive concomitant drugs which are also P-gp substrates. PMID:23527191

  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. Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a P-Glycoprotein cDNA.

    Directory of Open Access Journals (Sweden)

    Kristen M Pluchino

    Full Text Available The efflux transporter P-glycoprotein (P-gp is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

  6. 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

    line, IPEC-J2 MDR1, retained its high transepithelilal resistance (>15,000 Ω·cm(2)), which translated into low permeability of the small hydrophilic tracer, mannitol (P diazepam, displayed high permeability resulting in a dynamic range of 1500 (PDiazepam...

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Nanoparticle mediated P-glycoprotein silencing for improved drug delivery across the blood-brain barrier: a siRNA-chitosan approach.

    Directory of Open Access Journals (Sweden)

    Jostein Malmo

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

  10. In silico model for P-glycoprotein substrate prediction: insights from molecular dynamics and in vitro studies

    Science.gov (United States)

    Prajapati, Rameshwar; Singh, Udghosh; Patil, Abhijeet; Khomane, Kailas S.; Bagul, Pravin; Bansal, Arvind K.; Sangamwar, Abhay T.

    2013-04-01

    P-glycoprotein (P-gp) is a plasma membrane efflux transporter belonging to ATP-binding cassette superfamily, responsible for multidrug resistance in tumor cells. Over-expression of P-gp in cancer cells limits the efficacy of many anticancer drugs. A clear understanding of P-gp substrate binding will be advantageous in early drug discovery process. However, substrate poly-specificity of P-gp is a limiting factor in rational drug design. In this investigation, we report a dynamic trans-membrane model of P-gp that accurately identified the substrate binding residues of known anticancer agents. The study included homology modeling of human P-gp based on the crystal structure of C. elegans P-gp, molecular docking, molecular dynamics analyses and binding free energy calculations. The model was further utilized to speculate substrate propensity of in-house anticancer compounds. The model demonstrated promising results with one anticancer compound (NSC745689). As per our observations, the molecule could be a potential lead for anticancer agents devoid of P-gp mediated multiple drug resistance. The in silico results were further validated experimentally using Caco-2 cell lines studies, where NSC745689 exhibited poor permeability ( P app 1.03 ± 0.16 × 10-6 cm/s) and low efflux ratio of 0.26.

  11. A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle

    Directory of Open Access Journals (Sweden)

    Yannick Pauchet

    2016-12-01

    Full Text Available Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.

  12. Stereoselective transport of hydrophilic quaternary drugs by human MDR1 and rat Mdr1b P-glycoproteins

    OpenAIRE

    Guido J E J Hooiveld; Heegsma, Janette; van Montfoort, Jessica E; Jansen, Peter L M; Meijer, Dirk K.F; Müller, Michael

    2002-01-01

    The present study was performed to evaluate and compare the ability of human MDR1-, and rat Mdr1b- and Mdr2-P-glycoproteins to transport hydrophilic monoquaternary drugs. Transport studies were performed with plasma membrane vesicles isolated from MDR1-, Mdr1b-, or Mdr2-overexpressing insect cells.As model substrates we used the N-methylated derivatives of the diastereomers quinidine and quinine, the monoquaternary compounds N-methylquinidine and N-methylquinine. Vincristine, an established M...

  13. Screening for the P-Glycoprotein Inhibitory Pump Activity of Plant ...

    African Journals Online (AJOL)

    7(1) 16-22. Screening for the P-Glycoprotein Inhibitory Pump Activity of Plant Extracts that are Used in. Tanzanian Traditional Medicine ... 6G as the fluorescent probe and reserpine, a known inhibitor of P-glycoprotein pump, was used as a reference drug. .... The resistant s ublines are isolated and r e-incubated with higher ...

  14. 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)

  15. Molecular Imaging of P-glycoprotein in Chemoresistant Tumors Using a Dual-Modality PET/Fluorescence Probe.

    Science.gov (United States)

    Wang, Mengzhe; Mao, Chengqiong; Wang, Hui; Ling, Xueying; Wu, Zhanhong; Li, Zibo; Ming, Xin

    2017-10-02

    Overexpression of P-glycoprotein (Pgp) has been considered a primary cause for multidrug resistance in a variety of cancers for three decades. However, clinical translation of Pgp targeted therapeutics has been hindered by lack of patient preselection based on the Pgp presence in tumors. We aim to develop a molecularly targeted probe for imaging tumoral Pgp in vivo with positron emission tomography (PET) and fluorescence, and to provide a tool for preselecting the patients with tumoral Pgp expression. Thus, a Pgp monoclonal antibody 15D3 was chemically modified with IRDye800 (IR800) and DOTA chelator. The specificity of the antibody conjugates DOTA-Pab-IR800 was verified in Pgp-expressing 3T3-MDR1 and control 3T3 cells. After radiolabeling with 64Cu, the probe was applied in small animal PET imaging of Pgp in a mouse xenograft model of NCI/ADR-Res cells, which are chemoresistant through overexpression of Pgp. Quantification analysis of the PET images demonstrated that the tumor uptake of the radioactive probe was 9.9 ± 1.4, 12.1 ± 1.2, and 10.5 ± 1.0%ID/g at 4, 24, and 48 h post injection. The tumor-to-muscle ratio was 20.9 at 48 h post injection based on biodistribution studies. Fluorescence imaging was performed following PET experiments, and it demonstrated excellent tumor accumulation of this dual-modality probe in the NCI/ADR-Res tumors. Further, an image-guided surgery was successfully performed using the fluorescence modality of the probe, demonstrating potential utility of this probe in image-guided surgical removal of Pgp-positive drug resistant tumors in the patients. In conclusion, this study clearly demonstrated that the Pgp-targeted antibody probe, 64Cu-DOTA-Pab-IR800, could provide a promising diagnosis tool for detection of Pgp-expressing tumors in vivo.

  16. NVP-TAE684 reverses multidrug resistance (MDR) in human osteosarcoma by inhibiting P-glycoprotein (PGP1) function.

    Science.gov (United States)

    Ye, Shunan; Zhang, Jianming; Shen, Jacson; Gao, Yan; Li, Ying; Choy, Edwin; Cote, Gregory; Harmon, David; Mankin, Henry; Gray, Nathanael S; Hornicek, Francis J; Duan, Zhenfeng

    2016-02-01

    Increased expression of P-glycoprotein (PGP1) is one of the major causes of multidrug resistance (MDR) in cancer, including in osteosarcoma, which eventually leads to the failure of cancer chemotherapy. Thus, there is an urgent need to develop effective therapeutic strategies to override the expression and function of PGP1 to counter MDR in cancer patients. In an effort to search for new chemical entities targeting PGP1-associated MDR in osteosarcoma, we screened a 500+ compound library of known kinase inhibitors with established kinase selectivity profiles. We aimed to discover potential drug synergistic effects among kinase inhibitors and general chemotherapeutics by combining inhibitors with chemotherapy drugs such as doxorubicin and paclitaxel. The human osteosarcoma MDR cell lines U2OSR2 and KHOSR2 were used for the initial screen and secondary mechanistic studies. After screening 500+ kinase inhibitors, we identified NVP-TAE684 as the most effective MDR reversing agent. NVP-TAE684 significantly reversed chemoresistance when used in combination with doxorubicin, paclitaxel, docetaxel, vincristine, ET-743 or mitoxantrone. NVP-TAE684 itself is not a PGP1 substrate competitive inhibitor, but it can increase the intracellular accumulation of PGP1 substrates in PGP1-overexpressing cell lines. NVP-TAE684 was found to inhibit the function of PGP1 by stimulating PGP1 ATPase activity, a phenomenon reported for other PGP1 inhibitors. The application of NVP-TAE684 to restore sensitivity of osteosarcoma MDR cells to the cytotoxic effects of chemotherapeutics will be useful for further study of PGP1-mediated MDR in human cancer and may ultimately benefit cancer patients. © 2015 The British Pharmacological Society.

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

    Directory of Open Access Journals (Sweden)

    A. V. Shulkin

    2013-01-01

    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.

  18. 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.

  19. 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

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

    Directory of Open Access Journals (Sweden)

    Zsolt Bikadi

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

  1. In vitro P-glycoprotein assays to predict the in vivo interactions of P-glycoprotein with drugs in the central nervous system.

    Science.gov (United States)

    Feng, Bo; Mills, Jessica B; Davidson, Ralph E; Mireles, Rouchelle J; Janiszewski, John S; Troutman, Matthew D; de Morais, Sonia M

    2008-02-01

    Thirty-one structurally diverse marketed central nervous system (CNS)-active drugs, one active metabolite, and seven non-CNS-active compounds were tested in three P-glycoprotein (P-gp) in vitro assays: transwell assays using MDCK, human MDR1-MDCK, and mouse Mdr1a-MDCK cells, ATPase, and calcein AM inhibition. Additionally, the permeability for these compounds was measured in two in vitro models: parallel artificial membrane permeation assay and apical-to-basolateral apparent permeability in MDCK. The exposure of the same set of compounds in brain and plasma was measured in P-gp knockout (KO) and wild-type (WT) mice after subcutaneous administration. One drug and its metabolite, risperidone and 9-hydroxyrisperidone, of the 32 CNS compounds, and 6 of the 7 non-CNS drugs were determined to have positive efflux using ratio of ratios in MDR1-MDCK versus MDCK transwell assays. Data from transwell studies correlated well with the brain-to-plasma area under the curve ratios between P-gp KO and WT mice for the 32 CNS compounds. In addition, 3300 Pfizer compounds were tested in MDR1-MDCK and Mdr1a-MDCK transwell assays, with a good correlation (R(2) = 0.92) between the efflux ratios in human MDR1-MDCK and mouse Mdr1a-MDCK cells. Permeability data showed that the majority of the 32 CNS compounds have moderate to high passive permeability. This work has demonstrated that in vitro transporter assays help in understanding the role of P-gp-mediated efflux activity in determining the disposition of CNS drugs in vivo, and the transwell assay is a valuable in vitro assay to evaluate human P-gp interaction with compounds for assessing brain penetration of new chemical entities to treat CNS disorders.

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

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    Frances Jane Sharom

    2014-03-01

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

  3. HDAC2 was involved in placental P-glycoprotein regulation both in vitro and vivo.

    Science.gov (United States)

    Duan, Hongyu; Zhou, Kaiyu; Zhang, Yi; Yue, Peng; Wang, Tao; Li, Yifei; Qiu, Dajian; Wu, Jinlin; Hua, Yimin; Wang, Chuan

    2017-10-01

    Placental P-glycoprotein (P-gp) plays a significant role in regulating drugs' transplacental transfer rates. Investigations on placental P-gp regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the epigenetic regulation of placental P-gp is rare. Our previous study has demonstrated that HDACs inhibition could up-regulate placental P-gp and HDAC1/2/3 might be involved in this process. The present study was carried out to further explore whether HDAC1/2/3 were indeed involved in the regulation of placental P-gp or not and screen out the subtype engaged in this process. BeWo and JAR cells were transfected with HDAC1/2/3 specific siRNA. After 48 h of transfection, cells were harvested for real-time quantitative PCR (qRT-PCR), Western blot, immunofluorescence and fluorescent dye efflux assay to evaluate P-gp expression, localization, and efflux activity, respectively. Hdac2 siRNA was intraperitoneally injected to pregnant mice every 48 h from E7.5 to E15.5 and digoxin was administered by gavages 1 h prior to euthanasia at E16.5. Placental Hdac1/2/3 and P-gp expression were determined by qRT-PCR and Western blot. Maternal plasma and fetal-unit digoxin concentrations were detected by enzyme-multiplied immunoassay. In vitro, HDAC2 inhibition could significantly elevate P-gp expression and reduce intracellular accumulation of P-gp substrates (DiOC2 (3) and Rh 123) both in BeWo and JAR, while knockdown of HDAC1/3 had no influence on P-gp expression and its efflux activity. Additionally, in vivo, Hdac2 silencing in pregnant mice also elevated placental P-gp expression and decreased digoxin transplacental transfer rate. HDAC2 inhibition could result in induction of placental P-gp expression and functionality both in vitro and in vivo. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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    Crowe, Andrew, E-mail: a.p.crowe@curtin.edu.au; Tan, Ai May

    2012-05-01

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

  5. P-glycoprotein (ABCB1 and Oxidative Stress: Focus on Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Giulia Sita

    2017-01-01

    Full Text Available ATP-binding cassette (ABC transporters, in particular P-glycoprotein (encoded by ABCB1, are important and selective elements of the blood-brain barrier (BBB, and they actively contribute to brain homeostasis. Changes in ABCB1 expression and/or function at the BBB may not only alter the expression and function of other molecules at the BBB but also affect brain environment. Over the last decade, a number of reports have shown that ABCB1 actively mediates the transport of beta amyloid (Aβ peptide. This finding has opened up an entirely new line of research in the field of Alzheimer’s disease (AD. Indeed, despite intense research efforts, AD remains an unsolved pathology and effective therapies are still unavailable. Here, we review the crucial role of ABCB1 in the Aβ transport and how oxidative stress may interfere with this process. A detailed understanding of ABCB1 regulation can provide the basis for improved neuroprotection in AD and also enhanced therapeutic drug delivery to the brain.

  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.

    2016-01-01

    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. In vitro transport profile of carbamazepine, oxcarbazepine, eslicarbazepine acetate, and their active metabolites by human P-glycoprotein.

    Science.gov (United States)

    Zhang, Chunbo; Zuo, Zhong; Kwan, Patrick; Baum, Larry

    2011-10-01

    Antiepileptic drugs (AEDs) are widely used not only in the treatment of epilepsy but also as treatments for psychiatric disorders. Pharmacoresistance of AEDs in the treatment of epilepsy and psychiatric disorders is a serious problem. Transport of antiepileptic drugs by P-glycoprotein (Pgp, ABCB1, or MDR1), which is overexpressed in the blood-brain barrier, may be a mechanism for resistance of AEDs. For most AEDs, conflicting evidence precludes consensus on whether they are substrates of Pgp. The objective of this study was to evaluate whether analogs and metabolites of the AED carbamazepine are substrates of human Pgp. Polarized cell lines MDCKII and LLC transfected with the human MDR1 gene were used in the bidirectional transport assay and concentration equilibrium transport assay. The expression of Pgp was detected by real-time polymerase chain reaction (PCR) and immunofluorescent staining. Rhodamine-123 uptake was also determined. Pgp did not transport carbamazepine, but it did transport its active metabolite carbamazepine-10,11-epoxide. Pgp also pumped eslicarbazepine acetate and oxcarbazepine, as well as their active metabolite (S)-licarbazepine. Transport of the drugs was in the order of ESL>OXC>S-LC>CBZ-E in concentration equilibrium conditions. The transport of these drugs was blocked by Pgp inhibitors tariquidar and verapamil. All carbamazepine analogs or metabolites tested are Pgp substrates, except for carbamazepine. These data suggest that resistance to carbamazepine, oxcarbazepine, or eslicarbazepine acetate may be attributed to increased efflux function of Pgp because they or their active metabolites are Pgp substrates. Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.

  8. 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.

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

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    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.

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

    Science.gov (United States)

    Maia, Raquel C; Vasconcelos, Flavia C; Souza, Paloma S; Rumjanek, Vivian M

    2018-01-07

    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.

  11. Delivery of P-glycoprotein substrates using chemosensitizers and nanotechnology for selective and efficient therapeutic outcomes.

    Science.gov (United States)

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

    2012-07-10

    As a result of its broad substrate specificity and critical localization in excretory and barrier function tissues, P-glycoprotein (P-gp) plays major roles in the pharmacokinetics, safety and efficacy profiles of numerous drugs. P-gp is often responsible for the failure of many chemical treatments against cancer, immunosuppressive, infectious and neurodegenerative diseases. Among the therapeutic approaches to circumvent P-gp function, advances in the design of new chemical P-gp modulators to interact specifically with P-gp have yielded few clinical successful reports. Members of a class of components that were initially developed as surface active agents showed promising results with regard to the modulation of P-gp. These components include surfactants and amphiphilic co-polymers. Alternatively, colloidal systems were developed to facilitate drug uptake in resistant cells. This approach is based on the encapsulation of drugs, which masks them from the biological environment and prevents their transport by P-gp using the surfactants released from the nanocarrier. Likewise, a novel and synergistic strategy is currently being explored and involves nanocarrier-mediated transport and controlled release of both P-gp substrates and P-gp modulators. In this review, we discuss recent results obtained by direct modulation with chemosensitizers and the available nanotechnology to modulate P-gp function. In this manuscript, we also discuss unexplored pathways for future therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding

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    Aller, Stephen G.; Yu, Jodie; Ward, Andrew; Weng, Yue; Chittaboina, Srinivas; Zhuo, Rupeng; Harrell, Patina M.; Trinh, Yenphuong T.; Zhang, Qinghai; Urbatsch, Ina L.; Chang, Geoffrey; (Scripps); (TTU)

    2009-04-22

    P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of -6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

  13. Role of P-glycoprotein inhibitors in ceramide-based therapeutics for treatment of cancer.

    Science.gov (United States)

    Morad, Samy A F; Davis, Traci S; MacDougall, Matthew R; Tan, Su-Fern; Feith, David J; Desai, Dhimant H; Amin, Shantu G; Kester, Mark; Loughran, Thomas P; Cabot, Myles C

    2017-04-15

    The anticancer properties of ceramide, a sphingolipid with potent tumor-suppressor properties, can be dampened via glycosylation, notably in multidrug resistance wherein ceramide glycosylation is characteristically elevated. Earlier works using the ceramide analog, C6-ceramide, demonstrated that the antiestrogen tamoxifen, a first generation P-glycoprotein (P-gp) inhibitor, blocked C6-ceramide glycosylation and magnified apoptotic responses. The present investigation was undertaken with the goal of discovering non-anti-estrogenic alternatives to tamoxifen that could be employed as adjuvants for improving the efficacy of ceramide-centric therapeutics in treatment of cancer. Herein we demonstrate that the tamoxifen metabolites, desmethyltamoxifen and didesmethyltamoxifen, and specific, high-affinity P-gp inhibitors, tariquidar and zosuquidar, synergistically enhanced C6-ceramide cytotoxicity in multidrug resistant HL-60/VCR acute myelogenous leukemia (AML) cells, whereas the selective estrogen receptor antagonist, fulvestrant, was ineffective. Active C6-ceramide-adjuvant combinations elicited mitochondrial ROS production and cytochrome c release, and induced apoptosis. Cytotoxicity was mitigated by introduction of antioxidant. Effective adjuvants markedly inhibited C6-ceramide glycosylation as well as conversion to sphingomyelin. Active regimens were also effective in KG-1a cells, a leukemia stem cell-like line, and in LoVo human colorectal cancer cells, a solid tumor model. In summary, our work details discovery of the link between P-gp inhibitors and the regulation and potentiation of ceramide metabolism in a pro-apoptotic direction in cancer cells. Given the active properties of these adjuvants in synergizing with C6-ceramide, independent of drug resistance status, stemness, or cancer type, our results suggest that the C6-ceramide-containing regimens could provide alternative, promising therapeutic direction, in addition to finding novel, off-label applications

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

    Science.gov (United States)

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

    2015-01-01

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

  15. The effect of pomelo mix ethyl acetate extract on CYP3A6 and P-glycoprotein gene transcripts in rabbits.

    Science.gov (United States)

    Irshaid, Yacoub M; Zihlef, Malek A; Zmeili, Suheil M; Al-Antary, Eman T; Zmaily, Mais G; Al-Embideen, Somya N; Amireh, Abdallah O

    2014-07-01

    Pomelo fruit juice and pomelo ethylacetate extract have been shown to increase the bioavailability of some CYP3A substrates. The purpose of this study was to investigate if this effect might be contributed to by changes in CYP3A and p-glycoprotein mRNAs levels in the liver and proximal small intestine. The ethyl acetate extract of pomelo mix was administered for 7 days to 10 rabbits. Nine rabbits were administered tap water for 7 days. The administration was through oral intubation to the stomach. On the 8(th) day, the rabbits were sacrificed, and the liver and the proximal 15 cm of the small intestine were dissected. Total RNA was extracted from the specimens and cDNA was prepared by quantitative real-time-polymerase chain reaction (RT-PCR) using specific primers. The ethyl acetate extract of pomelo mix reduced the mRNA expression of CYP3A6 almost 5-folds in the intestine and 2-folds in the liver. In contrast, a 1-fold increase to the p-glycoprotein mRNA expression was observed under the same experimental conditions. In conclusion, the ethyl acetate extract of pomelo mix reduced the mRNA expression of CYP3A6 in both intestine and liver but to different degrees, while the p-glycoprotein mRNA expression was not reduced.

  16. 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.

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

    Science.gov (United States)

    Raaphorst, Renske M; Savolainen, Heli; Cantore, Mariangela; van de Steeg, Evita; van Waarde, Aren; Colabufo, Nicola A; Elsinga, Philip H; Lammertsma, Adriaan A; Windhorst, Albert D; Luurtsema, Gert

    2017-09-20

    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 [(11)C]verapamil, (R)-N-[(18)F]fluoroethylverapamil, (R)-O-[(18)F]fluoroethyl-norverapamil, [(18)F]MC225 and [(18)F]MC224 and we included also two new molecules [(18)F]MC198 and [(18)F]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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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.

  20. Can celecoxib affect P-glycoprotein-mediated drug efflux? A microPET study

    NARCIS (Netherlands)

    De Vries, Erik F. J.; Doorduin, Janine; Vellinga, Namkje A. R.; Van Waarde, Aren; Dierckx, Rudi A.; Klein, Hans C.

    Introduction: P-glycoprotein (Pgp) is an efflux pump that protects vital organs like the brain from toxic substances, but which is also associated with therapy resistance. The anti-inflammatory drug celecoxib potentiates the efficacy of several cytostatic and neurotropic drugs that are known Pgp

  1. Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

    NARCIS (Netherlands)

    Konings, WN; Poelarends, GJ

    2002-01-01

    Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural

  2. 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...

  3. P-Glycoprotein Function at the Blood-Brain Barrier: Effects of Age and Gender

    NARCIS (Netherlands)

    van Assema, D.M.E.; Lubberink, J.M.; Boellaard, R.; Schuit, R.C.; Windhorst, A.D.; Scheltens, P.; Lammertsma, A.A.; van Berckel, B.N.M.

    2012-01-01

    Purpose: P-glycoprotein (Pgp) is an efflux transporter involved in transport of several compounds across the blood-brain barrier (BBB). Loss of Pgp function with increasing age may be involved in the development of age-related disorders, but this may differ between males and females. Pgp function

  4. 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...

  5. Role of P-Glycoprotein Inhibitors in the Bioavailability Enhancement of Solid Dispersion of Darunavir

    Directory of Open Access Journals (Sweden)

    Saleha Rehman

    2017-01-01

    Full Text Available Objective. The aim of the present study was to improve bioavailability of an important antiretroviral drug, Darunavir (DRV, which has low water solubility and poor intestinal absorption through solid dispersion (SD approach incorporating polymer with P-glycoprotein inhibitory potential. Methods. A statistical approach where design of experiment (DoE was used to prepare SD of DRV with incorporation of P-glycoprotein inhibitors. Using DoE, different methods of preparation, like melt, solvent evaporation, and spray drying method, utilizing carriers like Kolliphor TPGS and Soluplus were evaluated. The optimized SD was characterized by DSC, FTIR, XRD, and SEM and further evaluated for enhancement in absorption using everted gut sac model, effect of food on absorption of DRV, and in vivo prospect. Results and Discussion. DSC, FTIR, XRD, and SEM confirmed the amorphicity of drug in SD. Oral bioavailability studies revealed better absorption of DRV when given with food. Absorption studies and in vivo study findings demonstrated great potential of Kolliphor TPGS as P-glycoprotein inhibitor for increasing intestinal absorption and thus bioavailability of DRV. Conclusion. It is concluded that SD of DRV with the incorporation of Kolliphor TPGS was potential and promising approach in increasing bioavailability of DRV as well as minimizing its extrusion via P-glycoprotein efflux transporters.

  6. Blonanserin, a novel atypical antipsychotic agent not actively transported as substrate by P-glycoprotein.

    Science.gov (United States)

    Inoue, Tomoko; Osada, Kenichi; Tagawa, Masaaki; Ogawa, Yuriko; Haga, Toshiaki; Sogame, Yoshihisa; Hashizume, Takanori; Watanabe, Takashi; Taguchi, Atsushi; Katsumata, Takashi; Yabuki, Masashi; Yamaguchi, Noboru

    2012-10-01

    Although blonanserin, a novel atypical antipsychotic agent with dopamine D(2)/serotonin 5-HT(2A) antagonistic properties, displays good brain distribution, the mechanism of this distribution has not been clarified. P-glycoprotein [(P-gp) or multidrug resistance protein 1 (MDR1)] is an efflux transporter expressed in the brain and plays an important role in limiting drug entry into the central nervous system (CNS). In particular, P-gp can affect the pharmacokinetics and efficacy of antipsychotics, and exacerbate or soothe their adverse effects. In this study, we conducted in vitro and in vivo experiments to determine whether blonanserin is a P-gp substrate. Risperidone and its active metabolite 9-hydroxyrisperidone, both of which are P-gp substrates, were used as reference drugs. Affinity of blonanserin, risperidone, and 9-hydroxyrisperidone for P-gp was evaluated by in vitro transcellular transport across LLC-PK1, human MDR1 cDNA-transfected LLC-PK1 (LLC-MDR1), and mouse Mdr1a cDNA-transfected LLC-PK1 (LLC-Mdr1a). In addition, pharmacokinetic parameters in the brain and plasma (B/P ratio) of test compounds were measured in mdr1a/1b knockout (KO) and wild-type (WT) mice. The results of in vitro experiments revealed that P-gp does not actively transport blonanserin as a substrate in humans or mice. In addition, blonanserin displayed comparable B/P ratios in KO and WT mice, whereas B/P ratios of risperidone and 9-hydroxyrisperidone differed markedly in these animals. Our results indicate that blonanserin is not a P-gp substrate and therefore its brain distribution is unlikely to be affected by this transporter. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    S M D K Ganga Senarathna

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

  9. Exosomes mediate interepithelial transfer of functional P-glycoprotein in chronic rhinosinusitis with nasal polyps.

    Science.gov (United States)

    Nocera, Angela L; Miyake, Marcel M; Seifert, Philip; Han, Xue; Bleier, Benjamin S

    2017-09-01

    P-glycoprotein (P-gp) drives type-2 helper T-cell inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) through unknown posttranslational mechanisms of overexpression. A recent randomized clinical trial demonstrated that inhibition of P-gp was as effective as oral steroids and biologics in treating CRSwNP. Exosomes are 30- to 150-nm vesicles capable of intercellular membrane protein transfer. The aims of this study were 1) to determine whether CRSwNP mucus exosomes are enriched with P-gp, and 2) whether exosomal P-gp can be functionally transferred to autologous epithelial cells as a putative mechanism for the proinflammatory overexpression of P-gp in CRSwNP. Institutional review board-approved study in CRSwNP and control patients (n = 10 per group). P-gp content of purified mucus exosomes was characterized by transmission electron microscopy and enzyme-linked immunosorbent assay. Epithelial transfer of exosomal P-gp was determined by time-lapse fluorescent microscopy and calcein acetoxymethylester functional P-gp assay. CD63+/P-gp+ exosomes were detected in both groups. P-gp was significantly enriched in CRSwNP exosomes relative to control (median 198.5; interquartile range 123.6-270.5 vs. 74.4; 41.3-95.0 pcg P-gp/10(9) exosomes, P = 0.002). Exosomes were absorbed by epithelial cells within 10 minutes, resulting in a significant increase in P-gp activity in CRSwNP patients relative to control (P = 0.006). Here we demonstrate the presence and P-gp enrichment of mucus-derived exosomes, or rhinosomes, in CRSwNP. These rhinosomes are capable of rapid intercellular transfer of P-gp, leading to increased P-gp function within recipient cells. This represents a novel mechanism for maintaining P-gp overexpression in CRSwNP, and more generally for interepithelial transfer of other proteins between mucosal epithelial cells. NA. Laryngoscope, 127:E295-E300, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  10. 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.

  11. Emerging significance of flavonoids as P-glycoprotein inhibitors in cancer chemotherapy.

    Science.gov (United States)

    Bansal, Tripta; Jaggi, Manu; Khar, Roop K; Talegaonkar, Sushama

    2009-01-01

    Chemotherapy forms the mainstay of cancer treatment particularly for patients who do not respond to local excision or radiation treatment. However, cancer treatment by drugs is seriously limited by P-glycoprotein (P-gp) associated multi-drug resistance (MDR) in various tumor cells. On the other hand, it is now widely recognized that P-gp also influences drug transport across various biological membranes. P-gp transporter is widely present in the luminal surface of enterocytes, biliary canalicular surface of hepatocytes, apical surface of proximal tubular cells of kidney, endothelial cells of blood brain barrier, etc. thus affecting absorption, distribution, metabolism and excretion of xenobiotics. Clinical significance of above mentioned carrier is appreciated from the fact that more than fifty percent of existing anti-cancer drugs undergo inhibitable and saturable P-gp mediated efflux. Consequently, there is an increasing trend to optimize pharmacokinetics, enhance antitumour activity and reduce systemic toxicity of existing anti-cancer drugs by inhibiting P-gp mediated transport. Although a wide variety of P-gp inhibitors have been discovered, research efforts are underway to identify the most appropriate one. Flavonoids (polyphenolic herbal constituents) form the third generation, non-pharmaceutical category of P-gp inhibitors. The effects produced by some of these components are found to be comparable to those of well-known P-gp inhibitors verapamil and cyclosporine. Identification of effective P-gp modulator among herbal compounds have an added advantage of being safe, thereby making them ideal candidates for bioavailability enhancement, tissue-penetration (e.g. blood brain barrier (BBB)), decreasing biliary excretion and multi-drug resistance modulating agents. The dual effects, i.e. P-gp modulation and antitumor activity, of these herbal derivatives may synergistically act in cancer chemotherapy. This paper presents an overview of the investigations on the

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

    Science.gov (United States)

    Knop, Jana; Misaka, Shingen; Singer, Katrin; Hoier, Eva; Müller, Fabian; Glaeser, Hartmut; König, Jörg; Fromm, Martin F

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  14. 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

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

    Directory of Open Access Journals (Sweden)

    Lixia Tian

    2013-11-01

    Full Text Available 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.

  16. 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...

  17. Increased oral availability and brain accumulation of the ALK inhibitor crizotinib by coadministration of the P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) inhibitor elacridar.

    NARCIS (Netherlands)

    Tang, S.C.; Nguyen, L.N.; Sparidans, R.W.; Wagenaar, E.; Beijnen, J.H.; Schinkel, A.H.

    2014-01-01

    Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non-small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in

  18. Use of P-glycoprotein gene probes to investigate anthelmintic resistance in Haemonchus contortus and comparison with Onchocerca volvulus

    NARCIS (Netherlands)

    Kwa, M.S.G.; Okoli, M.N.; Schulz-Key, H.; Okongkwo, P.O.; Roos, M.H.

    1998-01-01

    A P-glycoprotein gene probe from the sheep parasitic nematode Haemonchus contortus was developed and used to analyse restriction fragment length polymorphisms between susceptible isolates and isolates resistant to either benzimidazole; levamisole and benzimidazole; or benzimidazole, ivermectin and

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

    Science.gov (United States)

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

    2014-12-15

    A novel series of triazol-N-ethyl-tetrahydroisoquinoline based compounds were designed and synthesized via click chemistry. Most of the synthesized compounds showed P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal activities. Among them, compound 7 with little cytotoxicity towards GES-1 cells (IC50 >80μM) and K562/A02 cells (IC50 >80μM) exhibited more potency than verapamil (VRP) on increasing anticancer drug accumulation in K562/A02 cells. Moreover, compound 7 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 7 in reversing MDR revealed that it could remarkably increase the intracellular accumulation of both rhodamine-123 (Rh123) and adriamycin (ADM) in K562/A02 cells as well as inhibit their efflux from the cells. These results suggested that compound 7 showed more potency than the classical P-gp inhibitor VRP under the same conditions, which may be a promising P-gp-mediated MDR modulator for further development. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Possible Involvement of the Drug Transporters P Glycoprotein and Multidrug Resistance-Associated Protein Mrp2 in Disposition of Azithromycin

    OpenAIRE

    Sugie, Masami; Asakura, Emiko; Zhao, Ying Lan; Torita, Shoko; Nadai, Masayuki; Baba, Kenji; Kitaichi, Kiyoyuki; Takagi, Kenji; Takagi, Kenzo; HASEGAWA, Takaaki

    2004-01-01

    P glycoprotein and multidrug resistance-associated protein 2 (Mrp2), ATP-dependent membrane transporters, exist in a variety of normal tissues and play important roles in the disposition of various drugs. The present study seeks to clarify the contribution of P glycoprotein and/or Mrp2 to the disposition of azithromycin in rats. The disappearance of azithromycin from plasma after intravenous administration was significantly delayed in rats treated with intravenous injection of cyclosporine, a...

  1. 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.

  2. 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.

    Science.gov (United States)

    Liu, Qian; Hou, Jinfeng; Chen, Xiaoguang; Liu, Gengtao; Zhang, Dan; Sun, Hua; Zhang, Jinlan

    2014-01-01

    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.

  3. A P-glycoprotein gene serves as a component of the protective mechanisms against 2-tridecanone and abamectin in Helicoverpa armigera.

    Science.gov (United States)

    Xiang, Min; Zhang, Lei; Lu, Yao; Tang, Qiuling; Liang, Pei; Shi, Xueyan; Song, Dunlun; Gao, Xiwu

    2017-09-05

    P-glycoprotein (P-gp) exists in animals, fungi and bacteria and likely evolved as a defense mechanism against harmful substances. Here a cDNA (4054bp) encoding a putative P-glycoprotein gene from Helicoverpa armigera was cloned and named HaPgp1. This putative HaPgp1 sequence encoded a protein of 1253 amino acids with a molecular mass of approximately 137kDa. qPCR analyses demonstrated that the expression of HaPgp1 was significantly higher in 4th instar larvae when compared to other developmental stages. HaPgp1 transcripts were more abundant in the head and fat bodies than in other tissues. Compared with the control, the expression of HaPgp1 reach a peak at 12h after the treatment by 2-tridecanone in all tissues. However, the expression of HaPgp1 increased from 12h to 48h after treatment with abamectin in all tissues. Immunohistochemistry analyses also verified that 2-tridecanone and abamectin can induce the increase of HaPgp1 expression. RNAi of HaPgp1 significantly raised the mortality rate of larvae treated by 2-tridecanone and abamectin, as compared to control larvae fed with GFP dsRNA. These results illustrate the possible involvement of HaPgp1 as a component of the protective mechanisms to plant secondary chemicals such as 2-tridecanone and to certain classes of insecticides, like abamectin. Copyright © 2017. Published by Elsevier B.V.

  4. Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines

    NARCIS (Netherlands)

    Zaman, G. J.; Versantvoort, C. H.; Smit, J. J.; Eijdems, E. W.; de Haas, M.; Smith, A. J.; Broxterman, H. J.; Mulder, N. H.; de Vries, E. G.; Baas, F.

    1993-01-01

    Human cells can become multidrug resistant (MDR) by an increase in the activity of the MDR1 P-glycoprotein or by other, as yet unknown mechanisms, referred to as non-P-glycoprotein mediated MDR (non-Pgp MDR). S. P. C. Cole et al. [Science (Washington DC), 258: 1650-1654, 1992] recently reported that

  5. ANALYSIS OF THE EXPRESSION OF MRP, THE GENE FOR A NEW PUTATIVE TRANSMEMBRANE DRUG TRANSPORTER, IN HUMAN MULTIDRUG RESISTANT LUNG-CANCER CELL-LINES

    NARCIS (Netherlands)

    ZAMAN, GJR; VERSANTVOORT, CHM; SMIT, JJM; EIJDEMS, EWHM; DEHAAS, M; SMITH, AJ; BROXTERMAN, HJ; MULDER, NH; DEVRIES, EGE; BAAS, F; BORST, P

    1993-01-01

    Human cells can become multidrug resistant (MDR) by an increase in the activity of the MDR1 P-glycoprotein or by other, as vet unknown mechanisms, referred to as non-P-glycoprotein mediated MDR (non-Pgp MDR). S. P. C. Cole et al. [Science (Washington DC), 258: 1650-1654, 1992] recently reported that

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Where is it and how does it get there – intracellular localization and traffic of P-glycoprotein

    Directory of Open Access Journals (Sweden)

    Dong eFu

    2013-12-01

    Full Text Available P-glycoprotein (P-gp, an ATP-binding cassette (ABC, is able to transport structurally and chemically unrelated substrates. Overexpression of P-gp in cancer cells significantly decreases the intercellular amount of anticancer drugs, and results in multidrug resistance in cancer cells, a major obstacle in cancer chemotherapy. P-gp is mainly localized on the plasma membrane and functions as a drug efflux pump; however, P-gp is also localized in many intracellular compartments, such as endoplasmic reticulum, Golgi, endosomes and lysosomes. P-gp moves between the intracellular compartments and the plasma membrane in a microtubule-actin dependent manner. This review highlights our current understanding of 1 the intracellular localization of P-gp; 2 the trafficking and cycling pathways among the cellular compartments as well as between these compartments and the plasma membrane; and 3 the cellular factors regulating P-gp traffic and cycling. This review also presents a potential implication in overcoming P-gp-mediated multidrug resistance by targeting P-gp traffic and cycling pathways and impairing P-gp localization on the plasma membrane.

  8. Effects of P-Glycoprotein on the Transport of DL0410, a Potential Multifunctional Anti-Alzheimer Agent.

    Science.gov (United States)

    Pang, Xiaocong; Wang, Lin; Kang, De; Zhao, Ying; Wu, Song; Liu, Ai-Lin; Du, Guan-Hua

    2017-07-25

    In our study, we attempted to investigate the influences of P-glycoprotein (P-gp) on DL0410, a novel synthetic molecule for Alzheimer's disease (AD) treatment, for intestinal absorption and blood-brain barrier permeability in vitro and related binding mechanisms in silico. Caco-2, MDCK, and MDCK-MDR1 cells were utilized for transport studies, and homology modelling of human P-gp was built for further docking study to uncover the binding mode of DL0410. The results showed that the apparent permeability (Papp) value of DL0410 was approximately 1 × 10(-6) cm/s, indicating the low permeability of DL0410. With the presence of verapamil, the directional transport of DL0410 disappeared in Caco-2 and MDCK-MDR1 cells, suggesting that DL0410 should be a substrate of P-gp, which was also confirmed by P-gp ATPase assay. In addition, DL0410 could competitively inhibit the transport of Rho123, a P-gp known substrate. According to molecular docking, we also found that DL0410 could bind to the drug binding pocket (DBP), but not the nucleotide binding domain (NBD). In conclusion, DL0410 was a substrate as well as a competitive inhibitor of P-gp, and P-gp had a remarkable impact on the intestine and brain permeability of DL0410, which is of significance for drug research and development.

  9. 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

    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...

  10. 3D-QSAR modelling dataset of bioflavonoids for predicting the potential modulatory effect on P-glycoprotein activity.

    Science.gov (United States)

    Wongrattanakamon, Pathomwat; Lee, Vannajan Sanghiran; Nimmanpipug, Piyarat; Jiranusornkul, Supat

    2016-12-01

    The data is obtained from exploring the modulatory activities of bioflavonoids on P-glycoprotein function by ligand-based approaches. Multivariate Linear-QSAR models for predicting the induced/inhibitory activities of the flavonoids were created. Molecular descriptors were initially used as independent variables and a dependent variable was expressed as pFAR. The variables were then used in MLR analysis by stepwise regression calculation to build the linear QSAR data. The entire dataset consisted of 23 bioflavonoids was used as a training set. Regarding the obtained MLR QSAR model, R of 0.963, R (2)=0.927, [Formula: see text], SEE=0.197, F=33.849 and q (2)=0.927 were achieved. The true predictabilities of QSAR model were justified by evaluation with the external dataset (Table 4). The pFARs of representative flavonoids were predicted by MLR QSAR modelling. The data showed that internal and external validations may generate the same conclusion.

  11. 3D-QSAR modelling dataset of bioflavonoids for predicting the potential modulatory effect on P-glycoprotein activity

    Directory of Open Access Journals (Sweden)

    Pathomwat Wongrattanakamon

    2016-12-01

    Full Text Available The data is obtained from exploring the modulatory activities of bioflavonoids on P-glycoprotein function by ligand-based approaches. Multivariate Linear-QSAR models for predicting the induced/inhibitory activities of the flavonoids were created. Molecular descriptors were initially used as independent variables and a dependent variable was expressed as pFAR. The variables were then used in MLR analysis by stepwise regression calculation to build the linear QSAR data. The entire dataset consisted of 23 bioflavonoids was used as a training set. Regarding the obtained MLR QSAR model, R of 0.963, R2=0.927, Radj2=0.900, SEE=0.197, F=33.849 and q2=0.927 were achieved. The true predictabilities of QSAR model were justified by evaluation with the external dataset (Table 4. The pFARs of representative flavonoids were predicted by MLR QSAR modelling. The data showed that internal and external validations may generate the same conclusion.

  12. 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 Km and Vmax, for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC50) and the disassociation constant for an inhibitor/P-gp complex (Ki) 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.

  13. Funcionalidad de la glicoproteína P linfocitaria en la colitis ulcerosa P-glycoprotein functional activity in peripheral blood lymphocytes in ulcerative colitis

    Directory of Open Access Journals (Sweden)

    Catalina M. Cortada

    2009-10-01

    Full Text Available La glicoproteína gp-P, codificada por el MDR1 es una bomba de eflujo transmembrana capaz de movilizar una gran cantidad de fármacos de uso frecuente. Se expresa en la cara luminal del epitelio intestinal, en linfocitos y otros tejidos con función de barrera. MDR1 ha sido postulado como gen candidato en la colitis ulcerosa. El objetivo del presente trabajo fue evaluar la función de la gp-P en linfocitos aislados de sangre periférica de pacientes con colitis en actividad (n = 27 clasificados clínicamente como refractarios (n=16 o respondedores (n = 11 al tratamiento. Se estudió el eflujo de rodamina 123, sustrato de la glicoproteína P, en ausencia y presencia del inhibidor verapamilo (100 μM determinando la fluorescencia intracelular por citometría de flujo. Los resultados se expresaron evaluando el comportamiento de dos marcadores que corresponden al % de células que contienen máxima (M1/mínima (M2 concentración del colorante, reflejando inactividad/actividad de la bomba. Utilizando el test de Kruskal-Wallis y post test de Dunn, se observaron diferencias significativas entre refractarios versus respondedores (p P-glycoprotein (P-gp, encoded by MDR-1, is a transmembrane efflux pump that has been involved in relevant clinical drug transport. It is expressed in lymphocytes, luminal epithelium of colon and other tissues with barrier function. MDR1 was proposed as a candidate gene for ulcerative colitis. The aim of the present work was to investigate the role of P-gp in therapeutic response of ulcerative colitis by studying its functionality in lymphocytes isolated from peripheral blood. Samples were taken from 27 patients with active colitis classified clinically in refractory (n = 16 and responders (n = 11 to treatment. Rhodamine 123 (a fluorescent P-glycoprotein substrate efflux was studied by flow cytometry as absence and presence of an inhibitor (verapamil, 100 μM. Data were expressed evaluating the behaviour of two markers defined

  14. Imaging Multidrug Resistance P-glycoprotein Transport Function Using MicroPET with Technetium-94m-Sestamibi

    Directory of Open Access Journals (Sweden)

    Heather M. Bigott

    2005-01-01

    Full Text Available The best characterized mechanism of multidrug resistance (MDR in cancer involves the MDR1 efflux transporter P-glycoprotein (Pgp. The positron-emitting radiotracer hexakis (2-methoxyisobutylisonitrile-94mTc (94mTc-MIBI was synthesized and validated in cell transport studies as a substrate for MDR1 Pgp. In vivo small-scale PET imaging and biodistribution studies of mdr1a/1b (−/− gene deleted and wild-type mice demonstrated the use of 94mTc-MIBI to detect Pgp function. The reversal effect of a Pgp modulator was shown in tissue distribution studies of KB 3–1 (Pgp- and KB 8–5 (Pgp+ tumor-bearing nude mice. The current 94mTc-MIBI experiments parallel previous studies employing 99mTc-MIBI, showing essentially identical performance of the two technetium radiotracers and providing biological validation of 94mTc-MIBI for PET imaging of multidrug resistance.

  15. 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.

  16. Three- and four-class classification models for P-glycoprotein inhibitors using counter-propagation neural networks.

    Science.gov (United States)

    Thai, K-M; Huynh, N-T; Ngo, T-D; Mai, T-T; Nguyen, T-H; Tran, T-D

    2015-01-01

    P-glycoprotein (P-gp) is an ATP binding cassette (ABC) transporter that helps to protect several certain human organs from xenobiotic exposure. This efflux pump is also responsible for multi-drug resistance (MDR), an issue of the chemotherapy approach in the fight against cancer. Therefore, the discovery of P-gp inhibitors is considered one of the most popular strategies to reverse MDR in tumour cells and to improve therapeutic efficacy of commonly used cytotoxic drugs. Until now, several generations of P-gp inhibitors have been developed but they have largely failed in preclinical and clinical studies due to lack of selectivity, poor solubility and severe pharmacokinetic interactions. In this study, three models (SION, SIO, SIN) to classify specific 'true' P-gp inhibitors as well as three other models (CPBN, CPB1, CPN) to distinguish between P-gp inhibitors, CYP 3A inhibitors and co-inhibitors of these proteins with rather high accuracy values for the test set and the external set were generated based on counter-propagation neural networks (CPG-NN). Such three and four-class classification models helped provide more information about the bioactivities of compounds not only on one target (P-gp), but also on a combination of multiple targets (P-gp, CYP 3A).

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

    Science.gov (United States)

    Kikete, Siambi; Liang, Rongjia; Wang, Lili

    2017-01-01

    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. PMID:28250793

  18. An in silico approach for screening flavonoids as P-glycoprotein inhibitors based on a Bayesian-regularized neural network

    Science.gov (United States)

    Wang, Yong-Hua; Li, Yan; Yang, Sheng-Li; Yang, Ling

    2005-03-01

    P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, functions as a biological barrier by extruding cytotoxic agents out of cells, resulting in an obstacle in chemotherapeutic treatment of cancer. In order to aid in the development of potential P-gp inhibitors, we constructed a quantitative structure-activity relationship (QSAR) model of flavonoids as P-gp inhibitors based on Bayesian-regularized neural network (BRNN). A dataset of 57 flavonoids collected from a literature binding to the C-terminal nucleotide-binding domain of mouse P-gp was compiled. The predictive ability of the model was assessed using a test set that was independent of the training set, which showed a standard error of prediction of 0.146 ± 0.006 (data scaled from 0 to 1). Meanwhile, two other mathematical tools, back-propagation neural network (BPNN) and partial least squares (PLS) were also attempted to build QSAR models. The BRNN provided slightly better results for the test set compared to BPNN, but the difference was not significant according to F-statistic at p = 0.05. The PLS failed to build a reliable model in the present study. Our study indicates that the BRNN-based in silico model has good potential in facilitating the prediction of P-gp flavonoid inhibitors and might be applied in further drug design.

  19. 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.

  20. Computational predictive models for P-glycoprotein inhibition of in-house chalcone derivatives and drug-bank compounds.

    Science.gov (United States)

    Ngo, Trieu-Du; Tran, Thanh-Dao; Le, Minh-Tri; Thai, Khac-Minh

    2016-11-01

    The human P-glycoprotein (P-gp) efflux pump is of great interest for medicinal chemists because of its important role in multidrug resistance (MDR). Because of the high polyspecificity as well as the unavailability of high-resolution X-ray crystal structures of this transmembrane protein, ligand-based, and structure-based approaches which were machine learning, homology modeling, and molecular docking were combined for this study. In ligand-based approach, individual two-dimensional quantitative structure-activity relationship models were developed using different machine learning algorithms and subsequently combined into the Ensemble model which showed good performance on both the diverse training set and the validation sets. The applicability domain and the prediction quality of the developed models were also judged using the state-of-the-art methods and tools. In our structure-based approach, the P-gp structure and its binding region were predicted for a docking study to determine possible interactions between the ligands and the receptor. Based on these in silico tools, hit compounds for reversing MDR were discovered from the in-house and DrugBank databases through virtual screening using prediction models and molecular docking in an attempt to restore cancer cell sensitivity to cytotoxic drugs.

  1. Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics.

    Science.gov (United States)

    Pan, Lurong; Aller, Stephen G

    2015-01-20

    P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

  2. Effects of the antioxidant baicalein on the pharmacokinetics of nimodipine in rats: a possible role of P-glycoprotein and CYP3A4 inhibition by baicalein.

    Science.gov (United States)

    Cho, Young-Ah; Choi, Jun-Shik; Burm, Jin-Pil

    2011-01-01

    The reduced bioavailability of nimodipine after oral administration might not only be due to the metabolizing enzyme cytochrome P450 3A4(CYP3A4) but also to the P-glycoprotein efflux transporter in the small intestine. The aim of this study was to investigate the effects of baicalein on the pharmacokinetics of nimodipine in rats. The effect of baicalein on P-glycoprotein and CYP3A4 activity was evaluated. A single dose of nimodipine was administered intravenously (3 mg/kg) and orally (12 mg/kg) to rats in the presence and absence of baicalein (0.4, 2 and 8 mg/kg). Baicalein inhibited CYP3A4 enzyme activity in a concentration-dependent manner, with a 50% inhibition concentration (IC(50)) of 9.2 μM. In addition, baicalein significantly enhanced the cellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-glycoprotein. Baicalein significantly altered the pharmacokinetics of orally administered nimodipine. Compared to the oral control group given nimodipine alone, the area under the plasma concentration-time curve (AUC(0-∞)) and the peak plasma concentration (C(max)) of nimodipine significantly increased (p nimodipine in the presence of baicalein (2 and 8 mg/kg) was 31.0-35.3%, which was significantly enhanced (p nimodipine was 1.39- to 1.58-fold greater than that of the control group. The pharmacokinetics of intravenous nimodipine were not affected by baicalein in contrast to those of oral nimodipine. Baicalein significantly enhanced the oral bioavailability of nimodipine, which may be mainly due to inhibition of the CYP3A4-mediated metabolism of nimodipine in the small intestine and/or in the liver and the inhibition of the P-glycoprotein efflux pump in the small intestine by baicalein. The increase in oral bioavailability of nimodipine in the presence of baicalein should be taken into consideration as a potential drug interaction between nimodipine and baicalein.

  3. P-glycoproteins and hepatobiliary secretion: studies on cloning, function, and expression

    OpenAIRE

    Hooiveld, Guido Jacobus Etienne Johannes

    2000-01-01

    De lver produceert gal en is als de grootste klier van ons lichaam te beschouwen. Gal is een waterige vloeistof die van de lever via de galwegen naar de darmen loopt en daar een belangrijke rol speelt in de voedselvertering. Teven voert de lever via de gal een groot aantal stoffen uit naar de darmen.... Zie: Samenvatting

  4. Influence of P-glycoprotein modulation on plasma concentrations and pharmacokinetics of orally administered prednisolone in dogs.

    Science.gov (United States)

    Van der Heyden, Sara; Croubels, Siska; Gadeyne, Caroline; Ducatelle, Richard; Daminet, Sylvie; Murua Escobar, Hugo; Sterenczak, Katharina; Polis, Ingeborgh; Schauvliege, Stijn; Hesta, Myriam; Chiers, Koen

    2012-06-01

    To evaluate the impact of modulation of the membrane-bound efflux pump P-glycoprotein (P-gp) on plasma concentrations of orally administered prednisolone in dogs. 7 healthy adult Beagles. Each dog received 3 treatments (control [no treatment], rifampicin [100 mg/d, PO, for 21 days, as an inducer of P-gp], and ketoconazole [100 mg/d, PO, for 21 days, as an inhibitor of P-gp]). A single dose of prednisolone (1 mg/kg, PO) was administered on day 8 of each treatment period. There was a 7-day washout period between subsequent treatments. Plasma concentrations of prednisolone were determined by use of a validated liquid chromatography-tandem mass spectrometry method. Duodenum and colon biopsy specimens were obtained endoscopically from anesthetized dogs and assessed for P-gp protein labeling via immunohistochemical analysis and mRNA quantification via real-time PCR assay. Total fecal collection was performed for evaluation of effects of P-gp modulation on digestion of nutrients. Rifampicin treatment upregulated duodenal P-gp in dogs and significantly reduced the area under the plasma concentration-time curve of prednisolone. Ketoconazole typically downregulated expression of duodenal P-gp, with a subsequent increase in the area under the plasma concentration-time curve of prednisolone. There was a noticeable interindividual difference in response. Digestion of nutrients was not affected. Modulation of P-gp expression influenced plasma concentrations of prednisolone after oral administration in dogs. Thus, treatment response to prednisolone may be influenced by coadministration of P-gp-modulating medications or feed ingredients.

  5. Doxycycline as an inhibitor of p-glycoprotein in the alpaca for the purpose of maintaining avermectins in the CNS during treatment for parelaphostrongylosis.

    Science.gov (United States)

    Agbedanu, Prince N; Anderson, Kristi L; Brewer, Matthew T; Carlson, Steve A

    2015-09-15

    Meningeal worms (Parelaphostrongylus tenuis) are a common malady of alpacas, often refractory to conventional treatments. Ivermectin is a very effective anthelmintic used against a variety of parasites but this drug is not consistently effective against alpaca meningeal worms once the parasite has gained access to the CNS, even if used in a protracted treatment protocol. Ivermectin is not effective against clinical cases of P. tenuis, raising the possibility that the drug is not sustained at therapeutic concentrations in the central nervous system (CNS). A specific protein (designated as p-glycoprotein (PGP)) effluxes ivermectin from the brain at the blood-brain barrier, thus hampering the maintenance of therapeutic concentrations of the drug in the CNS. Minocycline is a synthetic tetracycline antibiotic with an excellent safety profile in all animals tested to date. Minocycline has three unique characteristics that could be useful for treating meningeal worms in conjunction with ivermectin. First, minocycline is an inhibitor of PGP at the blood-brain barrier and this inhibition could maintain effective concentrations of ivermectin in the brain and meninges. Second, minocycline protects neurons in vivo through a number of different mechanisms and this neuroprotection could alleviate the potential untoward neurologic effects of meningeal worms. Third, minocycline is a highly lipid-soluble drug, thus facilitating efficient brain penetration. We thus hypothesized that minocycline will maintain ivermectin, or a related avermectin approved in ruminants (abamectin, doramectin, or eprinomectin), in the alpaca CNS. To test this hypothesis, we cloned the gene encoding the alpaca PGP, expressed the alpaca PGP in a heterologous expression system involving MDCK cells, and measured the ability of minocycline to inhibit the efflux of avermectins from the MDCK cells; doxycycline was used as a putative negative control (based on studies in other species). Our in vitro studies

  6. 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 BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

  7. The P-glycoprotein (ABCB1) linker domain encodes high-affinity binding sequences to alpha- and beta-tubulins.

    Science.gov (United States)

    Georges, Elias

    2007-06-26

    P-Glycoprotein (or ABCB1) has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs. ABCB1 encodes a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of approximately 90 amino acids, the "linker domain" with as yet unknown function(s). In this report, we demonstrate a role for this domain in binding to other cellular proteins. Using overlapping hexapeptides that encode the entire amino acid sequence of the linker domain of human ABCB1, we show a direct and specific binding between sequences in the linker domain and several intracellular proteins. Three different polypeptide sequences [617EKGIYFKLVTM627 (LDS617-627), 657SRSSLIRKRSTRRSVRGSQA676 (LDS657-676), and 693PVSFWRIMKLNLT705 (LDS693-705)] in the linker domain interacted tightly with several proteins with apparent molecular masses of approximately 80, 57, and 30 kDa. Interestingly, only the 57 kDa protein (or P57) interacted with all three different sequences of the linker domain. Purification and partial N-terminal amino acid sequencing of P57 showed that it encodes the N-terminal amino acids of alpha- and beta-tubulins. The identity of the P57 interacting protein as tubulins was further confirmed by Western blotting using monoclonal antibodies to alpha- and beta-tubulin. Taken together, the results of this study provide the first evidence for ABCB1 protein interaction mediated by sequences in the linker domain. These findings are likely to provide further insight into the functions of ABCB1 in normal and drug resistant tumor cells.

  8. Evaluation of memory enhancing clinically available standardized extract of Bacopa monniera on P-glycoprotein and cytochrome P450 3A in Sprague-Dawley rats.

    Directory of Open Access Journals (Sweden)

    Rajbir Singh

    Full Text Available Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold and digoxin (1.3 and 1.2 fold, respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp.

  9. The orally administered P-glycoprotein inhibitor R101933 does not alter the plasma pharmacokinetics of docetaxel

    NARCIS (Netherlands)

    L. van Zuylen; A. Sparreboom (Alex); A. van der Gaast (Ate); M.E.L. van der Burg (Maria); V.M. van Beurden (Vera); C.J. Bol; R. Woestenborghs; P.A. Palmer; J. Verweij (Jaap)

    2000-01-01

    textabstractThis Phase I study was performed to assess the feasibility of combining docetaxel with the new P-glycoprotein inhibitor R101933 and to determine the dose limiting toxicity of this combination. Fifteen patients received oral R101933 alone at a dose escalated

  10. 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...

  11. Reduced plasma cholesterol and increased fecal sterol loss in multidrug resistance gene 2 P-glycoprotein-deficient mice

    NARCIS (Netherlands)

    Voshol, P. J.; Havinga, R.; Wolters, H.; Ottenhoff, R.; Princen, H. M.; Oude Elferink, R. P.; Groen, A. K.; Kuipers, F.

    1998-01-01

    BACKGROUND & AIMS: mdr2 P-glycoprotein (Pgp) deficiency in mice leads to the absence of biliary phospholipids and cholesterol in the presence of normal bile salt secretion. The aim of this study was to evaluate the importance of the biliary pathway in cholesterol homeostasis by determining the

  12. Reduced plasma cholesterol and increased fecal sterol loss in multidrug resistance gene 2 P-glycoprotein-deficient mice

    NARCIS (Netherlands)

    Voshol, PJ; Havinga, R; Wolters, H; Ottenhoff, R; Princen, HMG; Elferink, RPJO; Groen, AK; Kuipers, F

    Background & Aims: mdr2 P-glycoprotein (Pgp) deficiency in mice leads to the absence of biliary phospholipids and cholesterol in the presence of normal bile salt secretion. The aim of this study was to evaluate the importance of the biliary pathway in cholesterol homeostasis by determining the

  13. Positron emission tomography studies on binding of central nervous system drugs and P-glycoprotein function in the rodent brain

    NARCIS (Netherlands)

    Elsinga, Philip H.; Hendrikse, N. Harry; Bart, Joost; van Waarde, Aren; Vaalburg, Willem

    2005-01-01

    The permeability of the blood-brain barrier (BBB) is one of the factors determining the bioavailability of drugs in the brain. The BBB only allows passage of lipophilic drugs by passive diffusion. However, some lipophilic drugs hardly enter the brain. The transmembrane protein P-glycoprotein (P-gp)

  14. In-vitro evaluation of the P-glycoprotein interactions of a series of potentially CNS-active Amaryllidaceae alkaloids

    DEFF Research Database (Denmark)

    Eriksson, André Huss; Rønsted, Nina; Jäger, Anna Katharina

    2012-01-01

    Drug compounds interacting with the blood-brain barrier efflux transporter P-glycoprotein (P-gp) might have limited access to brain tissue. The aim of the present study was to evaluate whether nine potentially CNS-active Amaryllidaceae alkaloids of the crinine, lycorine and galanthamine types int...

  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. In vivo imaging of hepatobiliary transport function mediated by multidrug resistance associated protein and P-glycoprotein

    NARCIS (Netherlands)

    Hendrikse, NH; Kuipers, F; Meijer, C; Havinga, R; Bijleveld, CMA; van der Graaf, WTA; Vaalburg, W; de Vries, EGE

    Multidrug resistance associated proteins (MRPs) and P-glycoprotein (P-gp) are involved in hepatobiliary transport of various compounds. Our aim was (1) to define transporter specificity of the cholescintigraphic agents Tc-99m-HIDA and Tc-99m-MIBI, which are used clinically for myocardial perfusion

  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. 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. The impact of insulin on chemotherapeutic sensitivity to 5-fluorouracil in gastric cancer cell lines SGC7901, MKN45 and MKN28.

    Science.gov (United States)

    Wei, Zhao; Liang, Li; Junsong, Liu; Rui, Chen; Shuai, Chang; Guanglin, Qiu; Shicai, He; Zexing, Wang; Jin, Wang; Xiangming, Che; Shufeng, Wang

    2015-06-18

    The role of insulin in the pathogenesis of cancer has been increasingly emphasized because of the high incidence of obesity and metabolic syndrome and their correlated complication including cancer. This study aimed to explore the impact of insulin on chemoresistance to 5-fluorouracil in gastric cancer and the possible mechanisms. Tissue samples of gastric cancer and adjacent normal gastric mucosa from patients with or without obesity were performed immunohistochemical staining for P-glycoprotein. The follow-up was done after the surgical treatment. The effect of insulin on chemotherapeutic sensitivity of the three gastric cancer cell lines to 5-fluorouracil was evaluated by pre-incubation with insulin before administration of 5-fluorouracil. The expression of P-glycoprotein was determined by Western blotting. P-glycoprotein were overexpressed in tissues from patients who suffered gastric cancer and were higher in those simultaneously suffered gastric cancer and obesity. Addition of 1 μM insulin remarkably promoted the proliferation of SGC7901, MKN45 and MKN28 cells and decreased the cytotoxicity of 5-fluorouracil. In addition, the expression of P-glycoprotein was upregulated in SGC7901, MKN45 and MKN28 cells. Insulin improved the proliferation of gastric cancer cell lines and contributed to chemoresistance of gastric cancer cells to 5-fluorouracil which is likely to involve upregulation of P-glycoprotein.

  20. Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

    NARCIS (Netherlands)

    Pol, van der M.A.; Feller, N.; Roseboom, M; Moshaver, B; Westra, G; Broxterman, H.J.; Ossenkoppele, G.J.; Schuurhuis, G.J.

    2003-01-01

    50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by

  1. Co-delivery of Se nanoparticles and pooled SiRNAs for overcoming drug resistance mediated by P-glycoprotein and class III β-tubulin in drug-resistant breast cancers.

    Science.gov (United States)

    Zheng, Wenjing; Yin, Tiantian; Chen, Qingchang; Qin, Xiuying; Huang, Xiaoquan; Zhao, Shuang; Xu, Taoyuan; Chen, Lanmei; Liu, Jie

    2016-02-01

    Drug resistance mediated by P-glycoprotein (P-gp) and class III β-tubulin (β-tubulin III) is a major barrier in microtubule-targeting cancer chemotherapy. In this study, layered double hydroxide nanoparticles (LDHs) were employed to simultaneously deliver selenium (Se) and pooled small interfering RNAs (siRNAs) to achieve therapeutic efficacy. LDH-supported Se nanoparticles (Se@LDH) were compacted with siRNAs (anti-P-gp and anti-β-tubulin III) via electrostatic interactions, which could protect siRNA from degradation. Se@LDH showed excellent abilities to deliver siRNA into cells, including enhancing siRNA internalization, and promoting siRNA escape from endosomes. siRNA transfection experiments further confirmed a higher gene silencing efficiency of Se@LDH than LDH. Interestingly, we found Se@LDH may be a microtubule (MT) stabilizing agent which could inhibit cell proliferation by blocking cell cycle at G2/M phase, disrupting normal mitotic spindle formation and inducing cell apoptosis. When complexed with different specific siRNAs, Se@LDH/siRNA nanoparticles, especially the Se@LDH-pooled siRNAs, exhibit an efficient gene-silencing effect that significantly downregulate the expression of P-gp and β-tubulin III. Moreover, Se@LDH-pooled siRNAs could induce cell apoptosis, change cell morphology and increase cellular ROS levels through change the expression of Bcl-2/Bax, activation of caspase-3, PI3K/AKT/mTOR and MAPK/ERK pathways. These results suggested that co-delivery of Se and pooled siRNAs may be a promising strategy for overcoming the drug resistance mediated by P-gp and β-tubulin III in drug-resistant breast cancers. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  2. Functional assay and structure-activity relationships of new third-generation P-glycoprotein inhibitors.

    Science.gov (United States)

    Müller, Henrik; Pajeva, Ilza K; Globisch, Christoph; Wiese, Michael

    2008-03-01

    Twenty-eight compounds, including 24 structurally related derivatives of tariquidar synthesized in our laboratory, and four XR compounds, reported by Xenova group Ltd, were investigated by the Hoechst 33342 and Calcein AM functional assays for estimation of their inhibitory effects on the transport activity of P-glycoprotein (P-gp). A high correlation between the effects obtained in both assays was observed at the substrate concentrations used. The analyses of kinetics data from experiments at different substrate concentrations revealed non-competitive inhibition in the Calcein AM assay and competitive inhibition in the Hoechst 33342 assay. The 3D structures of the compounds were further aligned on Hoechst 33342 using flexible and pharmacophore alignments. The results suggested that inhibitors could interact with the H-binding site of P-gp and this could potentially be achieved by different ways of binding. The best 3D-QSAR models, generated by CoMFA and CoMSIA, yielded an internal predictive squared correlation coefficient higher than 0.8 and included electrostatic, steric, hydrogen bond acceptor, and hydrophobic fields. Validation of the models on an external test set of 30 XR compounds gave predictive squared correlation coefficients of up to 0.66. An excellent correspondence between the experimental and modeled activities of the test compounds was observed. The models can be used for prediction and rational design of new P-gp inhibitors.

  3. Intestinal absorption of pallidifloside D are limited by P-glycoprotein in mice.

    Science.gov (United States)

    Wang, Ming-Yu; Yang, Ming; Hou, Pi-Yong; Chen, Xiu-Bo; Li, Hong-Gang; Yan, Jiu-Xing; Zhang, Jun; Zhang, Yan-Wen; Wu, Xiao-Hui

    2017-08-03

    1. Pallidifloside D, a saponin glycoside constituent from the total saponins of Smilax riparia, had been proved to be very effective in hyperuricemic control. But it is poorly bioavailable after oral administration. Here, we determined the role of P-glycoprotein (P-gp) in the intestinal absorption of Pallidifloside D. 2. We found that Pallidifloside D significantly stimulated P-gp ATPase activity in vitro ATPase assay with a small EC50 value of 0.46 μM. 3. In the single-pass perfused mouse intestine model, the absorption of Pallidifloside D was not favored in the small intestine (duodenum, jejunum and ileum) with a P*w value of 0.35-0.78. By contrast, this compound was well-absorbed in the colon with a P*w value of 1.23. The P-gp inhibitors cyclosporine significantly enhanced Pallidifloside D absorption in all four intestinal segments (duodenum, jejunum, ileum and colon) and the fold change ranged from 5.5 to 15.3. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of Pallidifloside D by a 2.5-fold after oral administration. 4. These results suggest that P-gp-mediated efflux is a limiting factor for intestinal absorption of Pallidifloside D in mice.

  4. 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.

  5. Advances in P-glycoprotein-based approaches for delivering anticancer drugs: pharmacokinetic perspective and clinical relevance.

    Science.gov (United States)

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

    2014-01-01

    P-glycoprotein (P-gp) is a multi-specific efflux transporter belonging to ATP-binding cassette (ABC) transporter family, encoded by the ABCB1 gene, which significantly impacts the pharmacokinetics as well as multidrug resistance of anticancer drugs. This review explores how human P-gp transporters modulate the pharmacokinetics of anticancer drugs and emerging strategies to modulate their function. The key findings in direct modulation by various P-gp inhibitors on pharmacokinetics of various anticancer P-gp substrates are described. The role of pharmaceutical excipients as P-gp inhibitor with the focus on the recent development in novel drug delivery systems to modulate pharmacokinetics of anticancer drugs is also outlined. The concomitant use of anticancer P-gp substrate and P-gp inhibitor is an effective and safe way to enhance the bioavailability of anticancer drugs. The poor bioavailability and toxicity of anticancer drugs limit their therapeutic efficacy. These characteristics can be improved by using various nanocarriers which exhibited a high potential to bypass this efflux protein. The best combination of P-gp inhibitor and substrate anticancer drug in a single nanocarrier formulation is a future challenge and is still probably some years away from the marketplace.

  6. 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...

  7. 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.

  8. Dexverapamil to modulate vinblastine resistance in metastatic renal cell carcinoma

    NARCIS (Netherlands)

    G.H.J. Mickisch; M.A. Noordzij (Marinus); A.v.d. Gaast (A. v d); P. Gebreamlack (P.); K.U. Köhrmann (K.); E. Mogler-Drautz (E.); N. Kupper (Nina); F.H. Schröder (Fritz)

    1995-01-01

    textabstractMultidrug resistance (MDR) in a variety of human tumours such as renal cell carcinoma (RCC) is thought to be caused by expression of the MDR1 gene and may be reversed by applying modern chemosensitisers such as dexverapamil, which inhibit the MDR1 gene product P-glycoprotein. This

  9. A short cross-linker activates human P-glycoprotein missing a catalytic carboxylate.

    Science.gov (United States)

    Loo, Tip W; Clarke, David M

    2017-12-01

    P-glycoprotein (P-gp) is an ATP-dependent drug pump that protects us from toxic agents and confers multidrug resistance. It has a tweezer-like structure with each arm consisting of a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates bind to sites within the TMDs to activate ATPase activity by promoting a tweezer-like closing of the gap between the NBDs. The catalytic carboxylates may be critical for NBD movements because the E556Q(NBD1) or E1201Q(NBD2) mutation inhibited drug-stimulated ATPase activity. If the catalytic carboxylates were components of the mechanism to bring the NBDs together, then we predicted that insertion of a flexible cross-linker between the arms would increase ATPase activity of the mutants. We found that cross-linking (between L175C(TMD1) and N820C(TMD2)) with a short flexible cross-linker (7.8Å maximum) restored high levels of drug-stimulated ATPase activity of the E556Q or E1201Q mutants. Cross-linking with a longer cross-linker (22Å maximum) however, did not restore activity. Cross-linking could not rescue all ATPase deficient mutants. For example, cross-linking L175C/N820C with short or long cross-linkers did not activate the H-loop mutants H587A or H1232A or the Walker A K433M or K1076M mutants. The results suggest that the E556 and E1201 catalytic carboxylates are part of a spring-like mechanism that is required to facilitate movements between the open and closed conformations of P-gp during ATP hydrolysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. P-glycoprotein-like protein, a possible genetic marker for ivermectin resistance selection in Onchocerca volvulus.

    Science.gov (United States)

    Bourguinat, Catherine; Ardelli, Bernadette F; Pion, Sébastien D S; Kamgno, Joseph; Gardon, Jacques; Duke, Brian O L; Boussinesq, Michel; Prichard, Roger K

    2008-04-01

    Ivermectin (IVM) is the only safe drug for mass-treatment of onchocerciasis. IVM resistance has been reported in gastrointestinal nematode parasites of animals. A reduction in response to IVM in Onchocerca volvulus could have significant consequences for the onchocerciasis control programs. We have found evidence that, in O. volvulus, repeated IVM treatment selects for specific alleles, of P-glycoprotein-like protein (PLP), a half-sized ABC transporter. In this study, O. volvulus samples were derived from a clinical trial in Cameroon, in which patients were sampled before, and following 3 years (1994-1997) of IVM treatments. There were four treatment groups: 150 microg/kg (1 x p.a. or 4 x p.a.) and 800 microg/kg (1 x p.a. or 4 x p.a.). DNA of O. volvulus macrofilariae was genotyped over a 476bp region of the PLP gene and at two control genes. Of the six polymorphic positions found in the PLP amplicon, three of them showed significant selection after 4 x p.a. treatment with IVM (total of 13 IVM treatments) in female worms, and one of the same single nucleotide polymorphisms (SNPs) showed significant selection in the male worms. One of the selected SNPs in the female worms caused an amino acid coding change in the putative protein sequence. We found a clear selection of some genotypes, a high SNPs association and a loss of polymorphism following 4 x p.a. treatment with IVM. These PLP SNPs and genotypes could be useful markers to follow selection for IVM resistance in the field.

  11. Can celecoxib affect P-glycoprotein-mediated drug efflux? A microPET study

    Energy Technology Data Exchange (ETDEWEB)

    Vries, Erik F.J. de [Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, PO Box 30.001, 9700 RB Groningen (Netherlands)], E-mail: e.f.j.de.vries@ngmb.umcg.nl; Doorduin, Janine; Vellinga, Namkje A.R.; Waarde, Aren van; Dierckx, Rudi A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, PO Box 30.001, 9700 RB Groningen (Netherlands); Klein, Hans C. [Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, PO Box 30.001, 9700 RB Groningen (Netherlands); Department of Psychiatry, University Medical Center Groningen, University of Groningen, PO Box 30.001, 9700 RB Groningen (Netherlands)

    2008-05-15

    Introduction: P-glycoprotein (Pgp) is an efflux pump that protects vital organs like the brain from toxic substances, but which is also associated with therapy resistance. The anti-inflammatory drug celecoxib potentiates the efficacy of several cytostatic and neurotropic drugs that are known Pgp substrates. To clarify whether Pgp is involved in the sensitizing effect of celecoxib, we investigated in vivo whether celecoxib is a substrate of Pgp and whether it can affect the efflux activity of the pump. Methods: In control rats and in rats treated with the Pgp modulator cyclosporin A (CsA), cerebral accumulation of radiolabeled [{sup 11}C]celecoxib was investigated by ex vivo biodistribution and micro-positron emission tomography imaging. In addition, the effect of unlabeled celecoxib and CsA (positive control) on the cerebral uptake of the Pgp substrate [{sup 11}C]verapamil was studied. Results: [{sup 11}C]Celecoxib uptake in rat brain was relatively high and homogeneously distributed. Treatment of rats with CsA only marginally increased cerebral tracer uptake, which is most likely due to reduced tracer clearance from plasma. [{sup 11}C]Verapamil brain uptake was more than 10-fold higher after treatment with CsA. In contrast, a high dose of celecoxib increased cerebral [{sup 11}C]verapamil uptake only twofold, which was accompanied by a similar increase in tracer concentration in plasma. Conclusions: This study shows that celecoxib is not a substrate of Pgp and does not substantially affect the Pgp-mediated efflux of [{sup 11}C]verapamil. Therefore, celecoxib-induced augmentation of the efficacy of chemotherapeutic and neurotropic drugs must be due to another mechanism than modulation of Pgp-mediated drug efflux.

  12. In silico analysis of the binding of anthelmintics to Caenorhabditis elegans P-glycoprotein 1

    Directory of Open Access Journals (Sweden)

    Marion A. David

    2016-12-01

    Full Text Available Macrocyclic lactones (ML are important anthelmintics used in animals and humans against parasite nematodes, but their therapeutic success is compromised by the spread of ML resistance. Some ABC transporters, such as p-glycoproteins (Pgps, are selected and overexpressed in ML-resistant nematodes, supporting a role for some drug efflux proteins in ML resistance. However, the role of such proteins in ML transport remains to be clarified at the molecular level. Recently, Caenorhabditis elegans Pgp-1 (Cel-Pgp-1 has been crystallized, and its drug-modulated ATPase function characterized in vitro revealed Cel-Pgp-1 as a multidrug transporter. Using this crystal structure, we have developed an in silico drug docking model in order to study the binding of ML and other anthelmintic drugs to Cel-Pgp-1. All tested ML bound with high affinity in a unique site, within the inner chamber of the protein, supporting that ML may be transported by Cel-Pgp-1. Interestingly, interacting residues delineate a ML specific fingerprint involving H-bonds, including T1028. In particular, benzofurane and spiroketal moieties bound to specific sub-sites. When compared with the aglycone ML, such as moxidectin and ivermectin aglycone, avermectin anthelmintics have significant higher affinity for Cel-Pgp-1, likely due to the sugar substituent(s that bind to a specific area involving H-bonds at Y771. Triclabendazole, closantel and emodepside bound with good affinities to different sub-sites in the inner chamber, partially overlapping with the ML binding site, suggesting that they could compete for Cel-Pgp-1-mediated ML transport. In conclusion, this work provides novel information on the role of nematode Pgps in transporting anthelmintics, and a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of clinically-relevant nematode Pgps, to improve anthelmintic therapeutics.

  13. Trimethoxybenzanilide-based P-glycoprotein modulators: an interesting case of lipophilicity tuning by intramolecular hydrogen bonding.

    Science.gov (United States)

    Tardia, Piero; Stefanachi, Angela; Niso, Mauro; Stolfa, Diana Antonella; Mangiatordi, Giuseppe Felice; Alberga, Domenico; Nicolotti, Orazio; Lattanzi, Gianluca; Carotti, Angelo; Leonetti, Francesco; Perrone, Roberto; Berardi, Francesco; Azzariti, Amalia; Colabufo, Nicola Antonio; Cellamare, Saverio

    2014-08-14

    One of the principal reasons for the chemotherapy failure is the overexpression of drug efflux pumps, ABCB1 (also known as MDR1 or P-gp) and ABCC1 (also known as MRP1), whose inhibition remains a priority to circumvent drug resistance. We have recently shown a clear trend between lipophilicity and P-glycoprotein inhibitory activity for a class of galloyl-based modulators targeting P-glycoprotein and MRP1. Herein we report a new series of polymethoxy benzamides, whose lipophilicity was modulated through the establishment of an intramolecular hydrogen bond (IMHB) which allows reaching of P-gp inhibitory activity at the submicromolar IC50 level. The present study provides a strong rationale for candidates in the presence of IMHB as a key element for a high P-gp inhibitory activity.

  14. Possible involvement of the drug transporters P glycoprotein and multidrug resistance-associated protein Mrp2 in disposition of azithromycin.

    Science.gov (United States)

    Sugie, Masami; Asakura, Emiko; Zhao, Ying Lan; Torita, Shoko; Nadai, Masayuki; Baba, Kenji; Kitaichi, Kiyoyuki; Takagi, Kenji; Takagi, Kenzo; Hasegawa, Takaaki

    2004-03-01

    P glycoprotein and multidrug resistance-associated protein 2 (Mrp2), ATP-dependent membrane transporters, exist in a variety of normal tissues and play important roles in the disposition of various drugs. The present study seeks to clarify the contribution of P glycoprotein and/or Mrp2 to the disposition of azithromycin in rats. The disappearance of azithromycin from plasma after intravenous administration was significantly delayed in rats treated with intravenous injection of cyclosporine, a P-glycoprotein inhibitor, but was normal in rats pretreated with intraperitoneal injection erythromycin, a CYP3A4 inhibitor. When rats received an infusion of azithromycin, cyclosporine and probenecid, a validated Mrp2 inhibitor, significantly decreased the steady-state biliary clearance of azithromycin to 5 and 40% of the corresponding control values, respectively. However, both inhibitors did not alter the renal clearance of azithromycin, suggesting the lack of renal tubular secretion of azithromycin. Tissue distribution experiments showed that azithromycin is distributed largely into the liver, kidney, and lung, whereas both inhibitors did not alter the tissue-to-plasma concentration ratio of azithromycin. Significant reduction in the biliary excretion of azithromycin was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. An in situ closed-loop experiment showed that azithromycin was excreted from the blood into the gut lumen, and the intestinal clearance of azithromycin was significantly decreased by the presence of cyclosporine in the loop. These results suggest that azithromycin is a substrate for both P glycoprotein and Mrp2 and that the biliary and intestinal excretion of azithromycin is mediated via these two drug transporters.

  15. 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...

  16. Fexofenadine, a Putative In Vivo P-glycoprotein Probe, Fails to Predict Clearance of the Substrate Tacrolimus in Renal Recipients.

    Science.gov (United States)

    Vanhove, T; Bouillon, T; de Loor, H; Annaert, P; Kuypers, Drj

    2017-12-01

    Whether the combined use of probe drugs for CYP3A4 and P-glycoprotein can clarify the relative contribution of these proteins to pharmacokinetic variability of a dual substrate like tacrolimus has never been assessed. Seventy renal recipients underwent simultaneous 8-h pharmacokinetic profiles for tacrolimus, the CYP3A4 probe midazolam, and the putative P-glycoprotein probe fexofenadine. Patients were genotyped for polymorphisms in CYP3A5, CYP3A4, ABCB1, ABCC2 and SLCO2B1, -1B1, and 1B3. Carriers of the ABCB1 2677G>A polymorphism displayed lower fexofenadine Cmax (-66%; P = 0.012) and a trend toward higher clearance (+157%; P = 0.078). Predictors of tacrolimus clearance were CYP3A5 genotype, midazolam clearance, hematocrit, weight, and age (R2 = 0.61). Fexofenadine pharmacokinetic parameters were not predictive of tacrolimus clearance. In conclusion, fexofenadine pharmacokinetics varied considerably between renal recipients but most of this variability remained unexplained, with only minor effects of genetic polymorphisms. Fexofenadine cannot be used to assess in vivo CYP3A4-P-glycoprotein interplay in tacrolimus-treated renal recipients. © 2017 American Society for Clinical Pharmacology and Therapeutics.

  17. Animal Cell Expression Systems.

    Science.gov (United States)

    Butler, M; Reichl, Ing U

    2017-10-03

    The glycan profile of therapeutic recombinant proteins such as monoclonal antibodies is a critical quality attribute, which affects the efficacy of the final product. The cellular glycosylation process during protein expression is dependent upon a number of factors such as the availability of substrates in the media, the intracellular content of nucleotide sugars, and the enzyme repertoire of the host cells. In order to control the variability of glycosylation it is important to understand the critical process parameters and their acceptable range of values to enable reproducible production of proteins with a predetermined glycan profile providing the desired biological function or therapeutic effect. The depletion of critical nutrients such as glucose or galactose, which may occur toward the end of a culture process, can lead to truncated glycans. Terminal galactosylation and sialyation are particularly variable but may be controlled by the presence of some key media components. Ammonia accumulation, pH, and dissolved oxygen levels are also known to be key bioprocess parameters that affect the glycosylation of recombinant proteins. Specific enzyme inhibitors can be added to the media to drive the formation of selected and predetermined glycan profiles. Various attempts have been made to predict the glycan profiles of cellular expressed proteins and have led to metabolic models based upon knowledge of metabolic flux and the kinetics of individual glycosylation reactions.In contrast to single recombinant proteins, the glycan profiles of viral vaccines are far more complex and difficult to predict. The example of influenza A virus shows that hemagglutinin, the major antigenic determinant, has three to nine N-glycans, which may influence the antigenicity and efficacy of the vaccine. Glycosylation of the influenza A virus has been largely unmonitored in the past as production has been from eggs, where glycan profiles of antigens are difficult if not impossible to

  18. Glyceollin transport, metabolism, and effects on P-glycoprotein function in Caco-2 cells

    Science.gov (United States)

    Glyceollins are phytoalexins produced in soybeans from their isoflavone precursor daidzein. Their impressive anti-cancer and glucose normalization effects in rodents have generated interest in their therapeutic potential. The aim of the present studies was to begin to understand glyceollin intesti...

  19. P-glycoprotein and breast cancer resistance protein: two dominant transporters working together in limiting the brain penetration of topotecan.

    Science.gov (United States)

    de Vries, Nienke A; Zhao, Jin; Kroon, Emily; Buckle, Tessa; Beijnen, Jos H; van Tellingen, Olaf

    2007-11-01

    The brain is a pharmacologic sanctuary site, due to the presence of the blood-brain barrier (BBB). Whereas the effect of P-glycoprotein (P-gp) at the BBB is well established, the role of breast cancer resistance protein (BCRP) that is also expressed at the BBB is not. We have studied the effect of BCRP by administering topotecan to wild-type (WT), single Mdr1a/b(-/-) and Bcrp1(-/-), and compound Mdr1a/b(-/-)Bcrp1(-/-) knockout mice. Drug levels in plasma and tissues were determined by high-performance liquid chromatography. The area under the plasma and tissue concentration-time curve (AUC) of topotecan in brains of Mdr1a/b(-/-) and Bcrp1(-/-) mice was only 1.5-fold higher compared with WT mice, but in Mdr1a/b(-/-)Bcrp1(-/-) mice, where both transporters are absent, the AUC increased by 12-fold. The AUC in plasma was approximately 0.75-, 2.4-, and 3.7-fold higher in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice, respectively, resulting in 2.0-fold (P brain-to-plasma AUC ratios. Results using Mrp4(-/-) mice showed that this transporter had no effect on the brain penetration of topotecan. The P-gp/BCRP inhibitor elacridar fully inhibited P-gp-mediated transport of topotecan, whereas inhibition of Bcrp1-mediated transport by elacridar was minimal. Our results using Mdr1a/b(-/-)Bcrp1(-/-) mice clearly show the effect of Bcrp1 at the BBB and also show how two drug transporters act in concert to limit the brain penetration of topotecan. We expect that this finding will also apply to other drugs that are substrates of both P-gp and BCRP. Consequently, to improve the brain penetration of such compounds for targeting intracranial malignancies in patients, it will be essential to use potent inhibitors of both drug transporters.

  20. 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.

  1. Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Multidrug Resistance by a Novel Mechanism Involving the Hijacking of Lysosomal P-Glycoprotein (Pgp)*

    Science.gov (United States)

    Jansson, Patric J.; Yamagishi, Tetsuo; Arvind, Akanksha; Seebacher, Nicole; Gutierrez, Elaine; Stacy, Alexandra; Maleki, Sanaz; Sharp, Danae; Sahni, Sumit; Richardson, Des R.

    2015-01-01

    Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2′,7′-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport

  2. Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp).

    Science.gov (United States)

    Jansson, Patric J; Yamagishi, Tetsuo; Arvind, Akanksha; Seebacher, Nicole; Gutierrez, Elaine; Stacy, Alexandra; Maleki, Sanaz; Sharp, Danae; Sahni, Sumit; Richardson, Des R

    2015-04-10

    Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity

  3. Lung resistance-related protein as a predictor of clinical outcome in advanced testicular germ-cell tumours.

    NARCIS (Netherlands)

    Zurita, AJ; Diestra, JE; Condom, E; Muro, X Garcia Del; Scheffer, G.L.; Scheper, R.J.; Perez, J; Germa-Lluch, JR; Izquierdo, M.A.

    2003-01-01

    This study was undertaken to investigate the expression and predictive value for outcome of multidrug resistance-associated (MDR) proteins P-glycoprotein (Pgp), MRP1, BCRP, and LRP, in advanced testicular germ-cell tumours (TGCT). Paraffin-embedded sections from 56 previously untreated patients with

  4. Breast Cancer Resistance Protein and P-glycoprotein in Brain Cancer: Two Gatekeepers Team Up

    Science.gov (United States)

    Agarwal, Sagar; Hartz, Anika M.S.; Elmquist, William F.; Bauer, Björn

    2012-01-01

    Brain cancer is a devastating disease. Despite extensive research, treatment of brain tumors has been largely ineffective and the diagnosis of brain cancer remains uniformly fatal. Failure of brain cancer treatment may be in part due to limitations in drug delivery, influenced by the ABC drug efflux transporters P-gp and BCRP at the blood-brain and blood-tumor barriers, in brain tumor cells, as well as in brain tumor stem-like cells. P-gp and BCRP limit various anti-cancer drugs from entering the brain and tumor tissues, thus rendering chemotherapy ineffective. To overcome this obstacle, two strategies – targeting transporter regulation and direct transporter inhibition – have been proposed. In this review, we focus on these strategies. We first introduce the latest findings on signaling pathways that could potentially be targeted to down-regulate P-gp and BCRP expression and/or transport activity. We then highlight in detail the new paradigm of P-gp and BCRP working as a “cooperative team of gatekeepers” at the blood-brain barrier, discuss its ramifications for brain cancer therapy, and summarize the latest findings on dual P-gp/BCRP inhibitors. Finally, we provide a brief summary with conclusions and outline the perspectives for future research endeavors in this field. PMID:21827403

  5. Potent in vitro inhibition of CYP3A4 and P-glycoprotein by Rhodiola rosea.

    Science.gov (United States)

    Hellum, Bent H; Tosse, Anita; Hoybakk, Kathrine; Thomsen, Mette; Rohloff, Jens; Georg Nilsen, Odd

    2010-03-01

    Six clones of RHODIOLA ROSEA, obtained from plants originating from widely different areas in Norway, were investigated for their IN VITRO inhibitory potential on CYP3A4-mediated metabolism and P-gp efflux transport activity. Presumed active constituents in the ethanol extracts of the different clones were quantified. C-DNA baculovirus expressed CYP3A4 and Caco-2 cells were used for inhibitory assays, and as positive control inhibitors ketoconazole and verapamil were applied, respectively. A validated HPLC methodology was used to quantify the formation of 6-beta-OH-testosterone and scintillation counting was used to quantify the transport of (3)H-digoxin in Caco-2 cells. All clones showed potent inhibition of CYP3A4 and P-gp activities, with IC (50) values ranging from 1.7 to 3.1 microg/mL and from 16.7 to 51.7 microg/mL, respectively, being below that reported for other herbs and some known classic drug inhibitors, such as St. John's wort and fluoxetine. RHODIOLA ROSEA might thus be a candidate for clinically relevant drug interactions. The concentration of presumed biologically active constituents in the different clones varied considerably, but this variation was not related to the clones' inhibitory potential on CYP3A4 or P-gp activities. Other constituents might thus be responsible for the observed inhibitory properties. The place of origin seemed to be of minor importance for CYP3A4 or P-gp inhibition. Copyright Georg Thieme Verlag KG Stuttgart New York.

  6. Comparative tissue pharmacokinetics and efficacy of moxidectin, abamectin and ivermectin in lambs infected with resistant nematodes: Impact of drug treatments on parasite P-glycoprotein expression☆

    Science.gov (United States)

    Lloberas, Mercedes; Alvarez, Luis; Entrocasso, Carlos; Virkel, Guillermo; Ballent, Mariana; Mate, Laura; Lanusse, Carlos; Lifschitz, Adrian

    2012-01-01

    The high level of resistance to the macrocyclic lactones has encouraged the search for strategies to optimize their potential as antiparasitic agents. There is a need for pharmaco-parasitological studies addressing the kinetic-dynamic differences between various macrocyclic lactones under standardized in vivo conditions. The current work evaluated the relationship among systemic drug exposure, target tissue availabilities and the pattern of drug accumulation within resistant Haemonchus contortus for moxidectin, abamectin and ivermectin. Drug concentrations in plasma, target tissues and parasites were measured by high performance liquid chromatography. Additionally, the efficacy of the three molecules was evaluated in lambs infected with resistant nematodes by classical parasitological methods. Furthermore, the comparative determination of the level of expression of P-glycoprotein (P-gp2) in H. contortus recovered from lambs treated with each drug was performed by real time PCR. A longer persistence of moxidectin (P abamectin and ivermectin at day 2 post-treatment. However, the efficacy against H. contortus was 20.1% (ivermectin), 39.7% (abamectin) and 89.6% (moxidectin). Only the ivermectin treatment induced an enhancement on the expression of P-gp2 in the recovered adult H. contortus, reaching higher values at 12 and 24 h post-administration compared to control (untreated) worms. This comparative pharmacological evaluation of three of the most used macrocyclic lactones compounds provides new insights into the action of these drugs. PMID:24533290

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

    Directory of Open Access Journals (Sweden)

    Elodie Jouan

    2016-12-01

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

  8. 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 (Pinfection reduced absorption of orally administered enrofloxacin, 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.

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

    Science.gov (United States)

    Li, Yangfang; Wu, Qian; Li, Chen; Liu, Ling; Du, Kun; Shen, Jin; Wu, Yuqin; Zhao, Xiaofen; Zhao, Mei; Bao, Lingyun; Gao, Jin; Keep, Richard F; Xiang, Jianming

    2016-01-01

    While the blood-brain barrier (BBB) protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC) transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP) but not those overexpressing human P-gp (MDCKII-MDR cells) had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated.

  10. Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions.

    Science.gov (United States)

    Pinzón-Daza, Martha L; Cuellar-Saenz, Yenith; Nualart, Francisco; Ondo-Mendez, Alejandro; Del Riesgo, Lilia; Castillo-Rivera, Fabio; Garzón, Ruth

    2017-07-01

    P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1α activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1α activity under hypoxic conditions. However, HIF-1α activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 118: 1868-1878, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

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    Anna Maria Bianucci

    2012-06-01

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

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

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    Valeria Feinshtein

    2013-09-01

    Full Text Available Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD, a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp and Breast Cancer Resistance Protein (BCRP expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp for comparison. Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24–72 h exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3 and rhodamine123(rh123. Cyclosporine A (CsA served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3 and rh123 was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.

  13. Effects of polyethylene glycols on intestinal efflux pump expression and activity in Caco-2 cells

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    Darya Hodaei

    2015-09-01

    Full Text Available The present study was planned to investigate the influence of polyethylene glycols (PEGs on the activity and expression of P-glycoprotein (P-gp. Sub-toxic concentrations of PEGs in Caco-2 cells were determined using the MTT test assay. Then the measurement of Rhodamine-123 (Rho-123 uptake, a P-gp fluorescence substrate, in Caco-2 cells confronting PEG 400 (1% and 2% w/v, PEG 4000 (2% and 4% w/v, PEG 6000 (2% and 4% w/v, PEG 10000 (2% and 4% w/v, PEG 15000 (1% and 2% w/v, and PEG 35000 (2% and 4% w/v overnight was taken to elucidate whether non-toxic concentrations of PEGs are able to impact P-gp activity. Furthermore, western blotting was carried out to investigate P-gp protein expression. The results showed that PEG 400 at concentrations of 1% (w/v and 2% (w/v and PEG 6000 at the concentration of 4% (w/v are notably capable of blocking P-gp. Based on the obtained results it is concluded that the mentioned excipients could be used to obstruct P-gp efflux transporter in order to increase the bioavailability of co-administered substrate drug.

  14. 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.

  15. Prediction and characterization of P-glycoprotein substrates potentially bound to different sites by emerging chemical pattern and hierarchical cluster analysis.

    Science.gov (United States)

    Pan, Xianchao; Mei, Hu; Qu, Sujun; Huang, Shuheng; Sun, Jiaying; Yang, Li; Chen, Hua

    2016-04-11

    P-glycoprotein (P-gp), an ATP-binding cassette (ABC) multidrug transporter, can actively transport a broad spectrum of chemically diverse substrates out of cells and is heavily involved in multidrug resistance (MDR) in tumors. So far, the multiple specific binding sites remain a major obstacle in developing an efficient prediction method for P-gp substrates. Herein, emerging chemical pattern (ECP) combined by hierarchical cluster analysis was utilized to predict P-gp substrates as well as their potential binding sites. An optimal ECP model using only 3 descriptors was established with prediction accuracies of 0.80, 0.81 and 0.74 for 803 training samples, 120 test samples, and 179 independent validation samples, respectively. Hierarchical cluster analysis (HCA) of the ECPs of P-gp substrates derived 2 distinct ECP groups (ECPGs). Interestingly, HCA of the P-gp substrates based on ECP similarities also showed 2 distinct classes, which happened to be dominated by the 2 ECPGs, respectively. In the light of available experimental proofs and molecular docking results, the 2 distinct ECPGs were proved to be closely related to the binding profiles of R- and H-site substrates, respectively. The present study demonstrates, for the first time, a successful ECP model, which can not only accurately predict P-gp substrates, but also identify their potential substrate-binding sites. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 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.

  17. An investigation of heat shock protein 27 and P-glycoprotein mediated multi-drug resistance in breast cancer using liquid chromatography-tandem mass spectrometry-based targeted proteomics.

    Science.gov (United States)

    Xu, Feifei; Yang, Ting; Fang, Danjun; Xu, Qingqing; Chen, Yun

    2014-08-28

    One missing puzzle piece to study heat shock protein 27 (HSP27) in P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) was the amount of HSP27 and the extent of its phosphorylation in the biological context. Liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based targeted proteomics allows researchers to monitor associated proteins and their modification simultaneously and quantitatively. In this study, a targeted proteomics assay was first developed and validated for the quantification of HSP27 and its phosphorylated forms. Using this assay, the level of HSP27 was determined in non-tumoral cells MCF-10A, parental drug-sensitive cancer cells MCF-7/WT and drug-resistant cancer cells MCF-7/ADR. A decrease of HSP27 expression was observed in P-gp overexpressed MCF-7/ADR cells. A quantitative time-course analysis of both HSP27 and P-gp in doxorubicin (DOX)-treated MCF-7/WT cells also implied that HSP27 may participate in the P-gp modulation. Furthermore, stoichiometry of site-specific HSP27 phosphorylation indicated that DOX treatment rapidly induced the HSP27 phosphorylation at Ser82. Moreover, conventional analytical methods were also performed for a comparison. LC/MS/MS-based targeted proteomics turns out to be a promising quantification approach for the study of proteins in the preclinical and clinical environment. Unfortunately, rare studies applied this technology to detect multiple associated proteins or protein modification in one experiment. This study demonstrated the potential of LC/MS/MS-based targeted proteomics to understand the cell events in a more accurate context of biological system. By the quantitative time-course analysis of HSP27 and its phosphorylated forms at sites of Ser15 and Ser82, the possible role of HSP27 in P-gp mediated MDR was suggested. Further development of targeted proteomics in future may provide more insight into signal transduction pathways upon perturbation of a protein network or changes to a panel of proposed

  18. Reversal of P-glycoprotein overexpression by Ginkgo biloba extract in the brains of pentylenetetrazole-kindled and phenytoin-treated mice.

    Science.gov (United States)

    Zhang, Ce; Fan, Qing; Chen, Shu-Liang; Ma, Hui

    2015-08-01

    The purpose of this study was to investigate the combined effects of Ginkgo biloba extract and phenytoin (PHT) sodium as a dose regimen simulating the clinical treatment of patients with epilepsy, on P-glycoprotein (P-GP) overexpression in a pentylenetetrazole-kindled mouse model of epilepsy. Epilepsy was induced by intraperitoneal administration of pentylenetetrazole (40 mg/kg) for 7 days followed by intragastric administration of PHT (40 mg/kg) for 14 days. Thirty mice that developed seizures were randomly divided into three groups and administered PHT as well as the following treatments: saline (negative control); verapamil (20 mg/kg, positive control); and G. biloba (30 mg/kg). Seizure severity was recorded 30 minutes after treatment on Day 4 of drug administration, after which the mice were euthanized, and their brains isolated. Western blots and immunohistochemistry were performed to analyze the expression of P-GP and caspase-3, respectively, in the brain tissue. High-performance liquid chromatography was used to measure the concentrations of PHT in the brains of the treated mice. After 4 consecutive days of treatment, the seizure severity in the mice in the G. biloba extract group was more significantly reduced than the seizure severity in the saline control group, and a significant difference was observed between the G. biloba extract and verapamil control groups (p biloba extract and verapamil than it did in the saline-treated control group (p biloba extract and verapamil showed significantly increased brain PHT concentrations (p biloba extract group was significantly lower than that in the vehicle control group (p biloba. Therefore, this study showed that treatment with G. biloba extract in combination with PHT prevented the upregulation of P-GP expression in mice. Moreover, G. biloba extract decreased seizure severity in pentylenetetrazole-kindled/PHT-treated mice through a mechanism that might be related to the reduction of P-GP expression in the brain

  19. Drug resistance in cortical and hippocampal slices from resected tissue of epilepsy patients: no significant impact of P-glycoprotein and Multidrug resistance associated proteins.

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    Nora eSandow

    2015-02-01

    Full Text Available Drug resistant patients undergoing epilepsy surgery have a good chance to become sensitive to anticonvulsant medication, suggesting that the resected brain tissue is responsible for drug resistance. Here, we address the question whether P-glycoprotein (Pgp and multidrug resistance associated proteins (MRPs expressed in the resected tissue contribute to drug resistance in vitro. Effects of anti-epileptic drugs (carbamazepine, sodium valproate, phenytoin and two unspecific inhibitors of Pgp and MRPs (verapamil and probenecid on seizure-like events induced in slices from 35 hippocampal and 35 temporal cortex specimens of altogether 51 patients (161 slices were studied. Although in slice preparations the blood brain barrier is not functional, we found that seizure-like events predominantly persisted in the presence of anticonvulsant drugs (90% and also in the presence of verapamil and probenecid (86%. Following subsequent co-administration of antiepileptic drugs and drug transport inhibitors, seizure-like events continued in 63% of 143 slices. Drug sensitivity in slices was recognized either as transition to recurrent epileptiform transients (30% or as suppression (7%, particularly by perfusion with carbamazepine in probenecid containing solutions (43%, 9%. Summarizing responses to co-administration from more than one slice per patient revealed that suppression of seizure-like activity in all slices was only observed in 7 % of patients. Patients whose tissue was completely or partially sensitive (65 % presented with higher seizure frequencies than those with resistant tissue (35 %. However, corresponding subgroups of patients don’t differ with respect to expression rates of drug transporters. Our results imply that parenchymal MRPs and Pgp are not responsible for drug resistance in resected tissue.

  20. Sinomenine Sensitizes Multidrug-Resistant Colon Cancer Cells (Caco-2) to Doxorubicin by Downregulation of MDR-1 Expression

    Science.gov (United States)

    Liu, Zhen; Duan, Zhi-Jun; Chang, Jiu-Yang; Zhang, Zhi-feng; Chu, Rui; Li, Yu-Ling; Dai, Ke-Hang; Mo, Guang-quan; Chang, Qing-Yong

    2014-01-01

    Chemoresistance in multidrug-resistant (MDR) cells over expressing P-glycoprotein (P-gp) encoded by the MDR1 gene, is a major obstacle to successful chemotherapy for colorectal cancer. Previous studies have indicated that sinomenine can enhance the absorption of various P-gp substrates. In the present study, we investigated the effect of sinomenine on the chemoresistance in colon cancer cells and explored the underlying mechanism. We developed multidrug-resistant Caco-2 (MDR-Caco-2) cells by exposure of Caco-2 cells to increasing concentrations of doxorubicin. We identified overexpression of COX-2 and MDR-1 genes as well as activation of the NF-κB signal pathway in MDR-Caco-2 cells. Importantly, we found that sinomenine enhances the sensitivity of MDR-Caco-2 cells towards doxorubicin by downregulating MDR-1 and COX-2 expression through inhibition of the NF-κB signaling pathway. These findings provide a new potential strategy for the reversal of P-gp-mediated anticancer drug resistance. PMID:24901713

  1. Targeting MDR1-P-glycoprotein (MDR1-Pgp in immunochemotherapy of acute myeloid leukemia (AML

    Directory of Open Access Journals (Sweden)

    Maurizio Cianfriglia

    2013-06-01

    Full Text Available BACKGROUND: Monoclonal antibodies represent the fastest growing sector of pharmaceutical biotechnology and a number of antibody-based biopharmaceuticals have been approved for cancer treatment. However, in many cases the antibodies used for the treatment of tumors offer only a modest survival benefit to cancer patients. AIMS: In the present review-article we intend to analyze: i the curative regimen gemtuzumab ozogamicin (GO -mediate characterized by the absence of cytotoxic drugs MDR1-Pgp substrates to overcome the mechanism of action of this multidrug transporter, ii the safety and efficacy of MDR reversing strategy in AML outcome and, iii chemical and biological MDR modulators playing a dual relevant medical role as a therapeutic and MDR reversing agents but not yet entered in the clinical setting of AML. Since the similar multidrug transporter protein MDR1-Pgp and its down modulation factors may affect safety and efficacy of already generated antibody drug conjugates (ADCs a comprehensive overview of the most clinically representative immunoconjugates is reported. DISCUSSION: ADCs represent one of the most promising strategies to enhance the antitumor activity of antibodies. ADCs comprise an antibody (or an antibody fragment conjugated to a cytotoxic drug via a chemical linker. The therapeutic concept of ADCs is to use an antibody as a vehicle to selectively delivering a cytotoxic drug specifically to a tumor cell, in most cases by means of binding to target cell surface antigen. As a consequence, ADCs have significant potential for enhancing the antitumor activity of "naked" antibodies and reducing the systemic toxicity of the conjugated drugs.

  2. Modulation of Cytochrome P450, P-glycoprotein and Pregnane X Receptor by Selected Antimalarial Herbs-Implication for Herb-Drug Interaction.

    Science.gov (United States)

    Fasinu, Pius S; Manda, Vamshi K; Dale, Olivia R; Egiebor, Nosa O; Walker, Larry A; Khan, Shabana I

    2017-11-23

    Seven medicinal plants popularly used for treating malaria in West Africa were selected to assess herb-drug interaction potential through a series of in vitro methods. Fluorescent cytochrome P450 (CYP) assays were conducted using the recombinant CYP enzymes for CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 to assess the effect of the methanolic extracts on the metabolic activity of CYPs. Secondly, the inhibitory effect of the extracts was evaluated on P-glycoproteins (P-gp) using calcein-AM, a fluorescent substrate, in MDCK-II and hMDR1-MDCK-II cells. The inhibition of P-gp activity was determined as a reflection of increase in calcein-AM uptake. Additionally, the enzyme induction potential of the extracts was assessed through the modulation of PXR activity in HepG2 cells transiently transfected with pSG5-PXR and PCR5 plasmid DNA. Significant inhibition of CYP activity (IC50 herbs: A. muricata [CYP2C9, 3A4 and CYP2D6]; M. indica [CYP2C9]; M. charantia [CYP2C9 and CYP2C19]; P. amarus [CYP2C19, CYP2C9 and CYP3A4]; T. diversifolia [CYP2C19 and CYP3A4]. Extracts of four herbs (P. amarus, M. charantia, T. diversifolia and A. muricata) exhibited significant inhibition of P-gp with IC50 values (µg/mL) of 17 ± 1, 16 ± 0.4, 26 ± 1, and 24 ± 1, respectively. In addition, four herbs (A. mexicana, M. charantia, P. amarus and T. diversifolia) showed a >two-fold increase in induction in PXR activity. These findings suggest that these herbs may be capable of eliciting herb-drug interactions if consumed in high quantities with concomitant use of conventional therapies.

  3. Constitutive expression of the c-H-ras oncogene inhibits doxorubicin-induced apoptosis and promotes cell survival in a rhabdomyosarcoma cell line.

    Science.gov (United States)

    Nooter, K.; Boersma, A. W.; Oostrum, R. G.; Burger, H.; Jochemsen, A. G.; Stoter, G.

    1995-01-01

    Drugs used in anti-cancer chemotherapy are thought to exert their cytotoxic action by induction of apoptosis. Genes have been identified which can mediate or modulate this drug-induced apoptosis, among which are c-myc, p53 and bcl-2. Since expression of oncogenic ras genes is a frequent observation in human cancer, we investigated the effects of the c-H-ras oncogene on anti-cancer drug-induced apoptosis. Apoptosis induced by a 2 h doxorubicin exposure was measured by in situ nick translation and flow cytometry in a rat cell line (R2T24) stably transfected with the c-H-ras oncogene and in a control cell line (R2NEO) transfected only with the antibiotic resistance gene neo. Both cell lines (R2T24 and R2NEO) had nearly identical growth characteristics, including cell doubling time, distribution over the cell cycle phases and plating efficiency in soft agar. Doxorubicin exposure of the R2NEO cells led to massive induction of apoptosis. In contrast, R2T24 cells, expressing the c-H-ras oncogene, showed significantly less apoptosis after doxorubicin incubation. Doxorubicin induced approximately 3- to 5-fold less cytotoxicity in the R2T24 cells than in the R2NEO cells, as determined by clonogenic assay in soft agar. No difference was observed in intracellular doxorubicin accumulation between the two cell lines, indicating that the classical, P-glycoprotein-mediated multidrug resistance phenotype is not involved in the observed differences in drug sensitivity. In conclusion, our data show that constitutive expression of the c-H-ras oncogene suppresses doxorubicin-induced apoptosis and promotes cell survival, suggesting that human tumours with ras oncogene expression might be less susceptible to doxorubicin treatment. PMID:7880739

  4. Evaluation of (R)-[11C]verapamil as PET tracer of P-glycoprotein function in the blood-brain barrier : kinetics and metabolism in the rat

    NARCIS (Netherlands)

    Luurtsema, Gert; Molthoff, Carla F. M.; Schuit, Robert C.; Windhorst, Albert D.; Lammertsma, Adriaan A.; Franssen, Eric J F

    There is evidence that P-glycoprotein (P-gp) in the blood-brain barrier (BBB) may be involved in the aetiology of neurological disorders. For quantification of P-gp function in vivo, (R)-[11C]verapamil can be used as a positron emission tomography (PET) tracer, provided that a mathematical model

  5. BBB transport and P-glycoprotein functionality using MDR1A (-/-) and wild-type mice. Total brain versus microdialysis concentration profiles of rhodamine-123

    NARCIS (Netherlands)

    de Lange, ECM; de Bock, G; Schinkel, AH; de Boer, AG; Breimer, DD

    1998-01-01

    Purpose. The effect of P-glycoprotein (Pgp) on brain distribution using mdr1a (-/-) mice was investigated. Methods. Fluorescein (Flu) and FD-4 were used to check whether blood-brain barrier (BBB) integrity was maintained in mdr1a (-/-) mice. The Pgp substrate rhodamine-123 (R123) was infused and

  6. Impact of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) gene dosage on plasma pharmacokinetics and brain accumulation of dasatinib, sorafenib, and sunitinib.

    NARCIS (Netherlands)

    Tang, S.C.; Vries, N. de; Sparidans, R.W.; Wagenaar, E.; Beijnen, J.H.; Schinkel, A.H.

    2013-01-01

    Low brain accumulation of anticancer drugs due to efflux transporters may limit chemotherapeutic efficacy, necessitating a better understanding of the underlying mechanisms. P-glycoprotein (Abcb1a/1b) and breast cancer resistance protein (Abcg2) combination knockout mice often display

  7. Role of blood-brain barrier P-glycoprotein in limiting brain accumulation and sedative side-effects of asimadoline, a peripherally acting analgaesic drug

    NARCIS (Netherlands)

    Jonker, JW; Wagenaar, E; van Deemter, L; Gottschlich, R; Bender, HM; Dasenbrock, J; Schinkel, AH

    1 Studies with knockout mice lacking mdrla P-glycoprotein (P-gp) have previously shown that blood-brain barrier P-gp is important in preventing the accumulation of several drugs in the brain. 2 Asimadoline (EMD 61753) is a peripherally selective kappa-opioid receptor agonist which is under

  8. VALUE OF P-GLYCOPROTEIN, GLUTATHIONE-S-TRANSFERASE-PI, C-ERBB-2, AND P53 AS PROGNOSTIC FACTORS IN OVARIAN CARCINOMAS

    NARCIS (Netherlands)

    VANDERZEE, AGJ; HOLLEMA, H; SUURMEIJER, AJH; KRANS, M; SLUITER, WJ; WILLEMSE, PHB; AALDERS, JG; DEVRIES, EGE

    Purpose: To determine the prognostic value of immunostaining of P-glycoprotein (P-gp), glutathione S-transferase (GST) pi, c-erbB-2, and p53 in patients with advanced-stage ovarian carcinoma. Patients and Methods: Immunostaining of P-gp, GST pi, c-erbB-2, and p53 was performed on 89 primary tumors

  9. Data-driven homology modelling of P-glycoprotein in the ATP-bound state indicates flexibility of the transmembrane domains

    NARCIS (Netherlands)

    Stockner, T.; de Vries, S.J.|info:eu-repo/dai/nl/304837717; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Ecker, G.F.; Chiba, P.

    2009-01-01

    Human P-glycoprotein is an ATP-binding cassette transporter that plays an important role in the defence against potentially harmful molecules from the environment. It is involved in conferring resistance against cancer therapeutics and plays an important role for the pharmacokinetics of drugs. The

  10. P-Glycoprotein-Mediated Drug Interactions in Pregnancy and Changes in the Risk of Congenital Anomalies : A Case-Reference Study

    NARCIS (Netherlands)

    Daud, Aizati N.A.; Bergman, Jorieke E.H.; Bakker, Marian K.; Wang, Hao; Kerstjens-Frederikse, Wilhelmina S.; de Walle, Hermien E.K.; Groen, Henk; Bos, Jens H. J.; Hak, Eelko; Wilffert, Bob

    2015-01-01

    INTRODUCTION: Drug use in pregnancy is very common but may cause harm to the fetus. The teratogenic effect of a drug is partly dependent on the drug level in the fetal circulation, which is associated with the transport across the placenta. Many drugs are substrates of P-glycoprotein (P-gp), an

  11. Dietary cholesterol does not normalize low plasma cholesterol levels but induces hyperbilirubinemia and hypercholanemia in Mdr2 P-glycoprotein-deficient mice

    NARCIS (Netherlands)

    Voshol, P. J.; Koopen, N. R.; de Vree, J. M.; Havinga, R.; Princen, H. M.; Elferink, R. P.; Groen, A. K.; Kuipers, F.

    2001-01-01

    BACKGROUND/AIMS: Mdr2 P-glycoprotein deficiency in mice (Mdr2(-/-) leads to formation of cholesterol/cholesterol-depleted bile and reduced plasma HDL cholesterol. We addressed the questions: (1) does HDL in Mdr2(-/-) mice normalize upon phospholipid and/or cholesterol feeding, and (2): is the

  12. Dietary cholesterol does not normalize low plasma cholesterol levels but induces hyperbilirubinemia and hypercholanemia in Mdr2 P-glycoprotein-deficient mice

    NARCIS (Netherlands)

    Voshol, PJ; Koopen, NR; de Vree, JML; Havinga, R; Princen, HMG; Elferink, RPJO; Groen, AK; Kuipers, F

    Background/Aims: Mdr2 P-glycoprotein deficiency in mice (Mdr2((-/-))) leads to formation of cholesterol/cholesterol-depleted bile and reduced plasma HDL cholesterol. We addressed the questions: (1) does HDL in Mdr2((-/-)) mice normalize upon phospholipid and/or cholesterol feeding, and (2): is the

  13. Behavioral Effects and Central Nervous System Levels of the Broadly Available κ-Agonist Hallucinogen Salvinorin A Are Affected by P-Glycoprotein Modulation In Vivo

    Science.gov (United States)

    Caspers, Michael; Lovell, Kimberly M.; Kreek, Mary Jeanne; Prisinzano, Thomas E.

    2012-01-01

    Active blood-brain barrier mechanisms, such as the major efflux transporter P-glycoprotein (mdr1), modulate the in vivo/central nervous system (CNS) effects of many pharmacological agents, whether they are used for nonmedical reasons or in pharmacotherapy. The powerful, widely available hallucinogen salvinorin A (from the plant Salvia divinorum) is a high-efficacy, selective κ-opioid agonist and displays fast-onset behavioral effects (e.g., within 1 min of administration) and relatively short duration of action. In vitro studies suggest that salvinorin A may be a P-glycoprotein substrate; thus, the functional status of P-glycoprotein may influence the behavioral effects of salvinorin A or its residence in CNS after parenteral administration. We therefore studied whether a competing P-glycoprotein substrate (the clinically available agent loperamide; 0.032–0.32 mg/kg) or a selective P-glycoprotein blocker, tariquidar (0.32–3.2 mg/kg) could enhance unconditioned behavioral effects (ptosis and facial relaxation, known to be caused by κ-agonists in nonhuman primates) of salvinorin A, as well as its entry and residence in the CNS, as measured by cerebrospinal fluid sampling. Pretreatment with either loperamide or tariquidar dose-dependently enhanced salvinorin A-induced ptosis, but not facial relaxation. In a control study, loperamide and tariquidar were inactive when given as a pretreatment to ((+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69,593), a κ-agonist known to be a very poor P-glycoprotein substrate. Furthermore, pretreatment with tariquidar (3.2 mg/kg) also enhanced peak levels of salvinorin A in cerebrospinal fluid after intravenous administration. These are the first studies in vivo showing the sensitivity of salvinorin A effects to modulation by the P-glycoprotein transporter, a major functional component of the blood-brain barrier. PMID:22434677

  14. Reversal of P-glycoprotein overexpression by Ginkgo biloba extract in the brains of pentylenetetrazole-kindled and phenytoin-treated mice

    Directory of Open Access Journals (Sweden)

    Ce Zhang

    2015-08-01

    Full Text Available The purpose of this study was to investigate the combined effects of Ginkgo biloba extract and phenytoin (PHT sodium as a dose regimen simulating the clinical treatment of patients with epilepsy, on P-glycoprotein (P-GP overexpression in a pentylenetetrazole-kindled mouse model of epilepsy. Epilepsy was induced by intraperitoneal administration of pentylenetetrazole (40 mg/kg for 7 days followed by intragastric administration of PHT (40 mg/kg for 14 days. Thirty mice that developed seizures were randomly divided into three groups and administered PHT as well as the following treatments: saline (negative control; verapamil (20 mg/kg, positive control; and G. biloba (30 mg/kg. Seizure severity was recorded 30 minutes after treatment on Day 4 of drug administration, after which the mice were euthanized, and their brains isolated. Western blots and immunohistochemistry were performed to analyze the expression of P-GP and caspase-3, respectively, in the brain tissue. High-performance liquid chromatography was used to measure the concentrations of PHT in the brains of the treated mice. After 4 consecutive days of treatment, the seizure severity in the mice in the G. biloba extract group was more significantly reduced than the seizure severity in the saline control group, and a significant difference was observed between the G. biloba extract and verapamil control groups (p < 0.05. P-GP expression in the brain more significantly decreased in the mice treated with G. biloba extract and verapamil than it did in the saline-treated control group (p < 0.05. Compared with the saline-treated control group, the mice treated with G. biloba extract and verapamil showed significantly increased brain PHT concentrations (p < 0.05. Furthermore, caspase-3 expression in the brain tissue of the G. biloba extract group was significantly lower than that in the vehicle control group (p < 0.05; this finding demonstrated the neuroprotective effects of G. biloba

  15. Opioid analgesics and P-glycoprotein efflux transporters: a potential systems-level contribution to analgesic tolerance.

    Science.gov (United States)

    Mercer, Susan L; Coop, Andrew

    2011-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationships to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.

  16. Opioid Analgesics and P-glycoprotein Efflux Transporters: A Potential Systems-Level Contribution to Analgesic Tolerance

    Science.gov (United States)

    Mercer, Susan L.; Coop, Andrew

    2012-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationship development to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation. PMID:21050174

  17. A significant mechanism of molecular recognition between bioflavonoids and P-glycoprotein leading to herb-drug interactions.

    Science.gov (United States)

    Wongrattanakamon, Pathomwat; Nimmanpipug, Piyarat; Sirithunyalug, Busaban; Chansakaow, Sunee; Jiranusornkul, Supat

    2017-07-25

    Inhibition of P-glycoprotein (P-gp)'s function may conduct significant changes in the prescription drugs' pharmacokinetic profiles and escalate potential risks in taking place of drug/herb-drug interactions. Computational modeling was advanced to scrutinize some bioflavonoids which play roles in herb-drug interactions as P-gp inhibitors utilizing molecular docking and pharmacophore analyses. Twenty-five flavonoids were utilized as ligands for the modeling. The mouse P-gp (code: 4Q9H) was acquired from the PDB. The docking was operated utilizing AutoDock version 4.2.6 (Scripps Research Institute, La Jolla, CA) against the NBD2 of 4Q9H. The result illustrated the high correlation between the docking scores and observed activities of the flavonoids and the putative binding site of these flavonoids was proposed and compared with the site for ATP. To evaluate hotspot amino acid residues within the NBD2, Binding modes for the ligands were achieved using LigandScout to originate the NBD2-flavonoid pharmacophore models. The results asserted that these inhibitors competed with ATP for binding site in the NBD2 (as competitive inhibitors) including the hotspot residues which associated with electrostatic and van der Waals interactions with the flavonoids. In MD simulation of eight delegated complexes selected from the analyzed flavonoid subclasses, RMSD analysis of the trajectories indicated the residues were stable throughout the duration of simulations.

  18. Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein. Overexpression, purification, and role of cysteine 430.

    Science.gov (United States)

    Dayan, G; Baubichon-Cortay, H; Jault, J M; Cortay, J C; Deléage, G; Di Pietro, A

    1996-05-17

    Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glyco- protein were prepared on the basis of structure predictions. Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility. Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography. NBD1 efficiently interacted with nucleotides. Fluorescence methods showed that ATP bound at millimolar concentrations and its 2',3'-O-(2,4,6-trinitrophenyl) derivative at micromolar concentrations, while the 2'(3')-N-methylanthraniloyl derivative had intermediate affinity. Photoaffinity labeling was achieved upon irradiation with 8-azido-ATP. The domain exhibited ATPase activity with a Km for MgATP in the millimolar range, and ATP hydrolysis was competitively inhibited by micromolar 2',3'-O-(2,4,6-trinitrophenyl)-ATP. NBD1 contained a single cysteine residue, at position 430, that was derivatized with radiolabeled N-ethylmaleimide. Cysteine modification increased 6-fold the Kd for 2'(3')-N-methylanthraniloyl-ATP and prevented 8-azido-ATP photolabeling. ATPase activity was inhibited with a 5-fold increase in the Km for MgATP. The results suggest that chemical modification of Cys-430 is involved in the N-ethylmaleimide inhibition of whole P-glycoprotein by altering substrate interaction.

  19. Potential contribution of P-glycoproteins to macrocyclic lactone resistance in the cattle parasitic nematode Cooperia oncophora.

    Science.gov (United States)

    Demeler, Janina; Krücken, Jürgen; AlGusbi, Salha; Ramünke, Sabrina; De Graef, Jessie; Kerboeuf, Dominique; Geldhof, Peter; Pomroy, William E; von Samson-Himmelstjerna, Georg

    2013-03-01

    Resistance against macrocyclic lactones such as ivermectin is widespread among parasitic gastrointestinal nematodes of small ruminants and is rapidly increasing in cattle parasites. ABC transporters of the subfamily B, the so-called P-glycoproteins (Pgps) have been frequently implicated in ivermectin resistance and are a major cause of multi-drug resistance in protozoa and helminths. The Pgp inhibitor verapamil (VPL) dramatically enhanced susceptibility of the cattle parasitic nematode Cooperia oncophora to ivermectin in vitro as measured in a larval developmental assay and a larval migration inhibition assay using third stage larvae. Moreover, VPL completely restored susceptibility to ivermectin in a resistant isolate resulting in virtually identical dose-response curves of susceptible and resistant isolates in the presence of VPL. Further characterisation of the molecular mechanisms resulting in Pgp-mediated ivermectin resistance is still hampered by the lack of molecular and biochemical information for Pgps of parasitic nematodes. Using PCR with degenerate primers, fragments of four different C. oncophora Pgps could be amplified and the Conpgp-2, previously implicated in macrocyclic lactone resistance in Haemonchus contortus, and Conpgp-3 full-length cDNAs were obtained by RACE PCR. The pgp sequences presented here contribute important data required to systematically screen resistant C. oncophora isolates for up- or down-regulation of Pgps and for the detection of single nucleotide polymorphisms in Pgps to detect selection of specific Pgp alleles by anthelmintics as early as possible. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. 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.

  1. 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.

  2. 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

  3. Molecular dynamics simulation and molecular docking studies of 1,4-Dihydropyridines as P-glycoprotein's allosteric inhibitors.

    Science.gov (United States)

    Shahraki, Omolbanin; Zargari, Farshid; Edraki, Najmeh; Khoshneviszadeh, Mehdi; Firuzi, Omidreza; Miri, Ramin

    2017-02-13

    P-glycoprotein (P-gp) is a main factor contributing to multidrug resistance. The effect of this transporter protein on limiting the effectiveness of chemotherapy has been shown by various studies. In a previous report, we synthesized some 14-dihydropyridine (DHP) derivatives as inhibitors of human P-gp. In the present study, a computational approach has been exploited to reveal the main interactions between DHPs and P-gp. In order to do this, homology modeling was performed to obtain a model of the protein. Then, molecular dynamics simulation was used to refine the constructed model of P-gp in the presence of the lipids bilayer. Model validation was performed with several tools. Finally, molecular docking followed by MD simulation of ligand-protein complex was employed to elucidate the binding mode and the dynamical changes of protein with/without DHPs bound. The results emphasized that interaction of the residues Gln912, Ser909, Arg905, Ser474, Val472 with DHPs play a crucial role in the inhibitory of these ligands and this was in a relatively good accordance with the results reported in the experimental studies.

  4. Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models.

    Science.gov (United States)

    Zarrabi, Nawid; Ernst, Stefan; Verhalen, Brandy; Wilkens, Stephan; Börsch, Michael

    2014-03-15

    Single-molecule Förster resonance energy (smFRET) transfer has become a powerful tool for observing conformational dynamics of biological macromolecules. Analyzing smFRET time trajectories allows to identify the state transitions occuring on reaction pathways of molecular machines. Previously, we have developed a smFRET approach to monitor movements of the two nucleotide binding domains (NBDs) of P-glycoprotein (Pgp) during ATP hydrolysis driven drug transport in solution. One limitation of this initial work was that single-molecule photon bursts were analyzed by visual inspection with manual assignment of individual FRET levels. Here a fully automated analysis of Pgp smFRET data using hidden Markov models (HMM) for transitions up to 9 conformational states is applied. We propose new estimators for HMMs to integrate the information of fluctuating intensities in confocal smFRET measurements of freely diffusing lipid bilayer bound membrane proteins in solution. HMM analysis strongly supports that under conditions of steady state turnover, conformational states with short NBD distances and short dwell times are more populated compared to conditions without nucleotide or transport substrate present. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Phase I Trial of Lenalidomide and CCI-779 in Patients With Relapsed Multiple Myeloma: Evidence for Lenalidomide–CCI-779 Interaction via P-Glycoprotein

    Science.gov (United States)

    Hofmeister, Craig C.; Yang, Xiaoxia; Pichiorri, Flavia; Chen, Ping; Rozewski, Darlene M.; Johnson, Amy J.; Lee, Seungsoo; Liu, Zhongfa; Garr, Celia L.; Hade, Erinn M.; Ji, Jia; Schaaf, Larry J.; Benson, Don M.; Kraut, Eric H.; Hicks, William J.; Chan, Kenneth K.; Chen, Ching-Shih; Farag, Sherif S.; Grever, Michael R.; Byrd, John C.; Phelps, Mitch A.

    2011-01-01

    Purpose Multiple myeloma (MM) is an incurable plasma-cell neoplasm for which most treatments involve a therapeutic agent combined with dexamethasone. The preclinical combination of lenalidomide with the mTOR inhibitor CCI-779 has displayed synergy in vitro and represents a novel combination in MM. Patients and Methods A phase I clinical trial was initiated for patients with relapsed myeloma with administration of oral lenalidomide on days 1 to 21 and CCI-779 intravenously once per week during a 28-day cycle. Pharmacokinetic data for both agents were obtained, and in vitro transport and uptake studies were conducted to evaluate potential drug-drug interactions. Results Twenty-one patients were treated with 15 to 25 mg lenalidomide and 15 to 20 mg CCI-779. The maximum-tolerated dose (MTD) was determined to be 25 mg lenalidomide with 15 mg CCI-779. Pharmacokinetic analysis indicated increased doses of CCI-779 resulted in statistically significant changes in clearance, maximum concentrations, and areas under the concentration-time curves, with constant doses of lenalidomide. Similar and significant changes for CCI-779 pharmacokinetics were also observed with increased lenalidomide doses. Detailed mechanistic interrogation of this pharmacokinetic interaction demonstrated that lenalidomide was an ABCB1 (P-glycoprotein [P-gp]) substrate. Conclusion The MTD of this combination regimen was 25 mg lenalidomide with 15 mg CCI-779, with toxicities of fatigue, neutropenia, and electrolyte wasting. Pharmacokinetic and clinical interactions between lenalidomide and CCI-779 seemed to occur, with in vitro data indicating lenalidomide was an ABCB1 (P-gp) substrate. To our knowledge, this is the first report of a clinically significant P-gp–based drug-drug interaction with lenalidomide. PMID:21825263

  6. Activation of P-glycoprotein (Pgp)-mediated drug efflux by extracellular acidosis: in vivo imaging with {sup 68}Ga-labelled PET tracer

    Energy Technology Data Exchange (ETDEWEB)

    Thews, Oliver; Dillenburg, Wolfgang [University Medicine Mainz, Institute of Physiology and Pathophysiology, Mainz (Germany); Fellner, Marco; Roesch, Frank [University of Mainz, Institute of Nuclear Chemistry, Mainz (Germany); Buchholz, Hans-Georg; Bausbacher, Nicole; Schreckenberger, Mathias [University Medicine Mainz, Department of Nuclear Medicine, Mainz (Germany)

    2010-10-15

    In vitro it has been shown that the functional activity of P-glycoprotein (Pgp), an important drug transporter responsible for multidrug resistance, can be strongly increased by extracellular acidosis. Here mitogen-activated protein kinases (MAPK) (p38, ERK1/2) seem to play an important role for signal transduction. However, it is unclear whether these effects are also relevant in vivo. With the newly developed PET tracer Schiff base-based {sup 68}Ga-MFL6.MZ the functional Pgp activity was visualized under acidic conditions and during inhibition of MAPKs non-invasively by means of microPET in rat tumours. Tumours were acidified either by inspiratory hypoxia (8% O{sub 2}) or by injection of lactic acid. Inhibitors of the MAPK were injected intratumourally. With increasing tumour volume the tumour pH changed from 7.0 to 6.7 and simultaneously the Pgp activity increased almost linearly. When the tumour was acidified by direct lactic acid injection the PET tracer uptake was reduced by 20% indicating a higher transport rate out of the cells. Changing the inspiratory O{sub 2} fraction to 8% dynamically led to a reduction of extracellular pH and in parallel to a decrease of tracer concentration. While inhibition of the p38 pathway reduced the Pgp transport rate, inhibition of ERK1/2 had practically no impact. An acidic extracellular environment significantly stimulates the Pgp activity. The p38 MAPK pathway plays an important role for Pgp regulation in vivo, whereas ERK1/2 is of minor importance. From these results new strategies for overcoming multidrug resistance (e.g. reducing tumour acidosis, inhibition of p38) may be developed. (orig.)

  7. Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study.

    Science.gov (United States)

    Shahraki, Omolbanin; Edraki, Najmeh; Khoshneviszadeh, Mehdi; Zargari, Farshid; Ranjbar, Sara; Saso, Luciano; Firuzi, Omidreza; Miri, Ramin

    2017-01-01

    Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C4 and various carboxamide substituents at C3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells' sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C4 position and compounds with 4-chlorophenyl carboxamide at C3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 µM, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 µM, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal.

  8. Glucagon-like peptide 2 prevents down-regulation of intestinal multidrug resistance-associated protein 2 and P-glycoprotein in endotoxemic rats.

    Science.gov (United States)

    Arana, Maite Rocío; Tocchetti, Guillermo Nicolás; Zecchinati, Felipe; Londero, Ana Sofía; Dominguez, Camila; Perdomo, Virginia; Rigalli, Juan Pablo; Villanueva, Silvina Stella Maris; Mottino, Aldo Domingo

    2017-09-01

    Multidrug resistance-associated protein 2 (Mrp2, ABCC2) and P-glycoprotein (P-gp, ABCB1) constitute essential components of the intestinal biochemical barrier that prevent incorporation of food contaminants, drugs or toxic metabolites into the blood stream. Endotoxemia induced in rats by administration of bacterial lipopolysaccharide (LPS) results in elevated intestinal permeability and toxicity of xenobiotics in part associated with down-regulation of expression and activity of Mrp2 and P-gp. We evaluated the protective effect of glucagon-like peptide 2 (GLP-2), a peptide hormone with enterotrophic properties, on Mrp2 and P-gp alterations induced by single i.p. injection of LPS (5mg/kg b.wt.) to rats. Two different protocols of GLP-2 administration, namely prevention and reversion, were examined. The prevention protocol consisted of 7s.c. injections of GLP-2 (125μg/kg b.wt.) administered every 12h, starting 60h before LPS administration. The reversion protocol consisted of 2 doses of GLP-2, starting 3h after LPS injection. Intestinal samples were collected 24h after LPS administration and expression (protein and mRNA) and activity of Mrp2 were evaluated in proximal jejunum whereas those of P-gp were studied in ileum. GLP-2 completely neutralized down-regulation of expression of Mrp2 and P-gp and loss of their respective activities induced by LPS under prevention protocol. GLP-2 was also able to prevent internalization of both transporters from the apical membrane of the enterocyte to intracellular compartments, as detected by confocal microscopy. LPS induced an increase in IL-1β and oxidized glutathione tissue levels, which were also counterbalanced by GLP-2 administration. In contrast, the reversion protocol failed to attenuate Mrp2 and P-gp down-regulation induced by LPS. We conclude that GLP-2 can prevent down-regulation of intestinal expression and activity of Mrp2 and P-gp in endotoxemic rats and that IL-1β and oxidative stress constitute potential targets

  9. SMS regulates the expression and function of P-gp and MRP2 in Caco-2 cells.

    Science.gov (United States)

    Jin, Guiying; Li, Yang; Zhu, Yuwen; Du, Lisha; Yan, Junkai; Yang, Qing

    2016-12-01

    Sphingomyelin synthase (SMS) has two isoforms of SMS1 and SMS2, the last enzyme involved in the biosynthesis of sphingomyelin (SM), and has impact on the expression of membrane proteins. In the present study, we explored the potential effects of SMS on drug transporters, a special family of membrane proteins in human intestinal epithelial Caco-2 cells. The specific knockdown of SMS1 or SMS2 with siRNA in Caco-2 cells substantially decreased the expression and function of P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2) rather than other drug transporters MRP1, MRP3, PEPT1, OATP2B1, and BCRP. In the SMS1 stable overexpressed Caco-2 cell line, the expression levels of P-gp and MRP2 and transcription factor pregnane X receptor (PXR) were upregulated and the phosphorylation levels of signaling pathways janus protein tyrosine kinase 2 (JAK-2) and extracellular signal-regulated kinases (ERK) were also evidently increased; however, the upregulated mRNA expression levels of PXR, P-gp, and MRP2 were diminished by inhibiting the phosphorylation of ERK and JAK-2. Furthermore, the SMS1 overexpression in Caco-2 cells altered the expression levels of ERM proteins ezrin and moesin, which are closely connected to the function of drug transporters. In conclusion, we herein demonstrate for the first time that in Caco-2 cells SMS regulates the expression and function of drug transporters P-gp and MRP2, and their regulator PXR is mediated by phosphorylated ERK and JAK-2 signaling pathways.

  10. 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

  11. Inhibition of P-glycoprotein in the blood-brain barrier alters avermectin neurotoxicity and swimming performance in rainbow trout.

    Science.gov (United States)

    Kennedy, Christopher J; Tierney, Keith B; Mittelstadt, Matthew

    2014-01-01

    The importance of the blood brain barrier (BBB) and the contribution to its function by the efflux transporter P-glycoprotein (P-gp) in teleosts were examined using the P-gp substrates and central nervous system neurotoxins ivermectin (22,23-dihydroavermectin B1a+22,23-dihydroavermectin B1b) [IVM]) and emamectin benzoate (4″-deoxy-49″epimethylaminoavermectin B1 benzoate [EB]). Trout were injected intraperitoneally with 0.01-1.0 and 1-50mg/kg of IVM or EB, respectively either alone or in combination with cyclosporin A (CsA: a P-gp substrate) at 1mg/kg. IVM affected the swimming performance (critical swimming speed, burst swimming distance, and schooling) at significantly lower concentrations than EB. When fish were exposed to IVM or EB in the presence of CsA, alterations to swimming were increased, suggesting that competition for P-gp in the BBB by CsA increased IVM and EB penetration into the CNS and decreased swimming capabilities. The effect of co-administration of CsA on swimming-related toxicity was different between IVM and EB-treated fish; EB toxicity was increased to a greater extent than IVM toxicity. The greater chemosensitization effect of EB vs. IVM was examined using a P-gp competitive inhibition assay in isolated trout hepatocytes with rhodamine 123 as a substrate. At the cellular level, IVM was a more potent inhibitor of P-gp than EB, which allowed for a greater accumulation of R123 in hepatocytes. These results provide evidence for a role of P-gp in the BBB of fish, and suggest that this protein protects fish from environmental neurotoxins. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. 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.

  13. Effect of bosutinib on the absorption of dabigatran etexilate mesylate, a P-glycoprotein substrate, in healthy subjects.

    Science.gov (United States)

    Hsyu, Poe-Hirr; Pignataro, Daniela Soriano; Matschke, Kyle

    2017-01-01

    Bosutinib, a dual Src and Abl tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia, demonstrated concentration-dependent inhibitory effects on P-glycoprotein (P-gp)-mediated digoxin efflux in vitro, suggesting that bosutinib may inhibit P-gp substrates. The effect of bosutinib on dabigatran etexilate mesylate (EM) absorption, a P-gp substrate, was evaluated. In this open-label, randomized, single-dose, one-cohort, two-sequence, two-period crossover study, healthy, fed subjects received dabigatran EM (150 mg × 1 orally) alone or 1 h after receiving bosutinib tablets (100 mg × 5 orally). Dabigatran EM monotherapy and concurrent administration of dabigatran EM with bosutinib resulted in similar values for concentration time curves from time zero extrapolated to infinity (AUCinf), but slightly lower maximum plasma concentration (C max) values (AUCinf, 1182 and 1186 ng·h/mL, respectively; C max, 129.8 and 114.1 ng/mL). The time to maximum concentration for dabigatran was 2.99 and 3.99 h for combination therapy. The ratio of the adjusted geometric means (test/reference) of dabigatran AUCinf and C max (90 % confidence interval) were 101.4 % (89.6-114.9 %) and 89.7 % (77.8-103.4 %), respectively, following administration of dabigatran EM with bosutinib (test) relative to dabigatran EM administered alone (reference). Six subjects receiving combination treatment reported a total of seven adverse events (AEs) versus none for subjects receiving monotherapy alone. All AEs were mild to moderate and considered treatment related. These data demonstrate that single doses of bosutinib do not affect dabigatran exposure, suggesting that bosutinib is not a clinical inhibitor of P-gp. ClinicalTrials.gov NCT02102633. https://clinicaltrials.gov/ct2/show/NCT02102633?term=NCT02102633&rank=1.

  14. Is early sestamibi imaging in head and neck cancer affected by MDR status, p53 expression, or cell proliferation?

    Energy Technology Data Exchange (ETDEWEB)

    Leitha, Thomas E-mail: THOMAS.LEITHA@akh-wien.ac.at; Glaser, Christoph; Lang, Susanna

    1998-08-01

    A consensus has emerged that early imaging (within 20 min post-injection [p.i.] of sestamibi) has the highest diagnostic yield. Tc-99m-sestamibi uptake has been associated with P-glycoprotein-mediated multi-drug resistance (MDR), tumor vascularization, invasiveness, and cell proliferation. The aim of this study was to assess whether these parameters affect tumor uptake in current imaging protocols. Twenty-three patients with squamous cell carcinoma (SCC) of the mouth floor who were scheduled for surgery were imaged 5 min. p.i. with a triple-head gamma camera (360 deg., 3 deg./step SPECT, UHRPAR collimators). Tumor:nontumor tissue (TBR), tumor:gingiva (TGR), tumor:salivary gland (TSR), and tumor:nuchal muscle ratios (TNR) were calculated based on the cts/pix values of ROIs in the axial slices. The expression of the MDR gene was determined histochemically from biopsies. Cell proliferation was quantitated by the histochemical analysis of the Ki-67 protein (Ki-67 index); additionally, the p53 tumor suppressor gene (p53 index), a posttranslational stabilizer of the cell cycle at the G1 stage, was assessed. No significant differences were found for the uptake indices between MDR+ and MDR- tumors. Moreover, no significant correlation between sestamibi uptake and the Ki-67 and p53 indices could be demonstrated. The diagnostic information content of currently applied imaging protocols for sestamibi in SCC of the mouth floor is not affected by tumor-specific properties.

  15. Is early sestamibi imaging in head and neck cancer affected by MDR status, p53 expression, or cell proliferation?

    Science.gov (United States)

    Leitha, T; Glaser, C; Lang, S

    1998-08-01

    A consensus has emerged that early imaging (within 20 min post-injection [p.i.] of sestamibi) has the highest diagnostic yield. Tc-99m-sestamibi uptake has been associated with P-glycoprotein-mediated multi-drug resistance (MDR), tumor vascularization, invasiveness, and cell proliferation. The aim of this study was to assess whether these parameters affect tumor uptake in current imaging protocols. Twenty-three patients with squamous cell carcinoma (SCC) of the mouth floor who were scheduled for surgery were imaged 5 min. p.i. with a triple-head gamma camera (360 degrees, 3 degrees/step SPECT, UHRPAR collimators). Tumor:nontumor tissue (TBR), tumor:gingiva (TGR), tumor:salivary gland (TSR), and tumor:nuchal muscle ratios (TNR) were calculated based on the cts/pix values of ROIs in the axial slices. The expression of the MDR gene was determined histochemically from biopsies. Cell proliferation was quantitated by the histochemical analysis of the Ki-67 protein (Ki-67 index); additionally, the p53 tumor suppressor gene (p53 index), a posttranslational stabilizer of the cell cycle at the G1 stage, was assessed. No significant differences were found for the uptake indices between MDR+ and MDR- tumors. Moreover, no significant correlation between sestamibi uptake and the Ki-67 and p53 indices could be demonstrated. The diagnostic information content of currently applied imaging protocols for sestamibi in SCC of the mouth floor is not affected by tumor-specific properties.

  16. Tumor-specific expression of anti-mdr1 ribozyme selectively restores chemosensitivity in multidrug-resistant colon-adenocarcinoma cells.

    Science.gov (United States)

    Gao, Z; Gao, Z; Fields, J Z; Boman, B M

    1999-07-30

    P-glycoprotein (Pgp)-conferred multidrug resistance (MDR) is expressed in cancer and in normal colon tissues and has important physiological functions. In order to selectively reverse MDR in malignant tissue without disrupting the function of normal colonocytes, a retroviral vector (pCEAMR) containing anti-mdr1 ribozyme coupled to the carcino-embryonic-antigen (CEA) promoter was constructed and introduced into resistant colon-cancer cells (SW1116R) that produce CEA and into control resistant cells (HeLaK) that do not produce CEA. Anti-mdr1 ribozyme was expressed in SW1116R cells but not in HeLaK cells. Subsequently, the expression of mdr1 mRNA and Pgp decreased significantly in the transfected SW1116R cells, and was even lower than in parent non-resistant SW1116 cells. The functional ability of Pgp to facilitate rhodamine 123 (Rh123) efflux showed that the transfected SW1116R cells with low Pgp expression retained Rh123, whereas non-transfected SW1116R cells with high Pgp expression released the dye quickly. There was no difference in mdr1 mRNA or in Pgp between non-transfected and transfected HeLaK cells. Drug resistance to doxorubicin (DOX) decreased 93.1% in the transfected SW1116R cells, while no change in drug resistance occurred in the infected HeLaK cells. DOX could clearly inhibit the growth of transfected SW1116R tumors but had no effect on untransfected and on transfected HeLaK cells in vivo. These results indicate that our anti-mdr1 ribozyme is expressed only in CEA-producing colon-cancer cells and reverses their drug resistance selectively.

  17. P-glycoprotein function at the blood-brain barrier imaged using 11C-N-desmethyl-loperamide in monkeys.

    Science.gov (United States)

    Liow, Jeih-San; Kreisl, William; Zoghbi, Sami S; Lazarova, Neva; Seneca, Nicholas; Gladding, Robert L; Taku, Andrew; Herscovitch, Peter; Pike, Victor W; Innis, Robert B

    2009-01-01

    11C-Loperamide is an avid substrate for P-glycoprotein (P-gp), but it is rapidly metabolized to 11C-N-desmethyl-loperamide (11C-dLop), which is also a substrate for P-gp and thereby contaminates the radioactive signal in the brain. Should further demethylation of 11C-dLop occur, radiometabolites with low entry into the brain are generated. Therefore, we evaluated the ability of 11C-dLop to quantify the function of P-gp at the blood-brain barrier in monkeys. Six monkeys underwent 12 PET scans of the brain, 5 at baseline and 7 after pharmacologic blockade of P-gp. A subset of monkeys also underwent PET scans with 15O-water to measure cerebral blood flow. To determine whether P-gp blockade affected peripheral distribution of 11C-dLop, we measured whole-body biodistribution in 4 monkeys at baseline and after P-gp blockade. The concentration of 11C-dLop in the brain was low under baseline conditions and increased 5-fold after P-gp blockade. This increase was primarily caused by an increased rate of entry into the brain rather than a decreased rate of removal from the brain. With P-gp blockade, uptake of radioactivity among brain regions correlated linearly with blood flow, suggesting a high single-pass extraction. After correction for cerebral blood flow, the uptake of 11C-dLop was fairly uniform among brain regions, suggesting that the function of P-gp is fairly uniformly distributed in the brain. On whole-body imaging, P-gp blockade significantly affected distribution of radioactivity only to the brain and not to other visually identified source organs. The effective dose estimated for humans was approximately 9 microSv/MBq. PET with 11C-dLop can quantify P-gp function at the blood-brain barrier in monkeys. The single-pass extraction of 11C-dLop is high and requires correction for blood flow to accurately measure the function of this efflux transporter. The low uptake at baseline and markedly increased uptake after P-gp blockade suggest that 11C-dLop will be useful to

  18. Investigating the binding interactions of the anti-Alzheimer's drug donepezil with CYP3A4 and P-glycoprotein.

    Science.gov (United States)

    McEneny-King, Alanna; Edginton, Andrea N; Rao, Praveen P N

    2015-01-15

    The anti-Alzheimer's agent donepezil is known to bind to the hepatic enzyme CYP3A4, but its relationship with the efflux transporter P-glycoprotein (P-gp) is not as well elucidated. We conducted in vitro inhibition studies of donepezil using human recombinant CYP3A4 and P-gp. These studies show that donepezil is a weak inhibitor of CYP3A4 (IC50=54.68±1.00μM) whereas the reference agent ketoconazole exhibited potent inhibition (CYP3A4 IC50=0.20±0.01μM). P-gp inhibition studies indicate that donepezil exhibits better inhibition relative to CYP3A4 (P-gp EC50=34.85±4.63μM) although it was less potent compared to ketoconazole (P-gp EC50=9.74±1.23μM). At higher concentrations, donepezil exhibited significant inhibition of CYP3A4 (69%, 84% and 87% inhibition at 100, 250 and 500μM, respectively). This indicates its potential to cause drug-drug interactions with other CYP3A4 substrates upon co-administration; however, this scenario is unlikely in vivo due to the low therapeutic concentrations of donepezil. Similarly, donepezil co-administration with P-gp substrates or inhibitors is unlikely to result in beneficial or adverse drug interactions. The molecular docking studies show that the 5,6-dimethoxyindan-1-one moiety of donepezil was oriented closer to the heme center in CYP3A4 whereas in the P-gp binding site, the protonated benzylpiperidine pharmacophore of donepezil played a major role in its binding ability. Energy parameters indicate that donepezil complex with both CYP3A4 and P-gp was less stable (CDOCKER energies=-15.05 and -4.91kcal/mol, respectively) compared to the ketoconazole-CYP3A4 and P-gp complex (CDOCKER energies=-41.89 and -20.03kcal/mol, respectively). Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Lymphocyte P-glycoprotein variability in healthy individuals Variabilidad de la glicoproteína P linfocitaria en una población sana

    Directory of Open Access Journals (Sweden)

    Catalina M. Cortada

    2009-12-01

    Full Text Available The aim of the present work was to describe the distribution of lymphocyte P-glycoprotein activity on a population of healthy individuals, taking also into account sex and age. P-glycoprotein activity in lymphocytes was measured by the Rhodamine 123 efflux assay using flow cytometry, in the presence and absence of verapamil, a P-glycoprotein inhibitor. We obtained a range of P-glycoprotein activity from 1.04 to 3.79. The distribution of the activity in the population studied was better described by a bimodal model, according with the Kolmogorov-Smirnov test. The frequency adjusted to the following equation: F = 0.70 N (2.11; 0.43 + 0.30 N(3.29; 0.26, in which 0.70 and 0.30 represented the proportion of each group, and 0.43 and 0.26 were the standard deviations of the activity of each group, respectively. The study of the relationship between subjects´ age and P-glycoprotein activity showed no statistical significance. When healthy volunteers were separated according to sex, similar distributions were observed, although for men an increase in proportion of higher P-glycoprotein function group was observed. The variability observed in the population studied was important, with some volunteers with very scarce activity and some with a fourfold higher activity. Characterization of P-glycoprotein functionality in the population represents a useful contribution to the beginning of pharmacological treatments that consider its effect on pharmacokinetics and pharmacodynamics of individualized patients.El objetivo del presente trabajo fue describir la distribución de la actividad de la glicoproteína P linfocitaria en una población de individuos sanos, considerando a su vez el sexo y la edad. La funcionalidad de la glicoproteína P fue determinada mediante el ensayo de eflujo de Rodamina 123, en presencia y ausencia de verapamilo, un inhibidor competitivo de este transportador, determinando la fluorescencia intracelular remanente mediante citometr

  20. Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.

    Science.gov (United States)

    Mangia, Carlo; Vismarra, Alice; Kramer, Laura; Bell-Sakyi, Lesley; Porretta, Daniele; Otranto, Domenico; Epis, Sara; Grandi, Giulio

    2016-04-18

    Ticks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin. IRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84% and 92% with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected. This is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

  1. Design new P-glycoprotein modulators based on molecular docking and CoMFA study of α, β-unsaturated carbonyl-based compounds and oxime analogs as anticancer agents

    Science.gov (United States)

    Sepehri, Bakhtyar; Ghavami, Raouf

    2017-02-01

    In this research, molecular docking and CoMFA were used to determine interactions of α, β-unsaturated carbonyl-based compounds and oxime analogs with P-glycoprotein and prediction of their activity. Molecular docking study shown these molecules establish strong Van der Waals interactions with side chain of PHE-332, PHE-728 and PHE-974. Based on the effect of component numbers on squared correlation coefficient for cross validation tests (including leave-one-out and leave-many-out), CoMFA models with five components were built to predict pIC50 of molecules in seven cancer cell lines (including Panc-1 (pancreas cancer cell line), PaCa-2 (pancreatic carcinoma cell line), MCF-7 (breast cancer cell line), A-549 (epithelial), HT-29 (colon cancer cell line), H-460 (lung cancer cell line), PC-3 (prostate cancer cell line)). R2 values for training and test sets were in the range of 0.94-0.97 and 0.84 to 0.92, respectively, and for LOO and LMO cross validation test, q2 values were in the range of 0.75-0.82 and 0.65 to 0.73, respectively. Based on molecular docking results and extracted steric and electrostatic contour maps for CoMFA models, four new molecules with higher activity with respect to the most active compound in data set were designed.

  2. 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

  3. Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor

    Energy Technology Data Exchange (ETDEWEB)

    Gräns, Johanna; Wassmur, Britt; Fernández-Santoscoy, María [Department of Biological and Environmental Sciences, University of Gothenburg, Box 463, SE 405 30 Gothenburg (Sweden); Zanette, Juliano; Woodin, Bruce R.; Karchner, Sibel I. [Biology Department, MS #32, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Nacci, Diane E.; Champlin, Denise; Jayaraman, Saro [Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, United States Environmental Protection Agency, 27 Tarzwell Drive, Narragansett, RI 02882 (United States); Hahn, Mark E.; Stegeman, John J. [Biology Department, MS #32, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Celander, Malin C., E-mail: malin.celander@gu.se [Department of Biological and Environmental Sciences, University of Gothenburg, Box 463, SE 405 30 Gothenburg (Sweden)

    2015-02-15

    Highlights: • Basal levels of PXR and Pgp mRNA are lower in liver of fish from NBH than from SC. • Hepatic PXR, CYP3A and Pgp mRNA levels are induced by PCB in fish from NBH. • Both non-dioxin-like and dioxin-like PCBs induce PXR, CYP3A and Pgp in NBH fish. • Branchial PXR and CYP3A mRNA levels are induced by PCB 126 in fish from SC. • There is possible cross-talk between AhR and PXR signaling in killifish. - Abstract: Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of

  4. Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and β-catenin signaling

    Directory of Open Access Journals (Sweden)

    Cabot Myles C

    2010-06-01

    Full Text Available Abstract Background Drug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS. MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance. Results MDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES. MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series

  5. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore...... to examine whether human bladder tumor cells express VDR. Tumor biopsies were obtained from 26 patients with TCC. Expression of VDR was examined by immunohistochemical experiments. All tumors expressed VDR. Biopsies from advanced disease contained more VDR positive cells than low stage disease (p ....05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...

  6. 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...

  7. Dasatinib reverses the multidrug resistance of breast cancer MCF-7 cells to doxorubicin by downregulating P-gp expression via inhibiting the activation of ERK signaling pathway

    Science.gov (United States)

    Chen, Ting; Wang, Changyuan; Liu, Qi; Meng, Qiang; Sun, Huijun; Huo, Xiaokui; Sun, Pengyuan; Peng, Jinyong; Liu, Zhihao; Yang, Xiaobo; Liu, Kexin

    2015-01-01

    Multidrug resistance (MDR) is one of the major obstacles to the efficiency of cancer chemotherapy, which often results from the overexpression of drug efflux transporters such as P-glycoprotein (P-gp). In the present study, we determined the effect of dasatinib which was approved for imatinib resistant chronic myelogenous leukemia (CML) and (Ph+) acute lymphoblastic leukemia (ALL) treatment on P-gp-mediated MDR. Our results showed that dasatinib significantly increased the sensitivity of P-gp-overexpressing MCF-7/Adr cells to doxorubicin in MTT assays; thus lead to an enhanced cytotoxicity of doxorubicin in MCF-7/Adr cells. Additionally, dasatinib increased the intracellular accumulation, inhibited the efflux of doxorubicin in MCF-7/Adr cells, and significantly enhanced doxorubicin-induced apoptosis in MCF-7/Adr cells. Further studies showed that dasatinib altered the expression levels of mRNA, protein levels of P-gp, and the phosphorylation of signal–regulated kinase (ERK) both in time-dependent (before 24 h) and dose-dependent manners at concentrations that produced MDR reversals. In conclusion, dasatinib reverses P-gp-mediated MDR by downregulating P-gp expression, which may be partly attributed to the inhibition of ERK pathway. Dasatinib may play an important role in circumventing MDR when combined with other conventional antineoplastic drugs. PMID:25482933

  8. Expression of periostin in breast cancer cells.

    Science.gov (United States)

    Ratajczak-Wielgomas, Katarzyna; Grzegrzolka, Jedrzej; Piotrowska, Aleksandra; Matkowski, Rafal; Wojnar, Andrzej; Rys, Janusz; Ugorski, Maciej; Dziegiel, Piotr

    2017-10-01

    Periostin (POSTN) is a protein involved in multiple processes important for cancer development, both at the stage of cancer initiation and progression, as well as metastasis. The aim of this study was to determine the expression of POSTN in the cells of non-invasive ductal breast carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) and to correlate it with clinicopathological data. Immunohistochemical studies (IHC) were conducted on 21 cases of fibrocystic breast change (FC), 44 cases of DCIS and 92 cases of IDC. POSTN expression at mRNA (real-time PCR) and protein level (western blot analysis) was also confirmed in selected breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231 and BO2). Statistically significant higher level of POSTN expression in IDC and DCIS cancer cells compared to FC was noted. Also, the level of POSTN expression in the cytoplasm of IDC cells was shown to increase with the increasing degree of tumour malignancy (G) and significantly higher expression of POSTN was observed in each degree of tumour malignancy (G) relative to FC. Statistically significant higher POSTN expression was observed in tumours with estrogen receptor-negative (ER-) and progesterone receptor-negative (PR-) phenotypes in comparison to estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) cases, as well as significant negative correlation between POSTN expression in cancer cells and expression of ER and PR (p<0.05). Additionally, statistically significant differences in POSTN expression were shown between particular breast cancer cell lines, both at mRNA and protein level. Observed POSTN expression was the lowest in the case of MCF-7, and the highest in MDA-MB-231 and BO2 of the most aggressive potential clinically corresponding to G3 tumours. POSTN expression in the cytoplasm of IDC cancer cells may play an important role in cancer transformation mechanism.

  9. Effect of duration of exposure to verapamil on vincristine activity against multidrug-resistant human leukemic cell lines.

    Science.gov (United States)

    Cass, C E; Janowska-Wieczorek, A; Lynch, M A; Sheinin, H; Hindenburg, A A; Beck, W T

    1989-11-01

    Verapamil sensitizes multidrug-resistant cell lines to various heterocyclic anticancer drugs by inhibition of energy-dependent release of drug, presumably by interaction with membrane glycoproteins involved in drug efflux. This work assessed verapamil sensitization of human multidrug-resistant lymphocytic and myeloid leukemic cell lines (CEM/VLB100, HL-60/AR) to vincristine during exposures of short duration (4 h). When cells were transferred to drug-free medium immediately after simultaneous 4-h exposures to vincristine and verapamil, the antiproliferative activity of vincristine was not altered in CEM/VLB100 cells and was only moderately increased in HL-60/AR cells. In contrast, when cells were transferred to verapamil-containing medium, vincristine activity was greatly increased against both CEM/VLB100 and HL-60/AR cells. Verapamil enhanced accumulation and inhibited release of [3H]vincristine by CEM/VLB100 and HL-60/AR cells, indicating that the sensitization was due to an increase in cell-associated vincristine after transfer of cells to vincristine-free medium. Slot blot analysis of cellular RNA with the pMDR1 probe revealed high levels of expression of the mdr1 gene in CEM/VLB100 cells but no detectable expression in HL-60/AR cells. Consistent with this finding, polypeptides (Mr 170,000 to 180,000) that were recognized by a monoclonal antibody (C219) against P-glycoprotein were greatly overexpressed in CEM/VLB100 cells, but were expressed at low levels, if at all, in HL-60/AR cells. These results demonstrate the importance of duration of exposure to verapamil in reversing multidrug resistance, not only in cells that overexpress P-glycoprotein but also in cells, such as HL-60/AR, that express little, if any, P-glycoprotein.

  10. 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

  11. Molecular expression in transfected corneal endothelial cells

    Science.gov (United States)

    Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

    2017-10-01

    To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

  12. Inhibition of P-glycoprotein, multidrug resistance-associated protein 2 and cytochrome P450 3A4 improves the oral absorption of octreotide in rats with portal hypertension.

    Science.gov (United States)

    Sun, Xiao-Yu; Duan, Zhi-Jun; Liu, Zhen; Tang, Shun-Xiong; Li, Yang; He, Shou-Cheng; Wang, Qiu-Ming; Chang, Qing-Yong

    2016-12-01

    The aim of the present study was to increase the intestinal transport of octreotide (OCT) by targeting the first-pass impact to identify a potential method for decreasing portal vein pressure (PVP) using oral OCT. Thus, the bioavailability of intestinally absorbed OCT was evaluated in normal rats and rats with portal hypertension (PH) that had been administered P-glycoprotein/multidrug resistance-associated protein 2/cytochrome P450 3A4 (P-gp/MRP2/CYP3A4) inhibitors. The mRNA and protein expression levels of P-gp, MRP2 and CYP3A4 were evaluated in normal and PH rats with or without OCT and the inhibitors using RT-PCR, western blot and immunohistochemical analyses. The potential effects of the inhibitor administration on PVP were also examined. The results suggest that P-gp, MRP2 and CYP3A4 play important roles in prohibiting the enteral absorption of OCT, particularly under a PH environment. Moreover, inhibitors of P-gp, MRP2 and CYP3A4 decrease the first-pass effects of OCT and effectively reduce PVP under PH conditions. Therefore, the present results suggest P-gp, MRP2 and CYP3A4 are key factors in the intestinal absorption of OCT. The inhibition of P-gp, MRP2 and CYP3A4 can markedly decrease the first-pass effects of OCT, and their use may facilitate the use of orally administered OCT.

  13. IAA8 expression during vascular cell differentiation

    Science.gov (United States)

    Andrew T. Groover; Amy Pattishall; Alan M. Jones

    2003-01-01

    We report the characterization of a member of the auxin-induced IAA gene family from zinnia, designated zIAA8, which is expressed by mesophyll cells differentiating as tracheary elements in vitro. Transcription of zIAA8 is upregulated within 3 h after cell isolation in inductive medium,...

  14. Selecting surfactants for the maximum inhibition of the activity of the multidrug resistance efflux pump transporter, P-glycoprotein: conceptual development

    Directory of Open Access Journals (Sweden)

    Shireesh Apte

    2010-09-01

    Full Text Available Amphiphilic excipients, such as surfactants, have been shown to be inhibitors of the multidrug resistance (MDR efflux pump transporter protein, P glycoprotein (Pgp. In vitro studies using many surfactants have demonstrated that those with an optimum hydrophilic-lipophilic balance (HLB exhibit greater efflux pump inhibition than those that are either very hydrophobic, or very hydrophilic, although the correlation of HLB to Pgp inhibition activity remains weak. Using the data from multiple in vitro studies, a model has been conceptualized that underscores the attributes of both the HLB and the critical micellar concentration (CMC, occurring in tandem, and unable of being varied independently, as key determinants toward prediction of surfactant Pgp inhibition activity. The algorithm that formalizes this concept provides a ‘semi-rational’ method of choosing surfactants for a specific type of cancer for maximum inhibition of MDR.

  15. 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...... 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...... pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid...

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

    Science.gov (United States)

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

    2016-06-15

    The purpose of this study was to develop HM30181 mesylate salt (HM30181M)-loaded microcapsules as a novel P-glycoprotein inhibitor for enhancing the oral absorption of paclitaxel. The effect of various carriers including hydrophilic polymers and solvents on the solubility of HM30181M were evaluated. Among the hydrophilic polymers and solvents tested, HPMC and methylene chloride (and ethanol) provided the highest HM30181M solubility. Numerous HM30181M-loaded microcapsules were prepared with HPMC, silicon dioxide and acidifying agents using a spray-drying technique, and their solubility, dissolution and physicochemical properties were evaluated. Furthermore, a pharmacokinetic study was performed after oral administration of paclitaxel alone, simultaneously with HM30181M powder or HM30181M-loaded microcapsules to rats. Among the acidifying agents investigated, phosphoric acid provided the best improvement in the solubility and dissolution of HM30181M. Moreover, the microcapsule composed of HM30181M, HPMC, silicon dioxide and phosphoric acid at a weight ratio of 3:6:3:2 remarkably enhanced the solubility and dissolution of HM30181M compared with the HM30181M powder alone. The microcapsules were spherical in shape, had a reduced particle size of about 7μm, and contained HM30181M in an amorphous state. Furthermore, this microcapsule significantly enhanced HM30181M absorption, making it about 1.7-fold faster and 1.6-fold greater after simultaneous administration, leading to about 70- and 2-fold improved oral bioavailability of paclitaxel compared with paclitaxel alone and the simultaneous administration with HM30181M powder, respectively. Thus, this novel microcapsule could be a potential candidate for effective P-glycoprotein inhibition during oral administration of paclitaxel. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. P-glycoprotein has differential effects on the disposition of statin acid and lactone forms in mdr1a/b knockout and wild-type mice.

    Science.gov (United States)

    Chen, Cuiping; Lin, Jian; Smolarek, Teresa; Tremaine, Larry

    2007-10-01

    In the present study we examined the disposition of atorvastatin, lovastatin, and simvastatin in acid and lactone forms and pravastatin in acid form in multidrug-resistant gene (mdr1a/b) knockout (KO), and wild-type (WT) mice. Each statin was administered s.c. to mdr1a/b KO and WT mice at 3.0 mg/kg (n > or = 3 mice/time point). Blood, brain, and liver samples were harvested at 0, 0.5, 1.5, and 3 h postdose. Plasma and tissue concentrations of the acid and lactone (only the acid form was determined for pravastatin) were determined using a liquid chromatography-mass spectrometry method. Both lactone and acid were observed in plasma when lactones were administered, but only acids were detected when the acid forms were administered. The plasma and liver concentrations of acid or lactone were similar between the KO and WT mice. Two- to 23-fold higher concentrations were observed in liver than in plasma, suggesting potential uptake transporters involved. A significantly higher (p < 0.05) brain penetration in the KO compared with the WT mice was observed for lovastatin acid (but the brain/plasma ratio was low for both KO and WT mice) and lactone and simvastatin lactone but not for atorvastatin or pravastatin. The present results suggest that mouse P-glycoprotein does not affect the lactone-acid interconversion or liver-plasma distribution. Furthermore, P-glycoprotein plays a limited role in restricting the brain penetration of the acid forms of atorvastatin, pravastatin, simvastatin, lovastatin, and atorvastatin lactone but may limit the brain availability of the lactone forms of simvastatin and lovastatin.

  18. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  19. P-Glycoprotein Mediated Multidrug Resistance Reversal by Phytochemicals: A Review of SAR & Future Perspective for Drug Design.

    Science.gov (United States)

    Syed, Safiulla Basha; Coumar, Mohane Selvaraj

    2016-01-01

    A major impediment for cancer chemotherapy is the development of multidrug-resistance (MDR). Continuous use of chemotherapeutic drugs during cancer therapy induces the expression of PGlycoprotein (P-gp, MDR1), an ATP dependant transporter, which in turn reduces the intracellular accumulation of chemotherapeutic drugs leading to MDR. Extensive research over the years has identified several potential P-gp inhibitors, both synthetic as well as natural origin, to overcome the MDR during cancer chemotherapy. In this review, we discuss the cellular pathways involved and transcription factors regulating the expression of P-gp. A number of phytochemicals are reported to inhibit P-gp activity and MDR1 expression; the structure-activity relationship (SAR) among the phytochemicals for P-gp inhibition and the effect of these phytochemicals on cellular signaling pathways regulating P-gp expression are discussed in detail. Moreover, structural biology and mutagenesis studies on P-gp along with docking studies throw light on the structural requirements for P-gp inhibition. Insight provided in the review about the phytochemicals molecular mechanism and SAR could catalyze the design of potent P-gp inhibitors in the future and could help to overcome MDR in cancer chemotherapy.

  20. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

    Science.gov (United States)

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa

    2012-11-01

    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

  1. The influence of Osmunda regalis root extract on head and neck cancer cell proliferation, invasion and gene expression.

    Science.gov (United States)

    Schmidt, Marianne; Skaf, Josef; Gavril, Georgiana; Polednik, Christine; Roller, Jeanette; Kessler, Michael; Holzgrabe, Ulrike

    2017-12-04

    According to only a handful of historical sources, Osmunda regalis, the royal fern, has been used already in the middle age as an anti-cancer remedy. To examine this ancient cancer cure, an ethanolic extract of the roots was prepared and analysed in vitro on its effectiveness against head and neck cancer cell lines. Proliferation inhibition was measured with the MTT assay. Invasion inhibition was tested in a spheroid-based 3-D migration assay on different extracellular matrix surfaces. Corresponding changes in gene expression were analysed by qRT-PCR array. Induction of apoptosis was measured by fluorescence activated cell sorting (FACS) with the Annexin V binding method. The plant extract was analysed by preliminary phytochemical tests, liquid chromatography/mass spectroscopy (LC-MS) and thin layer chromatography (TLC). Anti-angiogenetic activity was determined by the tube formation assay. O. regalis extract revealed a growth inhibiting effect on the head and neck carcinoma cell lines HLaC78 and FaDu. The toxic effect seems to be partially modulated by p-glycoprotein, as the MDR-1 expressing HLaC79-Tax cells were less sensitive. O. regalis extract inhibited the invasion of cell lines on diverse extracellular matrix substrates significantly. Especially the dispersion of the highly motile cell line HlaC78 on laminin was almost completely abrogated. Motility inhibition on laminin was accompanied by differential gene regulation of a variety of genes involved in cell adhesion and metastasis. Furthermore, O. regalis extract triggered apoptosis in HNSCC cell lines and inhibited tube formation of endothelial cells. Preliminary phytochemical analysis proved the presence of tannins, glycosides, steroids and saponins. Liquid chromatography/mass spectroscopy (LC-MS) revealed a major peak of an unknown substance with a molecular mass of 864.15 Da, comprising about 50% of the total extract. Thin layer chromatography identified ferulic acid to be present in the extract. The

  2. Oncomirs Expression Profiling in Uterine Leiomyosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Bruna Cristine de Almeida

    2017-12-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. They play a key role in several biological processes. Their abnormal expression may lead to malignant cell transformation. This study aimed to evaluate the expression profile of 84 miRNAs involved in tumorigenesis in immortalized cells of myometrium (MM, uterine leiomyoma (ULM, and uterine leiomyosarcoma (ULMS. Specific cell lines were cultured and qRT-PCR was performed. Thirteen miRNAs presented different expression profiles in ULM and the same thirteen in ULMS compared to MM. Eight miRNAs were overexpressed, and five were underexpressed in ULM. In ULMS cells, five miRNAs exhibited an overexpression and eight were down-regulated. Six miRNAs (miR-1-3p, miR-130b-3p, miR-140-5p, miR-202-3p, miR-205-5p, and miR-7-5p presented a similar expression pattern in cell lines compared to patient samples. Of these, only three miRNAs showed significant expression in ULM (miR-1-3p, miR-140-5p, and miR-7-5p and ULMS (miR-1-3p, miR-202-3p, and miR-7-5p. Our preliminary approach identified 24 oncomirs with an altered expression profile in ULM and ULMS cells. We identified four differentially expressed miRNAs with the same profile when compared with patients’ samples, which strongly interacted with relevant genes, including apoptosis regulator (BCL2, epidermal growth factor receptor (EGFR, vascular endothelial growth factor A (VEGFA, insulin like growth factor 1 receptor (IGF1R,serine/threonine kinase (RAF1, receptor tyrosine kinase (MET, and bHLH transcription factor (MYCN. This led to alterations in their mRNA-target.

  3. Oncomirs Expression Profiling in Uterine Leiomyosarcoma Cells

    Science.gov (United States)

    Garcia, Natalia; Maffazioli, Giovana; Gonzalez dos Anjos, Laura; Chada Baracat, Edmund; Candido Carvalho, Katia

    2017-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. They play a key role in several biological processes. Their abnormal expression may lead to malignant cell transformation. This study aimed to evaluate the expression profile of 84 miRNAs involved in tumorigenesis in immortalized cells of myometrium (MM), uterine leiomyoma (ULM), and uterine leiomyosarcoma (ULMS). Specific cell lines were cultured and qRT-PCR was performed. Thirteen miRNAs presented different expression profiles in ULM and the same thirteen in ULMS compared to MM. Eight miRNAs were overexpressed, and five were underexpressed in ULM. In ULMS cells, five miRNAs exhibited an overexpression and eight were down-regulated. Six miRNAs (miR-1-3p, miR-130b-3p, miR-140-5p, miR-202-3p, miR-205-5p, and miR-7-5p) presented a similar expression pattern in cell lines compared to patient samples. Of these, only three miRNAs showed significant expression in ULM (miR-1-3p, miR-140-5p, and miR-7-5p) and ULMS (miR-1-3p, miR-202-3p, and miR-7-5p). Our preliminary approach identified 24 oncomirs with an altered expression profile in ULM and ULMS cells. We identified four differentially expressed miRNAs with the same profile when compared with patients’ samples, which strongly interacted with relevant genes, including apoptosis regulator (BCL2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), insulin like growth factor 1 receptor (IGF1R),serine/threonine kinase (RAF1), receptor tyrosine kinase (MET), and bHLH transcription factor (MYCN). This led to alterations in their mRNA-target. PMID:29295562

  4. GOLPH2 expression in renal cell cancer

    Directory of Open Access Journals (Sweden)

    Jung Klaus

    2008-11-01

    Full Text Available Abstract Background Renal cell carcinomas (RCC are among the most common and most lethal genitourinary malignancies. GOLPH2 (golgi phosphoprotein 2, GOLM1 has recently been proposed as a biomarker for hepatocellular and prostate cancer. In this study we analysed the expression patterns and the prognostic and diagnostic value of GOLPH2 in RCC. Methods GOLPH2 protein expression was analysed by immunohistochemistry in 104 clinically well characterized RCC cases in comparison with matched normal kidney tissue and in association with clinico-pathological parameters. Statistical analyses including Kaplan Meier analyses were performed with SPSS version 15.0. Results GOLPH2 was highly expressed in normal renal tubules and in almost half of RCC with a statistically significant predominance in the papillary and chromophobe histological subtypes. No other associations with clinico-pathological parameters were detectable. The Kaplan-Meier curves showed a weak trend for unfavourable prognosis of tumours with high GOLPH2 expression, but failed significance. Conclusion GOLPH2 protein is expressed in normal renal tissue (especially in distal tubular epithelia and is down-regulated in the majority of clear cell RCC. In papillary and chromophobe RCC GOLPH2 expression is consistently present. In contrast to its diagnostic value in hepatocellular and prostatic carcinomas, a prognostic or diagnostic value of GOLPH2 in RCC appears to be unlikely.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fuchs, Dominik [Research Group Molecular Neuro-Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany); Daniel, Volker; Sadeghi, Mahmoud; Opelz, Gerhard [Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany); Naujokat, Cord, E-mail: cord.naujokat@med.uni-heidelberg.de [Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany)

    2010-04-16

    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.

  6. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies that comp......It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies...... that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... survival might, therefore, act through such a matrix-to-cell suppression of apoptosis. Indeed, correlative mining of gene expression and patient survival databases suggests that poor survival in patients with metastatic cancer correlates highly with tumor expression of a common theme: the genes involved...

  7. Nanomaterials Enhanced Gene Expression in Yeast Cells

    Directory of Open Access Journals (Sweden)

    Su-Fang Chien

    2008-01-01

    Full Text Available Metal nanomaterials are shown to enhance gene expression for rice -galactosidase gene (-Gal in yeast cells. Au and Ag nanoparticles and their nanocomposites, silica-Au and silica-Ag, were prepared and characterized by UV-vis spectroscopy and TEM technique. The rice -galactosidase gene was cloned into the yeast chromosome, where the cloned cells were precultured and induced into a medium containing each of the testing nanomaterials. The nanomaterials were observed to incorporate inside the cells, and no cell death has been detected during the course of gene expression. The enzyme activity was determined by a synthetic substrate, p-nitrophenyl--D-galctopyranoside, and the yellow product yield was recorded in a spectrophotometer at 400 nm. When Au and Ag nanoparticles were incorporated with the culture, a 3–5 fold enhancement in -galactosidase was observed for intracellular activity as well as the secreted activity into the medium. The secreted protein was analyzed to have a pure form and displayed as a single protein band in the SDS-gel electrophoresis. The effects of size and chemical nature of nanomaterials on gene expression for the rice -galactosidase gene in yeast cells are discussed.

  8. Capsaicin pretreatment increased the bioavailability of cyclosporin in rats: involvement of P-glycoprotein and CYP 3A inhibition.

    Science.gov (United States)

    Zhai, Xue-jia; Shi, Fang; Chen, Fen; Lu, Yong-ning

    2013-12-01

    Capsaicin (CAP), the main ingredient responsible for the hot pungent taste of chilli peppers. This study investigated the effect of CAP on the pharmacokinetics of Cyclosporin A (CyA) in rats and the mechanism of this food-drug interaction. The results indicated that after 7 days of low or middle dose of CAP (0.3 or 1.0 mg/kg), the blood concentration of CyA was not significantly changed compared with that of vehicle-treated rats, whereas the blood concentration of CyA in high dose group (3.0 mg/kg) was significantly increased. The total clearance (CL/F) of CyA was decreased, and the bioavailability was significantly increased to about 1.44-fold of that in vehicle-treated rats after 7 days of high dose CAP treatment. At this time, the P-gp and CYP3A1/2 in the liver and intestine were decreased at both the mRNA and protein levels. These results demonstrated that chronic ingestion of high doses of CAP will increase the bioavailability of CyA to a significant extent in rats and the food-drug interaction between CAP and CyA appears to be due to modulation of P-gp and CYP3A gene expression by CAP, with differential dose-dependence. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  9. Polymorphisms in the multiple drug resistance protein 1 and in P-glycoprotein 1 are associated with time to event outcomes in patients with advanced multiple myeloma treated with bortezomib and pegylated liposomal doxorubicin

    NARCIS (Netherlands)

    G. Buda (Gabriele); D. Ricci (Deborah); C.C. Huang (Chris); R. Favis (Reyna); N. Cohen (Nadine); S.H. Zhuang (Sen); J-L. Harousseau (Jean-Luc); P. Sonneveld (Pieter); J. Bladé (Joan); R.Z. Orlowski (Robert)

    2010-01-01

    textabstractSingle nucleotide polymorphisms (SNPs) in the multiple drug resistance protein 1 (MRP1) and P-glycoprotein 1 (MDR1) genes modulate their ability to mediate drug resistance. We therefore sought to retrospectively evaluate their influence on outcomes in relapsed and/or refractory myeloma

  10. An antioxidant Trolox restores decreased oral absorption of cyclosporine A after liver ischemia-reperfusion through distinct mechanisms between CYP3A and P-glycoprotein in the small intestine.

    Science.gov (United States)

    Ikemura, Kenji; Inoue, Koichi; Mizutani, Hideki; Oka, Hisao; Iwamoto, Takuya; Okuda, Masahiro

    2012-09-05

    Oxidative stress is a critical mediator of various injuries following ischemia-reperfusion (I/R) associated with organ transplantation. Although oral bioavailability of cyclosporine A (CsA) was decreased by increased first-pass metabolism through CYP3A and P-glycoprotein (P-gp) specifically in the upper small intestine after liver I/R, the mechanism responsible for them remained to be clarified. In the present study, the effect of Trolox (an α-tocopherol analogue) on the decreased oral absorption of CsA through elevated intestinal CYP3A and P-gp after liver I/R and their regulations were investigated. Rats were subjected to 60 min of liver ischemia followed by 12h of reperfusion. Trolox was administered intravenously 5 min before reperfusion. Trolox diminished the increased malondialdehyde and total glutathione levels in plasma by liver I/R and concomitantly prevented the decreased area under the blood concentration-time curve of orally administered CsA as well as initial absorption rate of CsA from upper small intestine. The elevated CYP3A mRNA and activity in the upper small intestine as well as expression levels of P-gp in upper, middle, and lower small intestines after liver I/R were attenuated by Trolox administration. The elevations of CYP3A levels specifically in the upper small intestine of I/R rats were correlated with the lithocholic acid levels in the bile. These results demonstrate that Trolox ameliorates the decreased oral absorption of CsA through elevated intestinal CYP3A and P-gp by preventing oxidative stress, where the biliary lithocholic acid may be responsible for the elevated transcription of CYP3A specifically in the upper small intestine after liver I/R. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Ya. A. Udalova

    2009-01-01

    Full Text Available Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC. Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51; the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively.

  12. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

    Directory of Open Access Journals (Sweden)

    Duban-Deweer Sophie

    2010-11-01

    Full Text Available Abstract Background Brain capillary endothelial cells (BCECs form the physiological basis of the blood-brain barrier (BBB. The barrier function is (at least in part due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein. Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

  13. The Effect of P-Glycoprotein Inhibition and Activation on the Absorption and Serum Levels of Cyclosporine and Tacrolimus in Rats.

    Science.gov (United States)

    Yigitaslan, Semra; Erol, Kevser; Cengelli, Cigdem

    2016-01-01

    Permeability glycoprotein (P-glycoprotein or P-gp) plays an important role in the intestinal absorption of the immunosuppressive agents: cyclosporine and tacrolimus. The aim of this study was to determine how the intestinal absorption of cyclosporine and tacrolimus is affected when they are used with P-gp activating or inhibiting agents. In in vitro experiments, everted parts of rat small intestines were used to evaluate the effects of verapamil (a P-gp inhibitor) and rifampicin (a P-gp inducer) on the intestinal absorption of cyclosporine and tacrolimus. In in vivo experiments, the effects of verapamil and rifampicin on the plasma concentrations of cyclosporine and tacrolimus were evaluated. In in vitro experiments, the absorption of cyclosporine and tacrolimus from the small intestine increased in a time-dependent manner when the drugs were administered with or without verapamil or rifampicin. There was no difference in the absorption of cyclosporine ± verapamil/rifampicin between the jejunum and ileum; however, ileal absorption of tacrolimus + rifampicin was significantly higher than jejunal absorption (p tacrolimus were significantly increased when they were co-administered with verapamil (p < 0.001) and significantly decreased when co-administered with rifampicin (p < 0.05). P-gp may play an important role in the absorption of immunosupressive drugs, and it may contribute to drug-drug interactions thay may lead to inadequate drug response or toxicity.

  14. 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.

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

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    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.

  16. Evidence That P-glycoprotein Inhibitor (Elacridar)-Loaded Nanocarriers Improve Epidermal Targeting of an Anticancer Drug via Absorptive Cutaneous Transporters Inhibition.

    Science.gov (United States)

    Giacone, Daniela V; Carvalho, Vanessa F M; Costa, Soraia K P; Lopes, Luciana B

    2017-09-19

    Because P-glycoprotein (P-gp) plays an absorptive role in the skin, its pharmacological inhibition represents a strategy to promote cutaneous localization of anticancer agents that serve as its substrates, improving local efficacy while reducing systemic exposure. Here, we evaluated the ability of a nanoemulsion (NE) coencapsulating a P-gp inhibitor (elacridar) with the antitumor drug paclitaxel to promote epidermal targeting. Loaded NE displayed a nanometric size (45.2 ± 4.0 nm) and negative zeta potential (-4.2 ± 0.8 mV). Elacridar improved NE ability to inhibit verapamil-induced ATPase activity of P-gp; unloaded NE-inhibited P-gp when used at a concentration of 1500 μM, while elacridar encapsulation decreased this concentration by 3-fold (p p p P-gp inhibition and enabled epidermal targeting of paclitaxel, which in turn, can potentially reduce adverse effects associated with systemic exposure to anticancer therapy. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  17. 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.

  18. Aloe activated P-glycoprotein and CYP 3A: a study on the serum kinetics of aloe and its interaction with cyclosporine in rats.

    Science.gov (United States)

    Yang, Meng-Syuan; Yu, Chung-Ping; Huang, Ching-Ya; Chao, Pei-Dawn Lee; Lin, Shiuan-Pey; Hou, Yu-Chi

    2017-01-25

    Aloe, the leaf juice of Aloe vera, is a popular functional food worldwide. The major constituents of aloe are polyphenolic anthranoids such as aloin, aloe-emodin and rhein. Cyclosporine (CSP), an immunosuppressant with a narrow therapeutic window, is a probe substrate of P-glycoprotein (P-gp), an efflux pump, and CYP 3A4. This study first investigated the serum kinetics of aloe, then evaluated the modulation effects of aloe on P-gp and CYP 3A through an aloe-CSP interaction study in rats. The serum kinetic study showed that aloe-emodin glucuronides (G) and rhein sulfates/glucuronides (S/G) were major molecules in the bloodstream. The aloe-CSP interaction study showed that the systemic exposure to CSP was significantly decreased by either a single dose or multiple doses of aloe. The results of in vitro studies indicated that aloe activated P-gp and aloe metabolites activated CYP 3A4. In conclusion, aloe ingestion activated the functions of P-gp and CYP 3A in rats.

  19. Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy

    Directory of Open Access Journals (Sweden)

    Kim Kyung-Jong

    2008-08-01

    Full Text Available Abstract Background The membrane transporters such as P-glycoprotein (Pgp, the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines. Methods The MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses. Results The MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression but not SNU-668 (gastric, highest and SNU-C5 (gastric, no expression to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells. Conclusion These results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly

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

    Science.gov (United States)

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

    2011-11-01

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

  1. Expression of multidrug resistance proteins in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Swati Shukla

    2017-11-01

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

  2. Expression of multidrug resistance proteins in retinoblastoma.

    Science.gov (United States)

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

    2017-01-01

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

  3. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  4. Direct cell lysis for single-cell gene expression profiling

    Directory of Open Access Journals (Sweden)

    David eSvec

    2013-11-01

    Full Text Available The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA to be the best lysis agent, resulting in efficient cell lysis, high RNA stability and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single cells as well as samples composed of small numbers of cells.

  5. Cinnamaldehyde derivative (CB-PIC) sensitizes chemo-resistant cancer cells to drug-induced apoptosis via suppression of MDR1 and its upstream STAT3 and AKT signalling.

    Science.gov (United States)

    Yun, Miyong; Lee, Duckgue; Park, Moon-Nyeo; Kim, Eun-Ok; Sohn, Eun Jung; Kwon, Byung-Mog; Kim, Sung-Hoon

    2015-01-01

    Our group reported that cinnamaldehyde derivative, (E)-4-((2-(3-oxopop-1-enyl)phenoxy)methyl)pyridinium malonic acid (CB-PIC) induced apoptosis in hypoxic SW620 colorectal cancer cells via activation of AMP-activated protein kinase (AMPK) and extracellular signal regulated kinase (ERK). Herein, sensitizing effect of CB-PIC was investigated in resistant cancer cells such as paclitaxel (PT) resistant lung cancer cells (H460/PT), and Adriamycin (Adr) resistant breast cancer (MCF7/Adr) and colon cancer (HCT15/cos) cells. Various drug resistant cell lines were treated with CB-PIC, and the signalling pathway and functional assay were explored by Western blot, Rhodamine assay, FACS, RT-PCR and MTT assay. We found that CB-PIC effectively exerted cytotoxicity, increased sub G1 population and the cleaved form of poly (ADP-ribose) polymerase (PARP) and caspase 9 in drug resistant cancer cells. Furthermore, CB-PIC sensitized resistant cancer cells to adriamycin via downregulation of survival proteins such as survivin, Bcl-xL and Bcl-2, along with MDR1 suppression leading to accumulation of drug in the intracellular region. Of note, CB-PIC transcriptionally decreased MDR1 expression via suppression of STAT3 and AKT signalling in three resistant cancer cells with highly expressed P-glycoprotein. Nonetheless, CB-PIC did not affect transport activity of P-glycoprotein in a short time efflux assay, while epigallocatechin gallate (EGCG) accumulated Rhodamine 123 into intracellular region of cell by direct inhibition of MDR1 transport activity. These data demonstrate that CB-PIC suppresses the P-glycoprotein expression through inhibition of STAT3 and AKT signalling to overcome drug resistance in chemo-resistant cancer cells as a potent chemotherapeutic sensitizer. © 2015 S. Karger AG, Basel.

  6. Evaluation of P-glycoprotein (abcb1a/b) modulation of [(18)F]fallypride in MicroPET imaging studies.

    Science.gov (United States)

    Piel, Markus; Schmitt, Ulrich; Bausbacher, Nicole; Buchholz, Hans-Georg; Gründer, Gerhard; Hiemke, Christoph; Rösch, Frank

    2014-09-01

    [(18)F]Fallypride ([(18)F]FP) is an important and routinely used D2/D3 antagonist for quantitative imaging of dopaminergic neurotransmission in vivo. Recently it was shown that the brain uptake of the structurally related [(11)C]raclopride is modulated by P-glycoprotein (P-gp), an important efflux transporter at the blood-brain barrier. The purpose of this study was to determine whether the brain uptake of [(18)F]FP is influenced by P-gp. For examination of this possible modulation microPET studies were performed in a rat and a mouse model. Hence, [(18)F]FP was applied to Sprague Dawley rats, half of them being treated with the P-gp inhibitor cyclosporine A (CsA). In a second experimental series the tracer was applied to three different groups of FVB/N mice: wild type, P-gp double knockout (abcb1a/1b (-/-)) and CsA-treated mice. In CsA-treated Sprague Dawley rats [(18)F]FP showed an elevated standard uptake value in the striatum compared to the control animals. In FVB/N mice a similar effect was observed, showing an increasing uptake from wild type to CsA-treated and double knockout mice. Since genetically or pharmacologically induced reduction of P-gp activity increased the uptake of [(18)F]FP markedly, we conclude that [(18)F]FP is indeed a substrate of P-gp and that the efflux pump modulates its brain uptake. This effect - if true for humans - may have particular impact on clinical studies using [(18)F]FP for assessment of D2/3 receptor occupancy by antipsychotic drugs. This article is part of the Special Issue Section entitled 'Neuroimaging in Neuropharmacology'. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. In vivo evaluation of P-glycoprotein and breast cancer resistance protein modulation in the brain using [{sup 11}C]gefitinib

    Energy Technology Data Exchange (ETDEWEB)

    Kawamura, Kazunori [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan)], E-mail: kawamur@nirs.go.jp; Yamasaki, Tomoteru; Yui, Joji; Hatori, Akiko; Konno, Fujiko; Kumata, Katsushi; Irie, Toshiaki; Fukumura, Toshimitsu; Suzuki, Kazutoshi; Kanno, Iwao; Zhang Mingrong [Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan)

    2009-04-15

    Gefitinib (Iressa) is a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. Recent studies confirmed that gefitinib interacted with the breast cancer resistance protein (BCRP) at submicromolar concentrations, whereas other multidrug transporters, including P-glycoprotein (P-gp), showed much lower reactivity toward gefitinib. Recently, many tracers for positron emission tomography (PET) have been prepared to study P-gp function in vivo; however, PET tracers had not been evaluated for both P-gp and BCRP modulation in the brain. Therefore, we evaluated in vivo brain penetration-mediated P-gp and BCRP in mice using [{sup 11}C]gefitinib. Co-injection with gefitinib (over 50 mg/kg), a nonspecific P-gp modulator cyclosporin A (50 mg/kg), and the dual P-gp and BCRP modulator GF120918 (over 5 mg/kg) induced an increase in the brain uptake of [{sup 11}C]gefitinib in mice 30 min after injection. In the PET study of mice, the radioactivity level in the brain with co-injection of GF120918 (5 mg/kg) was three- to fourfold higher than that in control after initial uptake. The radioactivity level in the brain in P-gp and Bcrp knockout mice was approximately eightfold higher than that in wild-type mice 60 min after injection. In conclusion, [{sup 11}C]gefitinib is a promising PET tracer to evaluate the penetration of gefitinib into the brain by combined therapy with P-gp or BCRP modulators, and into brain tumors. Furthermore, PET study with GF120918 is a promising approach for evaluating brain penetration-mediated P-gp and BCRP.

  8. P-glycoprotein differentially affects escitalopram, levomilnacipran, vilazodone and vortioxetine transport at the mouse blood-brain barrier in vivo.

    Science.gov (United States)

    Bundgaard, Christoffer; Eneberg, Elin; Sánchez, Connie

    2016-04-01

    P-glycoprotein (P-gp)-mediated brain efflux of xenobiotics is a well-known process, which may result in suboptimal target engagement and consequently reduced efficacy of drugs exerting their therapeutic effects in the central nervous system. In the present study the role of P-gp in transport across the blood-brain barrier (BBB) was investigated with a series of newer antidepressants (levomilnacipran, vilazodone and vortioxetine) and a control substrate (escitalopram) using P-gp knock-out (KO) and P-gp competent wild-type (WT) mice. Brain and plasma exposure time-courses were measured after an acute subcutaneous dose and at steady-state obtained after subcutaneous drug infusion by osmotic minipumps. Following acute dosing, the brain-to-plasma KO/WT exposure enhancement ratios ((AUCbrain ko/AUCplasma ko)/(AUCbrain WT/AUCplasma WT)) were 5.8 (levomilnacipran), 5.4 (vilazodone), 3.1 (escitalopram) and 0.9 (vortioxetine), respectively. At steady-state, assessment of Kp,uu (unbound brain concentrations/unbound plasma concentrations) revealed a restriction in the brain distribution in WT mice for all compounds except vortioxetine. Levomilnacipran exhibited the most pronounced efflux with a Kp,uu-value of 0.038 in WT mice which was increased to 0.37 in KO mice. Based on both the acute and steady-state distribution data, the results suggest that levomilnacipran, vilazodone and escitalopram are susceptible to P-gp mediated efflux at the BBB in vivo in mice, whereas vortioxetine was practically devoid of being affected by P-gp in vivo. The functional impact of the drug transport-controlling role of P-gp at the BBB was demonstrated by in vivo cortical serotonin transporter occupancy of vilazodone, which exhibited a 20-fold higher plasma EC50 in WT mice compared to KOs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Comprehensive analog synthesis of (S)-valine thiazole peptidomimetic TTT-28 to understand enigmatic drug-binding sites of P-glycoprotein

    Science.gov (United States)

    Patel, Bhargav A.

    P-glycoprotein (P-gp) is considered an important therapeutic target for reversal of multidrug resistance (MDR) in cancer. It recognizes a diverse range of chemically and mechanistically dissimilar drugs. It has been postulated that the efflux by P-gp plays a major role in failure of chemotherapy. Hence, researchers have been trying to obtain a potent inhibitor of P-gp with specificity to tumor sites. In this pursuit, we previously were able to obtain a novel (S)-valine thiazole-derived peptidomimetic compound 1 ( TTT-28), which showed potent reversal of MDR in vitro as well as in vivo compared to verapamil, a well-known MDR modulator. We have also found that compound 1 triggers ATPase stimulation when incubated with P-gp alike verapamil, which implies its mechanism of action as competitive in nature. In this study, we attempted to understand structural requirements of ligands binding to a perplexing drug-binding site of P-gp and affecting its ATPase function. Toward this goal, we prepared a novel set of 64 analogues by fine tuning lead compound 1. These synthesized analogues were tested using ATPase activity assay. During the course of the study, a potent stimulator (1) of ATPase activity was transformed into an ATPase inhibitory leads such as compounds 43 , 57 and 113. The ATPase inhibitory activity of these compounds is predominantly contributed by the presence of a cyclohexyl group in place of the 2-aminobenzophenone moiety of ATPase activity stimulatory lead compound 1. Molecular modeling studies suggested a need for specific interactions with the drug-binding site of P-gp to induce different conformational states of P-gp to produce either stimulation or inhibition of ATPase activity. Collectively, this comprehensive synthesis work will facilitate further research towards P-gp inhibitor development.

  10. Comparative tissue pharmacokinetics and efficacy of moxidectin, abamectin and ivermectin in lambs infected with resistant nematodes: Impact of drug treatments on parasite P-glycoprotein expression

    OpenAIRE

    Lloberas, Mercedes; Alvarez, Luis; Entrocasso, Carlos; Virkel, Guillermo; Ballent, Mariana; Mate, Laura; Lanusse, Carlos; Lifschitz, Adrian

    2012-01-01

    The high level of resistance to the macrocyclic lactones has encouraged the search for strategies to optimize their potential as antiparasitic agents. There is a need for pharmaco-parasitological studies addressing the kinetic-dynamic differences between various macrocyclic lactones under standardized in vivo conditions. The current work evaluated the relationship among systemic drug exposure, target tissue availabilities and the pattern of drug accumulation within resistant Haemonchus co...

  11. Comparative tissue pharmacokinetics and efficacy of moxidectin, abamectin and ivermectin in lambs infected with resistant nematodes: Impact of drug treatments on parasite P-glycoprotein expression

    National Research Council Canada - National Science Library

    Lloberas, Mercedes; Alvarez, Luis; Entrocasso, Carlos; Virkel, Guillermo; Ballent, Mariana; Mate, Laura; Lanusse, Carlos; Lifschitz, Adrian

    2013-01-01

    .... The current work evaluated the relationship among systemic drug exposure, target tissue availabilities and the pattern of drug accumulation within resistant Haemonchus contortus for moxidectin, abamectin and ivermectin...

  12. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    OpenAIRE

    Kimitoshi Kohno; Noriaki Kitamura; Akihiro Kuma; Yoshihiro Yasuniwa; Takahiro Yamaguchi; Masaki Akiyama; Hiroto Izumi

    2011-01-01

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-li...

  13. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Energy Technology Data Exchange (ETDEWEB)

    Izumi, Hiroto, E-mail: h-izumi@med.uoeh-u.ac.jp; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Kuma, Akihiro; Kitamura, Noriaki; Kohno, Kimitoshi [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 (Japan)

    2011-10-19

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1), aurora kinase B (AURKB) and some minichromosome maintenance complex components (MCM). However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells) and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  14. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Directory of Open Access Journals (Sweden)

    Kimitoshi Kohno

    2011-10-01

    Full Text Available We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143 regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1, aurora kinase B (AURKB and some minichromosome maintenance complex components (MCM. However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  15. Forced Expression of ZNF143 Restrains Cancer Cell Growth.

    Science.gov (United States)

    Izumi, Hiroto; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Kuma, Akihiro; Kitamura, Noriaki; Kohno, Kimitoshi

    2011-10-19

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1), aurora kinase B (AURKB) and some minichromosome maintenance complex components (MCM). However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells) and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  16. CD25+ B-1a Cells Express Aicda

    Directory of Open Access Journals (Sweden)

    Hiroaki Kaku

    2017-06-01

    Full Text Available B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID, a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

  17. Implication of P-glycoprotein in formation of depression-prone personality: association study between the C3435T MDR1 gene polymorphism and interpersonal sensitivity.

    Science.gov (United States)

    Enokido, Masanori; Suzuki, Akihito; Sadahiro, Ryoichi; Matsumoto, Yoshihiko; Shibuya, Naoshi; Goto, Kaoru; Otani, Koichi

    2014-01-01

    Interpersonal sensitivity is defined as undue and excessive awareness of, and sensitivity to, the behavior and feelings of others. Previous studies suggested that interpersonal sensitivity is one of the vulnerable factors to depression, and that genetic factors and cortisol are involved in the formation of interpersonal sensitivity. On the other hand, P-glycoprotein, which is encoded by the multidrug resistance 1 (MDR1) gene, serves as a barrier to entry and as an active eliminator for xenobiotics and cellular metabolites including cortisol, which is implicated in multiple brain functions. In the present study, we examined the effects of the MDR1 C3435T polymorphism (rs1045642) on interpersonal sensitivity in healthy subjects. The subjects were 842 healthy Japanese volunteers (mean age = 26.7 years, male/female ratio = 490/352). The C3435T polymorphism of MDR1 gene was detected by a PCR method, and interpersonal sensitivity was assessed by the Interpersonal Sensitivity Measure (IPSM). The 2-factor analysis of covariance (ANCOVA) showed a significant main effect of the MDR1 genotype on the IPSM scores with a significant interaction between the genotype and gender. The subsequent 1-factor ANCOVA showed that in females the C/C genotype group had higher IPSM scores than the C/T genotype group (p < 0.001) and the T/T genotype group (p < 0.001), and the C/T genotype group had higher IPSM scores than the T/T genotype group (p = 0.014). In males no significant association was found between the MDR1 genotype and the IPSM scores. In allelic analyses using the χ(2) tests, the C allele frequency in females was significantly higher (p < 0.001) in the high IPSM group than in the low IPSM group, while there was no significant difference in the C allele frequency between the high and low IPSM groups in total subjects and males. The present study suggests that the C3435T polymorphism of the MDR1 gene affects the formation of a depression-prone personality trait in Japanese females.

  18. Enhanced oral bioavailability of felodipine by naringenin in Wistar rats and inhibition of P-glycoprotein in everted rat gut sacs in vitro.

    Science.gov (United States)

    Surya Sandeep, M; Sridhar, V; Puneeth, Y; Ravindra Babu, P; Naveen Babu, K

    2014-10-01

    The aim of this study was to investigate the effect of naringenin on the pharmacokinetics (PK) of felodipine in rats and membrane permeability across rat everted gut sacs in vitro. Rats were simultaneously co-administered with felodipine 10 mg/kg, p.o. and naringenin (25, 50 and 100 mg/kg, p.o.) for 15 consecutive days. Rats of the control groups received the corresponding volume of vehicle. Blood samples were withdrawn from retro-orbital plexus on first day in single dose PK study (SDS) and on 15th day in multiple dosing PK study (MDS). The PK parameters were calculated using Thermo kinetica. The co-administration of naringenin significantly elevated the Cmax and increased the AUCtotal of felodipine in dose-dependent manner. The Cmax of felodipine was increased from 173.25 ± 14.65 to 275.61 ± 44.62 and 223.26 ± 26.35 to 561.32 ± 62.53 ng/mL in SDS and MDS, respectively, at the dose of naringenin 100 mg/kg. The AUCtotal of felodipine was significantly (p < 0.001) increased from 2050.48 ± 60.57 to 3650.22 ± 78.61 and 3276.51 ± 325.61 to 7265.25 ± 536.11 (ng/mL/h) in SDS and MDS, respectively. The permeability of felodipine was increased in presence of naringenin and ritonavir (standard P-glycoprotein (P-gp) and Cytochrome P450 (CYP)3A4 inhibitor). Felodipine is a substrate of CYP3A4, and naringenin was reported to be a modulator of P-gp and CYP3A4. These results suggest that naringenin significantly increased the Cmax and AUC of felodipine is due to P-gp and CYP3A4 inhibition.

  19. Effect of an ethanol extract of Descurainia sophia seeds on Phase I and II drug metabolizing enzymes and P-glycoprotein activity in vitro.

    Science.gov (United States)

    Yi, Jin-Mu; Kim, Young Ah; Lee, You Jin; Bang, Ok-Sun; Kim, No Soo

    2015-12-18

    Descurainia sophia seeds have a variety of pharmacological functions and been widely used in traditional folk medicine. However, their effects on human drug metabolizing enzyme (DME) activities have not been elucidated. The present study investigated the inhibitory effects of an ethanol extract of D. sophia seeds (EEDS) on human Phase I/II (DMEs) and P-glycoprotein (p-gp) in vitro. The enzyme activities of human Phase I (cytochrome P450s, CYPs), Phase II (uridine diphosphate glucuronosyltransferases, UGTs) DMEs, and the drug transporter P-gp were determined in the presence of various concentrations of EEDS using commercially available luminogenic assay systems. The mode of enzyme inhibition and the inhibitory constant (Ki) value of EEDS were graphically determined with Lineweaver-Burk double reciprocal plots and secondary plots, respectively. The enzyme activity assays showed that EEDS moderately inhibited the CYP1A2, CYP2C9, and CYP2C19 isoforms with half maximal inhibitory concentrations (IC50) of 47.3, 25.8, and 38.7 μg/mL, respectively. Graphical analyses with Lineweaver-Burk double reciprocal plots and secondary plots indicated that EEDS competitively inhibited CYP2C9 with a Ki value of 19.8 μg/mL; however, it inhibited CYP2C9 and CYP2C19 in a mixed mode with Ki values of 5.2, and 11.9 μg/mL, respectively. Other Phase I (CYP2C8, CYP2D6, and CYP3A4) and Phase II (UGT1A1 and UGT2B7) enzymes as well as P-gp were weakly or negligibly affected by EEDS with concentrations up to 500 μg/mL. EEDS is a selective inhibitor of CYP1A2, CYP2C9, and CYP2C19 with moderate enzymatic inhibition. Clinically, full consideration should be given to a potential toxic adverse effect from a herb-drug interaction when drugs that are particularly susceptible to CYP1A2, CYP2C9, or CYP2C19-mediated metabolism are taken together with EEDS. Characterization of metabolic profiles of specific herbal drugs could help consumers and medical specialists to use them safely as a

  20. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Carolyn A. de Graaf

    2016-09-01

    Full Text Available Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

  1. Caveolin-1 expression in ovarian carcinoma is MDR1 independent.

    Science.gov (United States)

    Davidson, Ben; Goldberg, Iris; Givant-Horwitz, Vered; Nesland, Jahn M; Berner, Aasmund; Bryne, Magne; Risberg, Bjørn; Kopolovic, Juri; Kristensen, Gunnar B; Tropé, Claes G; van de Putte, Gregg; Reich, Reuven

    2002-02-01

    We studied the role of caveolin-1 in tumor progression and prognosis in serous ovarian carcinoma and the association between caveolin-1 and MDR1 expression. The study involved immunohistochemical analysis for caveolin-1 and P-glycoprotein (P-gp) expression in 75 effusions and 90 solid lesions from ovarian and primary peritoneal carcinoma; in situ hybridization for MDR1 messenger RNA (mRNA) expression in 62 effusions and all 90 tumors; and reverse transcription-polymerase chain reaction (RT-PCR) for caveolin-1 mRNA expression in 23 effusions. Immunohistochemical analysis localized caveolin-1 to the cell membrane in 43 effusions and 24 tumors. P-gp membrane expression was detected in 14 effusions and 11 tumors; MDR1 mRNA, in 20 effusions and 30 tumors. Caveolin-1 mRNA was expressed in 19 effusions. Caveolin-1 protein expression showed no association with that of P-gp protein or MDR1 mRNA. The expression of all markers was similar in carcinoma cells in pleural and peritoneal effusions. Caveolin-1 is a novel diagnostic marker for effusions; expression is moderately elevated in tumor cells in effusions, possibly owing to altered signal transduction and metabolism in cancer cells at this site. Expression seems MDR1 independent.

  2. Expression of the multidrug resistance protein MRP and the lung-resistance protein LRP in nasal NK/T cell lymphoma: further exploring the role of P53 and WT1 gene.

    Science.gov (United States)

    Huang, Wan-Ting; Huang, Chao-Cheng; Weng, Shao-Wen; Eng, Hock-Liew

    2009-02-01

    Nasal NK/T-cell lymphoma (NKTL) is relatively common in the adult men of Asia. Many patients with nasal NKTL have poor response to therapy. Some of them show P-glycoprotein over-expression. To investigate the expression of other multidrug resistance proteins (MDR) and possible regulatory factors in nasal NKTL, the clinical and pathological features are described. Thirty Chinese adults with nasal NKTL are presented. Immunohistochemical study was carried out to detect multidrug resistance-associated protein 1 (MRP) and lung resistance-related protein (LRP). The association between possible regulatory proteins (P53 and WT1) and MDR were explored. In situ hybridisation for Epstein-Barr virus (EBV) detection, polymerase chain reaction assay for T-cell receptor gene and direct sequencing for the P53 gene were performed. Seven (23.3%) and eight (26.7%) patients showed immunoreactivity of MRP and LRP, respectively. Positive staining for both markers was identified in 6.7% (2 cases). The EBV was detected in most cases (97%). Twenty-six (86.7%) cases expressed positive nuclear staining of P53. However, of the cases analysed for P53 mutation, none showed a mutaion at the hot spots studied. WT1 protein was not detected in the nasal NKTL. Our study reports expression of MRP and LRP in nasal NKTL. The over-expression of P53 is probably associated with high incidence of EBV infection and unlikely a regulatory protein for the expression of MRP and LRP. Further studies are necessary to validate the association between P53 mutation and expression of MRP and LRP in nasal NKTL.

  3. The biology of MDR1-P-glycoprotein (MDR1-Pgp in designing functional antibody drug conjugates (ADCs: the experience of gemtuzumab ozogamicin

    Directory of Open Access Journals (Sweden)

    Maurizio Cianfriglia

    2013-06-01

    Full Text Available BACKGROUND: The treatment of cancer remains a formidable challenge owing to the difficulties in differentiating tumor cells from healthy cells to ameliorate the disease without causing intolerable toxicity to patients. In addition, the emergence of MDR1-Pgp mediated multi-drug resistance (MDR it is a biological phenomenon that inhibits the curative potential of chemotherapeutic treatments. One way to improve the selectivity of therapeutic molecules in tumors would be to target them on the tumor site, thereby sparing normal tissues. AIMS: In this overview, we will discuss the biological factors influencing the safety and efficacy of the humanized mAb hP67.6 linked to the potent cytotoxic drug calicheamicin-gamma1 (gemtuzumab ozogamicin that target CD33 cell surface antigen expressed on AML cells. In addition, we highlight key aspects of MDR1-Pgp biology as a platform to understand its functional role in gemtuzumab ozogamicin immunotherapy which is tightly linked to an accurate assessment of the MDR status of AML cells. DISCUSSION: Several factors may affect the efficacy and safety of immunoconjugates. These include the common issues of chemical and antibody therapeutics such as specificity, heterogeneous target antigen expression and the complex pharmacokinetics profile of conveyed antibody. Further, the delivered drug may not be sufficient for providing therapeutic benefit, since the curative cytotoxic compound may be affected by intrinsic or acquired resistance of target cells. These and other potential problems, as well as the possible ways to overcome them will be discussed in this review by examining the biological factors involved in safety and efficacy of the first in class antibody drug conjugate (ADC gentuzumab ozogamicin. Despite this set-back, the extensive recorded data and the lessons learned from gentuzumab ozogamicin recently withdrawn from the market for safety concerns helped to pave the way for next generations of clinically

  4. Gene expression profiles in adenosine-treated human mast cells ...

    African Journals Online (AJOL)

    The role of mast cells in allergic diseases and innate immunity has been widely researched and much is known about the expression profiles of immune-related genes in mast cells after bacterial challenges. However, little is known about the gene expression profiles of mast cells in response to adenosine. Herein, we ...

  5. Identifying gene expression modules that define human cell fates

    OpenAIRE

    Germanguz, I; Listgarten, J; Cinkornpumin, J.; Solomon, A; Gaeta, X.; Lowry, W. E.

    2016-01-01

    Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in f...

  6. Effect of Y6, an epigallocatechin gallate derivative, on reversing doxorubicin drug resistance in human hepatocellular carcinoma cells.

    Science.gov (United States)

    Wen, Yan; Zhao, Rui-Qiang; Zhang, Yun-Kai; Gupta, Pranav; Fu, Li-Xiang; Tang, An-Zhou; Liu, Bu-Ming; Chen, Zhe-Sheng; Yang, Dong-Hua; Liang, Gang

    2017-05-02

    Cancer cells can acquire resistance to a wide variety of diverse and unrelated drugs, this phenomenon is termed multidrug resistance (MDR). Multidrug resistance has been an obstacle to the success of cancer chemotherapy. The present study investigated the reversal effect of Y6, a new compound obtained by chemically modifying the structure of epigallocatechin-3-gallate (EGCG) extracted from green tea. Y6 was proven to be effective in inhibiting cell proliferation and reversing drug resistance in doxorubicin (DOX) resistant human hepatocellular carcinoma cells (BEL-7404/DOX). BEL-7404/DOX cells were treated with either doxorubicin combination regimen (doxorubicin plus Y6 or epigallocatechin-3-gallate or verapamil separately) or doxorubicin alone. The results showed that cell proliferation was inhibited and late cell apoptosis increased in the combination treatment group, especially in the group treated with doxorubicin plus Y6. Further analysis revealed that the expressions of hypoxia-inducible factor-1α and multidrug resistance 1/P-glycoprotein decreased at both messenger RNA and protein levels by treatments with combined drugs compared to doxorubicin alone. Our results indicated that Y6, as a drug resistance reversal agent, increased the sensitivity of drug resistant cells to doxorubicin. The mechanisms of actions of Y6 in reversal effect were associated with the decreased expression of hypoxia-inducible factor-1α and multidrug resistance 1/P-glycoprotein.

  7. Physical disruption of cell-cell contact induces VEGF expression in RPE cells.

    Science.gov (United States)

    Farjood, Farhad; Vargis, Elizabeth

    2017-01-01

    To investigate the role of RPE cell-cell contact in vascular endothelial growth factor (VEGF) protein expression in cultures of primary human RPE (hRPE) cells and a human RPE cell line (ARPE-19). Two in vitro methods, scratching and micropatterning, were used to control the physical dissociation of RPE cell-cell junctions. Scratching was performed by scoring monolayers of RPE cells with a cell scraper. Micropatterning was achieved by using a stencil patterning method. Extracellular VEGF expression was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit. Immunocytochemistry (ICC) was performed to visualize the expression and localization of VEGF and intercellular proteins zonula occludens-1 (ZO-1), N-cadherin, β-catenin, and claudin-1 in RPE cultures. Higher expression of VEGF protein by cells on the edges of the scratched RPE layers was confirmed with ICC in short-term (1 day after confluency) and long-term (4 weeks after confluency) cultures. According to the ICC results, ZO-1, N-cadherin, β-catenin, and claudin-1 successfully localized to cell-cell junctions in long-term cultures of ARPE-19 and hRPE cells. However, unlike N-cadherin, β-catenin, and claudin-1, only ZO-1 localized junctionally in short-term cultures of both cell types. Moreover, removing cell-cell junctions by scratching resulted in the delocalization of ZO-1 from tight junctions to the cytoplasm. The loss of tight junction formation and the accumulation of ZO-1 in the cytoplasm correlated with increased VEGF expression. Micropatterning RPE cells on different sized circular patterns produced varying concentrations of cells with lost cell-cell junctions. When fewer cells formed intercellular junctions, increased extracellular VEGF secretion was observed from the ARPE-19 and hRPE cells. VEGF expression increases after physical disruption of RPE cell-cell connections. This increase in VEGF expression correlates with the loss of intercellular junctions and the localization of ZO-1 in

  8. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  9. Human brain imaging and radiation dosimetry of 11C-N-desmethyl-loperamide, a PET radiotracer to measure the function of P-glycoprotein.

    Science.gov (United States)

    Seneca, Nicholas; Zoghbi, Sami S; Liow, Jeih-San; Kreisl, William; Herscovitch, Peter; Jenko, Kimberly; Gladding, Robert L; Taku, Andrew; Pike, Victor W; Innis, Robert B

    2009-05-01

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that limits the distribution of drugs to several organs of the body. At the blood-brain barrier, P-gp blocks the entry of both loperamide and its metabolite, N-desmethyl-loperamide (N-dLop), and thereby prevents central opiate effects. Animal studies have shown that (11)C-dLop, compared with (11)C-loperamide, is an especially promising radiotracer because it generates negligible radiometabolites that enter the brain. The purposes of this study were to determine whether (11)C-dLop is a substrate for P-gp at the blood-brain barrier in humans and to measure the distribution of radioactivity in the entire body to estimate radiation exposure. Brain PET scans were acquired in 4 healthy subjects for 90 min and included concurrent measurements of the plasma concentration of unchanged radiotracer. Time-activity data from the whole brain were quantified using a 1-tissue-compartment model to estimate the rate of entry (K(1)) of radiotracer into the brain. Whole-body PET scans were acquired in 8 healthy subjects for 120 min. For brain imaging, after the injection of (11)C-dLop the concentration of radioactivity in the brain was low (standardized uptake value, approximately 15%) and stable after approximately 20 min. In contrast, uptake of radioactivity in the pituitary was about 50-fold higher than that in the brain. The plasma concentration of (11)C-dLop declined rapidly, but the percentage composition of plasma was unusually stable, with the parent radiotracer constituting 85% of total radioactivity after approximately 5 min. The rate of brain entry was low (K(1) = 0.009 +/- 0.002 mL.cm(-3).min(-1); n = 4). For whole-body imaging, as a measure of radiation exposure to the entire body the effective dose of (11)C-dLop was 7.8 +/- 0.6 muSv/MBq (n = 8). The low brain uptake of radioactivity is consistent with (11)C-dLop being a substrate for P-gp in humans and confirms that this radiotracer generates negligible quantities of

  10. Modeling bi-modality improves characterization of cell cycle on gene expression in single cells.

    Science.gov (United States)

    McDavid, Andrew; Dennis, Lucas; Danaher, Patrick; Finak, Greg; Krouse, Michael; Wang, Alice; Webster, Philippa; Beechem, Joseph; Gottardo, Raphael

    2014-07-01

    Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cell's phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%-17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome.

  11. [Drug resistance and its mechanism of intrinsic drug-resistant cell line GRC-1].

    Science.gov (United States)

    Kong, X; Ding, Y; Xia, T

    1997-11-01

    In order to probe the characteristics of drug resistance and its mechanisms of renal cell carcinoma, drug-resistant spectrum of renal cell carcinoma cell line GRC-1 was detected by in vitro MTT colorimetric assay, the mechanism of drug resistance in GRC-1 was also studied by the methods of both immunocytochemistry assay and flow fluorescence cytometry. The results demonstrated that GRC-1 was cross-resistant to adriamycin, vincrinstine, etoposide and carboplatinium, both mdr1 gene product P-glycoprotein and GST-pi which was an isozyme of glutathione S-transferases were expressed in GRC-1. The accumulation of net intracellular drugs of GRC-1 was less than that of drug sensitive breast cancer cell line MCF7, and the ability of pumping drugs out of cells was higher than that of MCF7. The results suggested that there is an intrinsic multidrug resistance in GRC-1 cell line, and both P-glycoprotein and glutathione systems play a role in the development of drug resistance for GRC-1. GRC-1 is an ideal target cell line for the study of drug resistance.

  12. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  13. Overcoming Multidrug Resistance in Human Cancer Cells by Natural Compounds

    OpenAIRE

    Nabekura, Tomohiro

    2010-01-01

    Multidrug resistance is a phenomenon whereby tumors become resistant to structurally unrelated anticancer drugs. P-glycoprotein belongs to the large ATP-binding cassette (ABC) transporter superfamily of membrane transport proteins. P-glycoprotein mediates resistance to various classes of anticancer drugs including vinblastine, daunorubicin, and paclitaxel, by actively extruding the drugs from the cells. The quest for inhibitors of anticancer drug efflux transporters has uncovered natural comp...

  14. Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Martins-Neves Sara R

    2012-04-01

    Full Text Available Abstract Background Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. Methods CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. Results The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. Conclusions MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma.

  15. Regulation of Mu Opioid Receptor Expression in Developing T Cells

    OpenAIRE

    Zhang, Lily; Belkowski, Judith Sliker; Briscoe, Tammi; Rogers, Thomas J.

    2012-01-01

    We have previously reported that functionally active μ-opioid receptors (MOR) are constitutively expressed at relatively low levels by developing T cells in the thymus. However, very little is known about the regulation of MOR expression by immature T cells. In this report, we first attempted to determine the effect of T cell receptor-induced T cell activation on the expression of MOR. We activated T cells with either the combination of anti-CD3 and CD28, or with superantigen, and observed a ...

  16. Advantages and Applications of CAR-Expressing Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Wolfgang eGlienke

    2015-02-01

    Full Text Available In contrast to donor T cells, natural killer (NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD. In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/ on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

  17. Increased expression of T-helper cell activation markers in ...

    African Journals Online (AJOL)

    Ehab

    part, by the actions of eosinophil-active cytokines derived predominantly from activated CD4 T-cells, of which interleukin-5 (IL-5) is uniquely eosinophil- specific 3,4. T-helper cells are a heterogeneous group of cells that can be divided into subpopulations on the basis of the expression of the cell surface markers. The.

  18. Investigation of factors responsible for cell line cytoplasmic expression differences

    Directory of Open Access Journals (Sweden)

    Finn Jonathan D

    2005-05-01

    Full Text Available Abstract Background Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line and COS-7 (a low expressing cell line. Results The main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes. Conclusion After exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.

  19. Gene expression signatures of human cell and tissue longevity.

    Science.gov (United States)

    Seim, Inge; Ma, Siming; Gladyshev, Vadim N

    2016-01-01

    Different cell types within the body exhibit substantial variation in the average time they live, ranging from days to the lifetime of the organism. The underlying mechanisms governing the diverse lifespan of different cell types are not well understood. To examine gene expression strategies that support the lifespan of different cell types within the human body, we obtained publicly available RNA-seq data sets and interrogated transcriptomes of 21 somatic cell types and tissues with reported cellular turnover, a bona fide estimate of lifespan, ranging from 2 days (monocytes) to a lifetime (neurons). Exceptionally long-lived neurons presented a gene expression profile of reduced protein metabolism, consistent with neuronal survival and similar to expression patterns induced by longevity interventions such as dietary restriction. Across different cell lineages, we identified a gene expression signature of human cell and tissue turnover. In particular, turnover showed a negative correlation with the energetically costly cell cycle and factors supporting genome stability, concomitant risk factors for aging-associated pathologies. In addition, the expression of p53 was negatively correlated with cellular turnover, suggesting that low p53 activity supports the longevity of post-mitotic cells with inherently low risk of developing cancer. Our results demonstrate the utility of comparative approaches in unveiling gene expression differences among cell lineages with diverse cell turnover within the same organism, providing insights into mechanisms that could regulate cell longevity.

  20. Gene expression profiling of Drosophila tracheal fusion cells.

    Science.gov (United States)

    Chandran, Rachana R; Iordanou, Ekaterini; Ajja, Crystal; Wille, Michael; Jiang, Lan

    2014-07-01

    The Drosophila trachea is a premier genetic system to investigate the fundamental mechanisms of tubular organ formation. Tracheal fusion cells lead the branch fusion process to form an interconnected tubular network. Therefore, fusion cells in the Drosophila trachea will be an excellent model to study branch fusion in mammalian tubular organs, such as kidneys and blood vessels. The fusion process is a dynamic cellular process involving cell migration, adhesion, vesicle trafficking, cytoskeleton rearrangement, and membrane fusion. To understand how these cellular events are coordinated, we initiated the critical step to assemble a gene expression profile of fusion cells. For this study, we analyzed the expression of 234 potential tracheal-expressed genes in fusion cells during fusion cell development. 143 Tracheal genes were found to encode transcription factors, signal proteins, cytoskeleton and matrix proteins, transporters, and proteins with unknown function. These genes were divided into four subgroups based on their levels of expression in fusion cells compared to neighboring non-fusion cells revealed by in situ hybridization: (1) genes that have relative high abundance in fusion cells, (2) genes that are dynamically expressed in fusion cells, (3) genes that have relative low abundance in fusion cells, and (4) genes that are expressed at similar levels in fusion cells and non-fusion tracheal cells. This study identifies the expression profile of fusion cells and hypothetically suggests genes which are necessary for the fusion process and which play roles in distinct stages of fusion, as indicated by the location and timing of expression. These data will provide the basis for a comprehensive understanding of the molecular and cellular mechanisms of branch fusion. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Identifying gene expression modules that define human cell fates.

    Science.gov (United States)

    Germanguz, I; Listgarten, J; Cinkornpumin, J; Solomon, A; Gaeta, X; Lowry, W E

    2016-05-01

    Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Spica prunellae and its marker compound rosmarinic acid induced the expression of efflux transporters through activation of Nrf2-mediated signaling pathway in HepG2 cells.

    Science.gov (United States)

    Wu, Jinjun; Zhu, Yuanfeng; Li, Fangyuan; Zhang, Guiyu; Shi, Jian; Ou, Rilan; Tong, Yunli; Liu, Yuting; Liu, Liang; Lu, Linlin; Liu, Zhongqiu

    2016-12-04

    Spica prunellae (SP) is a well-known traditional Chinese medicinal herb with properties of antihypertensive, antihyperglycemic, antiviral, anti-inflammatory, and antitumor activities. This herb is also popularly consumed as a food additive in some drinks or other food forms for treating pyreticosis. Rosmarinic acid (RA) is the marker compound from SP, which possesses anti-oxidative and anti-inflammatory functions. This study aims to investigate the regulatory effect of the water extract of SP (WESP) and RA on efflux transports (ETs), including P-glycoprotein (p-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) in HepG2 cell line. Results would provide beneficial information for the proper application of SP in clinics. HepG2 cells were treated with different doses of the tested drugs for 24 or 96h. MTT assay was used to examine cell viability. The protein and mRNA levels of the ETs were measured by using Western blot and real-time PCR, respectively. Reporter assay was used to study the antioxidant response element (ARE)-luciferin activity by using HepG2-C8 cells, which were generated by transfecting plasmid containing ARE-luciferin gene into HepG2 cells. The transport activities of ETs were tested by using substrate probes. WESP significantly (p<0.05) increased the expression of ETs in a dose-dependent manner. The increase caused by WESP was stronger than RA alone. Both WESP and RA promoted the translocation of nuclear factor E2-related factor-2 (Nrf2) from cytoplasm to the nucleus as well as significantly (p<0.05) enhanced the ARE-luciferin activity. WESP and RA also enhanced the efflux activity of P-gp and MRP2, accompanied by marked increase (p<0.05) in the intracellular ATP levels. WESP could significantly induce the expression of ETs through the activation of Nrf2-mediated signaling pathway in HepG2 cells. RA could be one of the active compounds responsible for the induction. WESP and RA also enhanced the efflux

  3. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Science.gov (United States)

    Pilbrough, Warren; Munro, Trent P; Gray, Peter

    2009-12-23

    Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise

  4. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

    Full Text Available Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean, approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations. Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50

  5. CD133 expression in chemo-resistant Ewing sarcoma cells

    Directory of Open Access Journals (Sweden)

    Kovar Heinrich

    2010-03-01

    Full Text Available Abstract Background Some human cancers demonstrate cellular hierarchies in which tumor-initiating cancer stem cells generate progeny cells with reduced tumorigenic potential. This cancer stem cell population is proposed to be a source of therapy-resistant and recurrent disease. Ewing sarcoma family tumors (ESFT are highly aggressive cancers in which drug-resistant, relapsed disease remains a significant clinical problem. Recently, the cell surface protein CD133 was identified as a putative marker of tumor-initiating cells in ESFT. We evaluated ESFT tumors and cell lines to determine if high levels of CD133 are associated with drug resistance. Methods Expression of the CD133-encoding PROM1 gene was determined by RT-PCR in ESFT tumors and cell lines. CD133 protein expression was assessed by western blot, FACS and/or immunostaining. Cell lines were FACS-sorted into CD133+ and CD133- fractions and proliferation, colony formation in soft agar, and in vivo tumorigenicity compared. Chemosensitivity was measured using MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxy-methoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium assays. Results PROM1 expression was either absent or extremely low in most tumors. However, PROM1 was highly over-expressed in 4 of 48 cases. Two of the 4 patients with PROM1 over-expressing tumors rapidly succumbed to primary drug-resistant disease and two are long-term, event-free survivors. The expression of PROM1 in ESFT cell lines was similarly heterogeneous. The frequency of CD133+ cells ranged from 2-99% and, with one exception, no differences in the chemoresistance or tumorigenicity of CD133+ and CD133- cell fractions were detected. Importantly, however, the STA-ET-8.2 cell line was found to retain a cellular hierarchy in which relatively chemo-resistant, tumorigenic CD133+ cells gave rise to relatively chemo-sensitive, less tumorigenic, CD133- progeny. Conclusions Up to 10% of ESFT express high levels of PROM1. In some tumors and cell

  6. TRPM5-expressing microvillous cells in the main olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Liman Emily R

    2008-11-01

    Full Text Available Abstract Background The main olfactory epithelium (MOE in the nasal cavity detects a variety of air borne molecules that provide information regarding the presence of food, predators and other relevant social and environmental factors. Within the epithelium are ciliated sensory neurons, supporting cells, basal cells and microvillous cells, each of which is distinct in morphology and function. Arguably, the least understood, are the microvillous cells, a population of cells that are small in number and whose function is not known. We previously found that in a mouse strain in which the TRPM5 promoter drives expression of the green fluorescent protein (GFP, a population of ciliated olfactory sensory neurons (OSNs, as well as a population of cells displaying microvilli-like structures is labeled. Here we examined the morphology and immunocytochemical properties of these microvillous-like cells using immunocytochemical methods. Results We show that the GFP-positive microvillous cells were morphologically diversified and scattered throughout the entire MOE. These cells immunoreacted to an antibody against TRPM5, confirming the expression of this ion channel in these cells. In addition, they showed a Ca2+-activated non-selective cation current in electrophysiological recordings. They did not immunoreact to antibodies that label cell markers and elements of the transduction pathways from olfactory sensory neurons and solitary chemosensory cells of the nasal cavity. Further, the TRPM5-expressing cells did not display axon-like processes and were not labeled with a neuronal marker nor did trigeminal peptidergic nerve fibers innervate these cells. Conclusion We provide morphological and immunocytochemical characterization of the TRPM5-expressing microvillous cells in the main olfactory epithelium. Our data demonstrate that these cells are non-neuronal and in terms of chemosensory transduction do not resemble the TRPM5-expressing olfactory sensory neurons

  7. Biological characteristics and chemosensitivity profile of four human anaplastic thyroid carcinoma cell lines.

    Science.gov (United States)

    Sekiguchi, M; Shiroko, Y; Arai, T; Kishino, T; Sugawara, I; Kusakabe, T; Suzuki, T; Yamashita, T; Obara, T; Ito, K; Hasumi, K

    2001-10-01

    Anaplastic thyroid carcinoma is a rapidly growing, aggressive neoplasm affecting the elderly which does not respond to most of the therapies. We established cultured cell lines from four untreated tumors. The cultures grew in a monolayer of spindle-shaped cells in three cell lines and of small polygonal cells in one line, having relatively long doubling times and chromosomal abnormalities. The xenotransplantation of the lines in athymic nude mice produced tumors with a histology similar to the original tumors. The immunocytochemical staining showed the expression of PCNA, HLA-class 1, cytokeratin, vimentin and FAS (fatty acid synthase) but not CEA, desmin or P-glycoprotein. The lines secreted TPA, IL-6, IL-8 and few or no thyroid-related hormones in the culture supernatant. One cell line produced G-CSF. The chemosensitivity assay revealed intrinsic drug resistance to nine out of 11 antineoplastic agents. The reverse transcriptase-polymerase chain reaction (RT-PCR) detected MRP (multidrug resistance-associated protein) mRNA but not mdr (multidrug resistance protein)-1 and mdr-3 mRNAs. This finding indicates that the multidrug resistance of these lines is mediated by a P-glycoprotein-unrelated mechanism. The RT-PCR also presented FAS mRNA in all the lines, and IL-6 and IL-8 mRNAs in some of the lines.

  8. High expression of markers of apoptosis in Langerhans cell histiocytosis

    DEFF Research Database (Denmark)

    Petersen, Bodil Laub; Lundegaard, Pia Rengtved; Bank, M I

    2003-01-01

    AIMS: Langerhans cell histiocytosis is a rare disease with clonal proliferation of dendritic histiocytes, occurring most frequently in infancy and early childhood. In the localized form (single system), the disease is self-limiting, but in the cases of multisystem disease a third of the patients...... develop organ dysfunction. In these cases the prognosis is poor. Our objective has been to study the immunohistochemical expression of Fas and Fas-ligand (Fas-L) in order to determine whether the level of expression of these proteins could predict the outcome of the disease. We also wanted to determine......53 and the number of cells in apoptosis detected with TUNEL. Langerhans cell histiocytosis cells showed strong expression of p53 and in some cases co-expression of Fas and Fas-L. The expression of Fas-L was significantly higher in infiltrates from patients with single-system disease. The actual...

  9. Explication of Definitional Description and Empirical Use of Fraction of Orally Administered Drugs Absorbed From the Intestine (Fa) and Intestinal Availability (Fg): Effect of P-glycoprotein and CYP3A on Fa and Fg.

    Science.gov (United States)

    Tanaka, Yuta; Kitamura, Yoshiaki; Maeda, Kazuya; Sugiyama, Yuichi

    2016-02-01

    Conventionally, it is believed that the fraction of orally administered drugs absorbed from the intestine (Fa) and intestinal availability (Fg) are independently determined by the apical membrane permeation and intestinal metabolism, respectively. However, the validity of this belief has not been well discussed, and Fa and Fg are often used without careful definition. In this review, Fa and Fg are mathematically described based on their definitions under the linear kinetics of metabolism and transport. Even considering with different models, intestinal metabolic enzymes such as cytochrome P450 3A affected both Fa and Fg, whereas apical efflux transporters including P-glycoprotein had no influence on Fg at least under the linear condition. To determine whether Fa and Fg calculated using different clinical methods are identical, empirical Fa and Fg were mathematically described based on "feces method" and "grapefruit juice method" and compared with their definitions. Fa and Fg obtained by the feces method corresponded with their definitions whereas the grapefruit juice method provided smaller Fa and larger Fg particularly for dual substrates of P-glycoprotein and cytochrome P450 3A with low membrane permeability. Our analyses suggest that the definitions and calculation methods of Fa and Fg should be considered when we intend to separately determine these values. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  10. Oct-4 expression maintained stem cell properties in prostate cancer ...

    African Journals Online (AJOL)

    The purpose of the present study is to isolate cancerous stem-like cells from normal healthy volunteers and prostate cancer patients (CD133+) which also express MDR1 and to ascertain the influence of Oct-4 on 'stem-ness' and differentiation of these CD133+ cells towards epithelium. Methods: CD133+ cells were isolated ...

  11. Regulatory natural killer cell expression in atopic childhood asthma ...

    African Journals Online (AJOL)

    Introduction: Different subsets of natural killer (NK) cells were found to play a role in pathogenesis of allergy. We sought to investigate the expression of regulatory NK cells (CD56+CD16+CD158+) in atopic children with bronchial asthma in order to outline the value of these cells as biomarkers of disease severity and/or ...

  12. Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells.

    Science.gov (United States)

    Peng, Weidan; Bao, Yunhua; Sawicki, Janet A

    2011-10-01

    To establish a method for efficient and relatively easy isolation of a cell population containing epithelial prostate stem cells, we developed two transgenic mouse models, K5/CFP and K18/RFP. In these models, promoters of the cytokeratin 5 (Krt5) and the cytokeratin 18 (Krt18) genes regulate cyan and red fluorescent proteins (CFP and RFP), respectively. CFP and RFP reporter protein fluorescence allows for visualization of K5(+) and K18(+) epithelial cells within the cellular spatial context of the prostate gland and for their direct isolation by FACS. Using these models, it is possible to test directly the stem cell properties of prostate epithelial cell populations that are positively selected based on expression of cytoplasmic proteins, K5 and K18. After validating appropriate expression of the K5/CFP and K18/RFP transgenes in the developing and adult prostate, we demonstrate that a subset of CFP-expressing prostate cells exhibits stem cell proliferation potential and differentiation capabilities. Then, using prostate cells sorted from double transgenic mice (K5/CFP + K18/RFP), we compare RNA microarrays of sorted K5(+)K18(+) basal and K5(-)K18(+) luminal epithelial cells, and identify genes that are differentially expressed. Several genes that are over-expressed in K5(+) cells have previously been identified as potential stem cell markers. These results suggest that FACS isolation of prostate cells from these mice based on combining reporter gene fluorescence with expression of potential stem cell surface marker proteins will yield populations of cells enriched for stem cells to a degree that has not been attained by using cell surface markers alone.

  13. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  14. [EGFR-expression in pulmonary neuroendocrine cell hyperplasia].

    Science.gov (United States)

    Kuhnen, C; Winter, B U

    2006-03-01

    15 cases of pulmonary neuroendocrine cell hyperplasia (carcinoid-tumorlets, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia/DIPNECH) and 20 neuroendocrine pulmonary tumors (10 carcinoid tumors, 5 large cell neuroendocrine, and 5 small cell neuroendocrine lung carcinomas) were immunohistochemically analyzed for the expression of epidermal growth factor receptor (EGFR, = HER-1). All cases of neuroendocrine cell hyperplasia exhibited a maximum EGFR expression (score 3 in 100% of cells) showing predominantly membranous, partly cytoplasmic staining. 4 ot the 10 carcinoid tumors were strongly positive for EGFR, whereas the other 6 were EGFR-negative. A total of 90% of large cell neuroendocrine and small cell neuroendocrine carcinomas were negative for EGFR. Overexpression of EGFR in pulmonary neuroendocrine cell hyperplasia might be significant for the pathogenesis of these lesions. As DIPNECH is characterized by clinical signs and symptoms including mild cough and obstructive functional impairment, a specific antagonistic therapeutic trial could aim at blocking EGFR/HER-1 or its subsequent signal transduction pathway.

  15. Differentiation of chronic lymphocytic leukemia B cells into immunoglobulin secreting cells decreases LEF-1 expression.

    Directory of Open Access Journals (Sweden)

    Albert Gutierrez

    Full Text Available Lymphocyte enhancer binding factor 1 (LEF-1 plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig secreting cells (ISC using the TLR9 agonist, CpG, together with cytokines (CpG/c. CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.

  16. [Expression of a human FL eukaryotic expressing plasmid mediated by lipofectamine in HFCL cells].

    Science.gov (United States)

    Ma, L J; Wang, G J; Li, L; Hong, X; Pei, X T

    2001-05-01

    Bone marrow stromal cell line-HFCL were transfected with the recombinant eukaryotic expressing vector-pIRESlneo/hFL by using liposome-mediated gene transfer method and get a stable expression. HFCL cells were transfected with the recombinant eukaryotic expressing vector-pIRESlneo/hFL by using liposome lipofectamine. Integration of hFL in the genome, transcription of hFL mRNA and expression of hFL protein in the transfected HFCL cells were assayed by Southern blot, Northern blot, Western blot and ELISA, the experiment of the human umbilical blood CD34+ cell multiplication. hFL cDNA was integrated into HFCL genome successfully, hFL mRNA was transcripted, hFL protein was expressed with (60.3 +/- 0.1) ng. 10(6) cell(-1) x d(-1) and the experiment of the human umbilical blood CD34+ cell multiplication shows that hFL has obvious biological activity in the supernatant. The recombinant plasmid is proved to be stably expressed in HFCL cells and obvious biological activity of hFL was detectable in the supernatant of the transfected cells.

  17. Engineering cells to improve protein expression.

    Science.gov (United States)

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J

    2014-06-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines.

    Directory of Open Access Journals (Sweden)

    Bisrat G Debeb

    Full Text Available BACKGROUND: The extraembryonic endoderm (ExEn defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines", which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE: Our findings (i suggest strongly that the ExEn precursor is a self-renewable entity, (ii indicate that active Oct4 gene expression (transcription plus translation is part of its molecular identity, and (iii provide an in vitro model of early ExEn differentiation.

  19. Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells.

    Science.gov (United States)

    Manokawinchoke, J; Sumrejkanchanakij, P; Subbalekha, K; Pavasant, P; Osathanon, T

    2016-12-01

    Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. CG5/Dx human breast cancer cell line: characterization of a new doxorubicin-resistant variant.

    Science.gov (United States)

    Gibelli, N; Zibera, C; Asti, A; Maestri, L; Bacchella, L; Pedrazzoli, P; Calligaro, A; Robustelli della Cuna, G

    1996-01-01

    By continuous exposure of CG5 human breast cancer cell line to increasing doxorubicin (Dx) concentrations, a multidrug-resistant (MDR) subline (CG5/Dx) was obtained. The resistant variant showed P-glycoprotein (P-gp) expression and a lower intracellular doxorubicin level than the parental cells. CG5/Dx cells were 19.4 fold more resistant to Dx than CG5 cells and showed a cross-resistance to some structurally related and unrelated compounds. Differences in kinetics, biological and ultrastructural features between the two cell lines were investigated. The CG5/Dx cells grew more slowly, produced higher CEA levels and showed a reduced progesterone receptor (PgR) content than the parental cells. Ultrastructural studies revealed differences involving, polyribosomes, rough endoplasmic reticulum, [mitochondria] and cytoskeleton.

  1. Choroid plexus epithelial monolayers – a cell culture model from porcine brain

    Directory of Open Access Journals (Sweden)

    Reichel Valeska

    2006-12-01

    Full Text Available Abstract Background The goal of the present study was to develop an in vitro choroid plexus (CP epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF and vice versa. Methods Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR, were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1 and multidrug resistance protein 1 (Mrp1, Abcc1 was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments. Results Choroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER values in the range of 100 to 150 Ωcm2. Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 ± 4.6 μl/cm2/h over 2–3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123. Conclusion It was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic

  2. Choroid plexus epithelial monolayers – a cell culture model from porcine brain

    Science.gov (United States)

    Baehr, Carsten; Reichel, Valeska; Fricker, Gert

    2006-01-01

    Background The goal of the present study was to develop an in vitro choroid plexus (CP) epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF) and vice versa. Methods Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR), were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1) and multidrug resistance protein 1 (Mrp1, Abcc1) was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments. Results Choroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER) values in the range of 100 to 150 Ωcm2. Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 ± 4.6 μl/cm2/h over 2–3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing) membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed) membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123. Conclusion It was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic transport across the blood

  3. Matrix metalloproteinase expression in keratocystic odontogenic tumors and primary cells.

    Science.gov (United States)

    Amm, Hope M; Casimir, Monee D; Clark, Dakota B; Sohn, Phillip; MacDougall, Mary

    2014-08-01

    Keratocystic odontogenic tumors (KCOTs) are locally invasive, rapidly proliferating cystic lesions of the jaw. The bone-invasive nature of these tumors has been previously associated with the expression of matrix metalloproteinases (MMPs), which degrade the extracellular matrix. The purpose of this study was to assess the expression and activity of MMPs in primary KCOT cells and tumor tissue. Four independently established KCOT primary cell populations were grown in Dulbecco's modified Eagle medium supplemented with 10% FBS and antibiotics. Primary cells were analyzed by qRT-PCR and immunohistochemistry (IHC), and for secretion of active MMPs. Primary tumor sections were analyzed by IHC. Of the 18 human MMPs examined, 9 were consistently expressed in primary KCOT cells. MMP-2 and MMP-14 were highly expressed in all KCOT populations, while MMP-1, 3, 11, 12, 16, 17, and 19 were moderately expressed. MMP-3, 11, 12, 16, 17 and 19 were shown to be expressed in KCOTs for the first time. No significant differences in MMPS profiles were found between syndromic (KCOT-3) and non-syndromic cell populations (KCOT-1/2/4). Protein expression of MMP-1, 11, 12, 14 and 16 was confirmed in each KCOT cell populations by IHC. KCOT-3 cells secreted active MMP-2 as determined by a gel zymography assay. Expression of MMP-1, 2, 3, 11, 12, 14, and 16 was confirmed in matching primary KCOT tumor sections representing syndromic and non-syndromic KCOTs. KCOT primary cell populations and tumors express a wide range of MMPs, which likely play a role in the bone-invasive nature of these tumors.

  4. Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells.

    Science.gov (United States)

    Boraas, Liana C; Guidry, Julia B; Pineda, Emma T; Ahsan, Tabassum

    2016-01-01

    Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this

  5. Impact of cell culture process changes on endogenous retrovirus expression.

    Science.gov (United States)

    Brorson, Kurt; De Wit, Christina; Hamilton, Elizabeth; Mustafa, Mehnaz; Swann, Patrick G; Kiss, Robert; Taticek, Ron; Polastri, Gian; Stein, Kathryn E; Xu, Yuan

    2002-11-05

    Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other

  6. Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144 during endothelial differentiation

    Directory of Open Access Journals (Sweden)

    Libermann Towia

    2008-05-01

    Full Text Available Abstract Background Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods Mouse embryonic stem (ES cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that

  7. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

    Science.gov (United States)

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado, Carlos D'Apparecida Santos

    2016-01-01

    Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

  8. Noise in Gene Expression Determines Cell Fate in Bacillus subtilis

    National Research Council Canada - National Science Library

    Hédia Maamar; Arjun Raj; David Dubnau

    2007-01-01

    .... In Bacillus subtilis, noise in ComK, the protein that regulates competence for DNA uptake, is thought to cause cells to transition to the competent state in which genes encoding DNA uptake proteins are expressed...

  9. Intraclonal protein expression heterogeneity in recombinant CHO cells

    National Research Council Canada - National Science Library

    Pilbrough, Warren; Munro, Trent P; Gray, Peter

    2009-01-01

    .... Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers...

  10. Analysis of allelic differential expression in human white blood cells

    National Research Council Canada - National Science Library

    Pant, P V Krishna; Tao, Heng; Beilharz, Erica J; Ballinger, Dennis G; Cox, David R; Frazer, Kelly A

    2006-01-01

    .... To identify human genes with allelic expression differences, we genotype DNA and examine mRNA isolated from the white blood cells of 12 unrelated individuals using oligonucleotide arrays containing 8406 exonic SNPs...

  11. [Construction of Trim6 eukaryotic expression vector and its expression in HEK293 cells].

    Science.gov (United States)

    Sun, Da-Kang; An, Xin-Ye; Hu, Feng-Ai; Li, Cai-Yu; Zheng, Jing

    2011-09-01

    To construct the recombinant eukaryotic expression vector pcDNA3.1 (+)-Trim6, and observe its expression in HEK293T cells in vitro. The total RNA was isolated from HeLa cells. After amplification with reverse transcription polymerase chain reaction (RT-PCR), the target sequences were cloned into the pcDNA3.1(+). The recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing. Then it was transfected into HEK293T cells.After 24 hours, the Trim6 expression was detected by Western blot. The results of the restriction enzyme digestion, PCR and sequencing confirmed the vector was constructed successfully, and it can express Trim6 protein in HEK293T cells. The vector is constructed successfully, which establishes the foundation for future research on the effect of Trim6.

  12. Cardiomyocyte expression and cell-specific processing of procholecystokinin

    DEFF Research Database (Denmark)

    Gøtze, Jens P.; Johnsen, Anders H.; Kistorp, Caroline

    2015-01-01

    Heart muscle cells produce peptide hormones such as natriuretic peptides. Developing hearts also express the gene for the classic intestinal hormone cholecystokinin (CCK) in amounts similar to those in the intestine and brain. However, cardiac expression of peptides other than natriuretic peptide...

  13. Expression of Podoplanin in Different Grades of Oral Squamous Cell ...

    African Journals Online (AJOL)

    Background: The expression of podoplanin is up‑regulated in a number of different human cancers, including squamous cell carcinoma of the oral cavity and its relationship with tumor invasion raises the possibility that podoplanin expression could be used as a biomarker for diagnosis and prognosis. Aim: The aim of the ...

  14. Increased expression of T-helper cell activation markers in ...

    African Journals Online (AJOL)

    Objective: To study the dynamic changes in peripheral blood CD4 cells expressing the activation markers naïve/memory (CD45RA/CD45RO) and interleukin–2 light chain receptor (CD25) in asthmatic children during and after resolution of acute asthma attacks and to determine whether the expression of these activation ...

  15. Differential expressed genes in ECV304 Endothelial-like Cells ...

    African Journals Online (AJOL)

    Background: Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through multiple mechanisms. Objective: With the application of DNA microarrays, we could monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad scale.

  16. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstr...

  17. Mechanosensory hair cells express two molecularly distinct mechanotransduction channels.

    Science.gov (United States)

    Wu, Zizhen; Grillet, Nicolas; Zhao, Bo; Cunningham, Christopher; Harkins-Perry, Sarah; Coste, Bertrand; Ranade, Sanjeev; Zebarjadi, Navid; Beurg, Maryline; Fettiplace, Robert; Patapoutian, Ardem; Mueller, Ulrich

    2017-01-01

    Auditory hair cells contain mechanotransduction channels that rapidly open in response to sound-induced vibrations. We report here that auditory hair cells contain two molecularly distinct mechanotransduction channels. One ion channel is activated by sound and is responsible for sensory transduction. This sensory transduction channel is expressed in hair cell stereocilia, and previous studies show that its activity is affected by mutations in the genes encoding the transmembrane proteins TMHS, TMIE, TMC1 and TMC2. We show here that the second ion channel is expressed at the apical surface of hair cells and that it contains the Piezo2 protein. The activity of the Piezo2-dependent channel is controlled by the intracellular Ca(2+) concentration and can be recorded following disruption of the sensory transduction machinery or more generally by disruption of the sensory epithelium. We thus conclude that hair cells express two molecularly and functionally distinct mechanotransduction channels with different subcellular distributions.

  18. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  19. CD34 expression in glioblastoma and giant cell glioblastoma.

    Science.gov (United States)

    Galloway, M

    2010-01-01

    This study aimed to determine whether CD34 is expressed in glioblastomas and giant cell glioblastomas, as this information may be of value when attempting to differentiate between giant cell glioblastomas and other relevant differential diagnoses such as pleomorphic xanthoastrocytomas with anaplastic features and anaplastic gangliogliomas. 11 giant cell glioblastomas and 16 non-giant cell glioblastomas were assessed with immunocytochemical staining for CD34. Standard immunocytochemical techniques were used, to reflect the staining patterns likely to be seen in routine diagnostic practice. Positive staining refers to staining of neoplastic cells. 73% of giant cell glioblastomas showed some degree of staining for CD34, and 55% showed strong widespread staining. 56% of non-giant cell glioblastomas showed some degree of CD34 staining, and 25% showed strong widespread staining. Both giant cell and non-giant cell glioblastomas frequently show CD34 expression by neoplastic cells, which may in some cases be strong and diffuse. Strong widespread staining of neoplastic cells for CD34 was more frequent in giant cell than non-giant cell glioblastomas, however this difference was not statistically significant. CD34 staining in isolation is unlikely to be of assistance in differentiating between giant cell glioblastoma and pleomorphic xanthoastrocytomas with anaplastic features or anaplastic gangliogliomas.

  20. Expression of stanniocalcin 1 in thyroid side population cells and thyroid cancer cells.

    Science.gov (United States)

    Hayase, Suguru; Sasaki, Yoshihito; Matsubara, Tsutomu; Seo, Daekwan; Miyakoshi, Masaaki; Murata, Tsubasa; Ozaki, Takashi; Kakudo, Kennichi; Kumamoto, Kensuke; Ylaya, Kris; Cheng, Sheue-yann; Thorgeirsson, Snorri S; Hewitt, Stephen M; Ward, Jerrold M; Kimura, Shioko

    2015-04-01

    Mouse thyroid side population (SP) cells consist of a minor population of mouse thyroid cells that may have multipotent thyroid stem cell characteristics. However the nature of thyroid SP cells remains elusive, particularly in relation to thyroid cancer. Stanniocalcin (STC) 1 and 2 are secreted glycoproteins known to regulate serum calcium and phosphate homeostasis. In recent years, the relationship of STC1/2 expression to cancer has been described in various tissues. Microarray analysis was carried out to determine genes up- and down-regulated in thyroid SP cells as compared with non-SP cells. Among genes up-regulated, stanniocalcin 1 (STC1) was chosen for study because of its expression in various thyroid cells by Western blotting and immunohistochemistry. Gene expression analysis revealed that genes known to be highly expressed in cancer cells and/or involved in cancer invasion/metastasis were markedly up-regulated in SP cells from both intact as well as partial thyroidectomized thyroids. Among these genes, expression of STC1 was found in five human thyroid carcinoma-derived cell lines as revealed by analysis of mRNA and protein, and its expression was inversely correlated with the differentiation status of the cells. Immunohistochemical analysis demonstrated higher expression of STC1 in the thyroid tumor cell line and thyroid tumor tissues from humans and mice. These results suggest that SP cells contain a population of cells that express genes also highly expressed in cancer cells including Stc1, which warrants further study on the role of SP cells and/or STC1 expression in thyroid cancer.

  1. HIV-1 induces DCIR expression in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Alexandra A Lambert

    2010-11-01

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  2. HIV-1 Induces DCIR Expression in CD4+ T Cells

    Science.gov (United States)

    Lambert, Alexandra A.; Imbeault, Michaël; Gilbert, Caroline; Tremblay, Michel J.

    2010-01-01

    The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4+ T cells found in the synovial tissue from rheumatoid arthritis (RA) patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons) and cells acutely infected in vitro (seen in both virus-infected and uninfected cells). Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals) and -independent intrinsic apoptotic pathways (involving the death effector AIF). Finally, we demonstrate that the higher surface expression of DCIR in CD4+ T cells is accompanied by an enhancement of virus attachment/entry, rep