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

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К. Yu. Khristenko

2016-01-01

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

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

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Yue-Hong Zhang

2017-07-01

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

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

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Gao W

2014-07-01

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

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.

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

Science.gov (United States)

Mo, Xianchao; Li, Weizhong

2014-05-01

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

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

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

2018-03-01

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

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

International Nuclear Information System (INIS)

Wang Ruoyu; Wang Hui; Fan Kai; Lv Shen

2007-01-01

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

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

DEFF Research Database (Denmark)

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

1993-01-01

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

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

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

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Lanying Pan

2015-02-01

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

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

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Lee Stephen D

2008-06-01

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

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

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

Science.gov (United States)

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

2015-06-01

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

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

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Sanja Perkovic

2012-09-01

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

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

International Nuclear Information System (INIS)

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

1995-01-01

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

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

DEFF Research Database (Denmark)

Brodin, Birger; Ozgür, Burak; Saaby, Lasse

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

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

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

Science.gov (United States)

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

2009-09-01

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

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

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Viera Bohacova

2018-05-01

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

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

International Nuclear Information System (INIS)

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

2001-01-01

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

Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

Science.gov (United States)

Xuan, X; Maeda, K; Mikami, T; Otsuka, H

1996-12-01

The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

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

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Renu Singh Dhanikula

2016-10-01

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

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

Science.gov (United States)

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

2008-01-01

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

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

Science.gov (United States)

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

2011-03-01

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

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

International Nuclear Information System (INIS)

Patel, Krupa J; Tannock, Ian F

2009-01-01

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

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

Science.gov (United States)

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

2014-02-05

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

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

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

2008-06-02

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

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

International Nuclear Information System (INIS)

Ivanina, Anna V.; Sokolova, Inna M.

2008-01-01

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

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

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Fu Juanjuan

2011-07-01

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

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

Science.gov (United States)

Crowe, Andrew; Bebawy, Mary

2012-08-15

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

P-glycoprotein targeted nanoscale drug carriers

KAUST Repository

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

2013-01-01

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

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

Science.gov (United States)

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.

Glycoprotein expression by adenomatous polyps of the colon

Science.gov (United States)

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

2008-03-01

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

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

Science.gov (United States)

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

1989-11-01

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

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

International Nuclear Information System (INIS)

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

2016-01-01

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-08-05

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin

Science.gov (United States)

André, Pascale; Spertini, Olivier; Guia, Sophie; Rihet, Pascal; Dignat-George, Françoise; Brailly, Hervé; Sampol, José; Anderson, Paul J.; Vivier, Eric

2000-01-01

Natural killer (NK) cells are components of the innate immune system that can recognize and kill virally infected cells, tumor cells, and allogeneic cells without prior sensitization. NK cells also elaborate cytokines (e.g., interferon-γ and tumor necrosis factor-α) and chemokines (e.g., macrophage inflammatory protein-1α) that promote the acquisition of antigen-specific immunity. NK cell differentiation is accompanied by the cell surface expression of a mucin-like glycoprotein bearing an NK cell-restricted keratan sulfate-related lactosamine carbohydrate, the PEN5 epitope. Here, we report that PEN5 is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1). The PEN5 epitope creates on PSGL-1 a unique binding site for L-selectin, which is independent of PSGL-1 tyrosine sulfation. On the surface of NK cells, the expression of PEN5 is coordinated with the disappearance of L-selectin and the up-regulation of Killer cell Ig-like Receptors (KIR). These results indicate that NK cell differentiation is accompanied by the acquisition of a unique carbohydrate, PEN5, that can serve as part of a combination code to deliver KIR+ NK cells to specific tissues. PMID:10725346

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

Science.gov (United States)

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

1996-01-01

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

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets

NARCIS (Netherlands)

Dercksen, M. W.; Weimar, I. S.; Richel, D. J.; Breton-Gorius, J.; Vainchenker, W.; Slaper-Cortenbach, C. M.; Pinedo, H. M.; von dem Borne, A. E.; Gerritsen, W. R.; van der Schoot, C. E.

1995-01-01

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

International Nuclear Information System (INIS)

Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

2009-01-01

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

Energy Technology Data Exchange (ETDEWEB)

Tiwari, Vaibhav [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Darmani, Nissar A.; Thrush, Gerald R. [Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

2009-12-18

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

Glycoprotein on cell surfaces

International Nuclear Information System (INIS)

Muramatsu, T.

1975-01-01

There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

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

DEFF Research Database (Denmark)

Nielsen, D; Maare, C; Eriksen, J

2001-01-01

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

Global analysis of glycoproteins identifies markers of endotoxin tolerant monocytes and GPR84 as a modulator of TNFα expression.

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Müller, Mario M; Lehmann, Roland; Klassert, Tilman E; Reifenstein, Stella; Conrad, Theresia; Moore, Christoph; Kuhn, Anna; Behnert, Andrea; Guthke, Reinhard; Driesch, Dominik; Slevogt, Hortense

2017-04-12

Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

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

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Jennifer Pasquier

2015-06-01

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

Nipah virus infection and glycoprotein targeting in endothelial cells

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Maisner Andrea

2010-11-01

Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

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.

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

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

2007-07-01

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

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

International Nuclear Information System (INIS)

Meng Zili; Shen Zhenya

2002-01-01

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

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

International Nuclear Information System (INIS)

Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L.

1989-01-01

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

The expression and serological reactivity of recombinant canine herpesvirus 1 glycoprotein D

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MarkéŽta Vaňkov‡á

2016-01-01

Full Text Available The aim of this work was to express recombinant glycoprotein D of canine herpesvirus 1 in bacterial cells and to evaluate its diagnostic sensitivity and specificity when compared to traditional serological methods. The gene fragment coding glycoprotein D of canine herpesvirus 1 was amplified by polymerase chain reaction, cloned into plasmid vector and expressed in Escherichia coli cells. Recombinant protein was then purified and used as an antigen in immunoblot for a detection of canine herpesvirus 1 specific antibodies. Antibody testing was performed on the panel of 100 canine sera by immunoblot with recombinant glycoprotein D as antigen and compared with indirect immunofluorescence assay. Serum samples were collected from 83 dogs with no history of canine herpesvirus 1 or reproductive disorders, and from 17 dogs from breeding kennels with a history of canine herpesvirus 1 related reproductive disorders. Sensitivity of glycoprotein D based immunoblot was 89.2% and specificity was 93%. Kappa value was calculated to be 0.8 between immunoblot and indirect immunofluorescence assay. Antibodies against canine herpesvirus 1 infection were detected in 33% of samples by immunoblot assay. Our study confirms that recombinant glycoprotein D expressed in bacterial cells could be used as a suitable and sensitive antigen for immunological tests and that herpesvirus infection seems to be common among the canine population in the Czech Republic.

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

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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.

An unusual dependence of human herpesvirus-8 Glycoproteins-induced cell-to-cell fusion on heparan sulfate

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Tiwari, Vaibhav; Darmani, Nissar A.; Thrush, Gerald R.; Shukla, Deepak

2009-01-01

Human herpes virus 8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: 1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; 2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, 3) coexpression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis. PMID:19747451

Evaluating the Effects of Cytomegalovirus Glycoprotein B on the Maturation and Function of Monocyte-derived dendritic cells

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Afsson shariat

2015-11-01

Full Text Available Background & Objectives: Interaction of cytomegalovirus glycoprotein B with toll-like receptors of dendritic cells leads to early signaling and innate immune responses. The aim of this study is to evaluate the effects of cytomegalovirus glycoprotein B on the maturation and function of monocyte-derived dendritic cells in treated groups in comparison with control groups. Materials & Methods: Blood samples were taken from 5 healthy volunteers. Following the generation of monocyte-derived dendritic cells on the fifth day of cell culture, half of the immature dendritic cells were treated with cytomegalovirus glycoprotein B, and the rest of them were induced to mature dendritic untreated cells and were used as the control group. The maturation and function of dendritic cells were evaluated in these two groups. Results: The gene expression level of toll-like receptor-4 significantly increased in the group treated with glycoprotein B (p < 0.05, whereas there were no significant differences in the expression rates of CD83, CD86, CD1a, and HLA-DR and the secretion of IL-23 from monocyte-derived dendritic cells between the treated groups and the controls. Conclusion: The increase in the gene expression of toll-like receptor-4 in monocyte-derived dendritic cells treated with cytomegalovirus glycoprotein B showed that cell contact is required to elicit cellular antiviral response and toll-like receptor activation. Thus, it is critical to recognize the viral and cellular determinants of the immune system in order to develop new therapeutic strategies against cytomegalovirus.

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

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

2017-01-01

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

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

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

2012-01-01

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

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

Science.gov (United States)

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

2012-03-01

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

Epitope diversity of N-glycans from bovine peripheral myelin glycoprotein P0 revealed by mass spectrometry and nano probe magic angle spinning 1H NMR spectroscopy

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Gutiérrez Gallego, R.; Jiménez Blanco, J.L.; Thijssen-van Zuylen, C.W.E.M.; Gotfredsen, C.H.; Voshol, H.; Duus, J.Ø.; Schachner, M.

2001-01-01

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most

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

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

2012-12-18

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

Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.

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Aricescu, A Radu; Owens, Raymond J

2013-06-01

Mammalian cells are rapidly becoming the system of choice for the production of recombinant glycoproteins for structural biology applications. Their use has enabled the structural investigation of a whole new set of targets including large, multi-domain and highly glycosylated eukaryotic cell surface receptors and their supra-molecular assemblies. We summarize the technical advances that have been made in mammalian expression technology and highlight some of the structural insights that have been obtained using these methods. Looking forward, it is clear that mammalian cell expression will provide exciting and unique opportunities for an integrative approach to the structural study of proteins, especially of human origin and medically relevant, by bridging the gap between the purified state and the cellular context. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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

2017-01-15

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

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

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Daniel G. Gerardi

2014-01-01

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

pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells

DEFF Research Database (Denmark)

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

1998-01-01

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

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

Science.gov (United States)

Singh, Manu Smriti; Lamprecht, Alf

2016-01-01

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

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

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Li-Li Liu

2017-01-01

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

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

Science.gov (United States)

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

2015-04-01

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

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

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Anja Brenn

2011-01-01

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

An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

KAUST Repository

Ali, Amal J.

2017-05-03

Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

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

Science.gov (United States)

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

2015-10-01

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

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

Science.gov (United States)

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

2013-02-01

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

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

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Yaqiong Zu

2014-08-01

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

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

International Nuclear Information System (INIS)

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

2005-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

KAUST Repository

Merzaban, Jasmeen S.

2011-06-09

Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin-/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved byWestern blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand- 1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL."E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL\\'s contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. © 2011 by The American Society of Hematology.

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...

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

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Aoranit Somno

2016-01-01

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

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

Science.gov (United States)

Ao, Jie; Chinnici, Jennifer L.; Maddi, Abhiram

2015-01-01

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix. PMID:26048011

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

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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.

P-glycoprotein activity and biological response

International Nuclear Information System (INIS)

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

2005-01-01

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

Annotating Human P-Glycoprotein Bioassay Data.

Science.gov (United States)

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

2012-08-01

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

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

Directory of Open Access Journals (Sweden)

Vanessa Lopes-Rodrigues

2016-11-01

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

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

Science.gov (United States)

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

2016-01-01

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

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

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Hongping Min

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

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

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

Science.gov (United States)

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

2014-11-01

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

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

NARCIS (Netherlands)

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

2016-01-01

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

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

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

International Nuclear Information System (INIS)

Cheng Bing; Liu Baoping; Han Xingmin

2003-01-01

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

Immunization with a dicistronic plasmid expressing a truncated form of bovine herpesvirus-1 glycoprotein D and the amino-terminal subunit of glycoprotein B results in reduced gB-specific immune responses

International Nuclear Information System (INIS)

Manoj, Sharmila; Babiuk, Lorne A.; Drunen Littel van-Hurk, Sylvia van den

2003-01-01

As an approach to create a divalent DNA vaccine, a truncated secreted version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD) and the amino-terminal subunit of glycoprotein B (gBb) were expressed from a dicistronic plasmid, designated pSLIAtgD-IRES-gBb. Intradermal immunization of mice with pSLIAtgD-IRES-gBb or a mixture of plasmids encoding tgD (pSLIAtgD) and gBb (pSLIAgBb) by needle injection or gene gun elicited strong tgD-specific immune responses. However, a significant reduction in gBb-specific immune responses was observed upon immunization of mice with pSLIAtgD-IRES-gBb or a mixture of pSLIAtgD and pSLIAgBb in comparison to immunization with pSLIAgBb alone. This reduction in gBb-specific immune responses induced by pSLIAtgD-IRES-gBb was due to production of low amounts of gBb from pSLIAtgD-IRES-gBb, inefficient processing and transport of gBb, and possibly competition for antigen-presenting cells by tgD and gBb. These results indicate that, although divalent plasmids may be used to express different antigens, the efficacy of vaccination with such plasmids may be influenced by the plasmid design and the characteristics of the expressed antigens

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

International Nuclear Information System (INIS)

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

2004-01-01

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

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

Science.gov (United States)

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

2016-01-01

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

Biological and immunogenic properties of rabies virus glycoprotein expressed by canine herpesvirus vector.

Science.gov (United States)

Xuan, X; Tuchiya, K; Sato, I; Nishikawa, Y; Onoderaz, Y; Takashima, Y; Yamamoto, A; Katsumata, A; Iwata, A; Ueda, S; Mikami, T; Otsuka, H

1998-01-01

In order to evaluate whether canine herpesvirus (CHV) could be used as a live vector for the expression of heterologous immunogenes, we constructed a recombinant canine herpesvirus (CHV) expressing glycoprotein (G protein) of rabies virus (RV). The gene of G protein was inserted within the thymidine kinase gene of CHV YP11mu strain under the control of the human cytomegalovirus immediate early promoter. The G protein expressed by the recombinant CHV was processed and transported to the cell surface as in RV infected cells, and showed the same biological activities such as low pH dependent cell fusion and hemadsorption. The antigenic authenticity of the recombinant G protein was confirmed by a panel of monoclonal antibodies specific for G protein. Dogs inoculated intransally with the recombinant CHV produced higher titres of virus neutralizing antibodies against RV than those inoculated with a commercial, inactivated rabies vaccine. These results suggest that the CHV recombinant expressing G protein can be used as a vaccine to control canine rabies and that CHV may be useful as a vector to develop live recombinant against other infectious diseases in dogs.

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

International Nuclear Information System (INIS)

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

2005-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2005-02-15

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

Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.

Science.gov (United States)

Allen, Cory; McDonald, Cari; Giannini, Caterina; Peng, Kah Whye; Rosales, Gabriela; Russell, Stephen J; Galanis, Evanthia

2004-11-01

Fusogenic membrane glycoproteins (FMG) such as the gibbon ape leukemia virus envelope (GALV) glycoprotein are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death. Nevertheless, ubiquitous expression of the GALV receptor, Pit-1, makes targeting desirable in order to increase the specificity of the observed cytopathic effect. Here we report on use of matrix metalloproteinase (MMP)-cleavable linkers to target the cytotoxicity of FMG-expressing adenoviral vectors against gliomas. Replication-defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the GALV glycoprotein linked to a blocking ligand (C-terminal extracellular domain of CD40 ligand) through either an MMP-cleavable linker (AdM40) or a non-cleavable linker (AdN40). Both viruses also co-expressed the green fluorescent protein (GFP) via an internal ribosomal entry site. The glioma cell lines U87, U118, and U251 characterized by zymography and MMP-2 activity assay as high, medium and low MMP expressors, respectively, the MMP-poor cell lines TE671 and normal human astrocytes were infected with AdM40 and AdN40 at different multiplicities of infection (MOIs) from 1-30. Fusion was quantitated by counting both number and size of syncytia. Infection of these cell lines with AdN40 did not result in fusion or cytotoxic cell death, despite the presence of infection, as demonstrated by GFP positivity, therefore indicating that the displayed CD40 ligand blocked GALV-induced fusion. Fusion was restored after infection of glioma cells with AdM40 at an MOI as low as 1 to an extent dependent on MMP expression and coxsackie adenovirus receptor (CAR) expression in the specific cell line. Western immunoblotting demonstrated the presence of the cleaved CD40 ligand in the supernatant of fused glioma cells. Use of the MMP

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.

The cell surface expressed nucleolin is a glycoprotein that triggers calcium entry into mammalian cells

International Nuclear Information System (INIS)

Losfeld, Marie-Estelle; Khoury, Diala El; Mariot, Pascal; Carpentier, Mathieu; Krust, Bernard; Briand, Jean-Paul; Mazurier, Joel; Hovanessian, Ara G.; Legrand, Dominique

2009-01-01

Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. It has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Several studies have demonstrated that surface nucleolin serves as a receptor for various extracellular ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the presence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon metabolic labeling of cells with [ 3 H]glucosamine. Besides its well-demonstrated role in the internalization of specific ligands, here we show that ligand binding to surface nucleolin could also induce Ca 2+ entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopeptide, which binds specifically surface nucleolin, triggers rapid and intense membrane Ca 2+ fluxes in various types of cells. The use of several drugs then indicated that Store-Operated Ca 2+ Entry (SOCE)-like channels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to surface nucleolin could be involved in the activation of signaling pathways by promoting Ca 2+ entry into cells

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.

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.

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

Science.gov (United States)

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

2014-12-01

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

Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

OpenAIRE

Puddington, L; Woodgett, C; Rose, J K

1987-01-01

The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

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

Directory of Open Access Journals (Sweden)

Cara Andrea

2007-03-01

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

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.

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

Science.gov (United States)

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

2018-01-01

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

Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

International Nuclear Information System (INIS)

Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G.

2007-01-01

The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of 3 H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

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Kamel El Omari

2013-01-01

Full Text Available Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1 at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

Science.gov (United States)

El Omari, Kamel; Iourin, Oleg; Harlos, Karl; Grimes, Jonathan M.; Stuart, David I.

2013-01-01

Summary Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed. PMID:23273918

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

International Nuclear Information System (INIS)

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

2016-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-08-15

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

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

OpenAIRE

Li-Li Dong; Ru Tang; Yu-Jia Zhai; Tejsu Malla; Kai Hu

2017-01-01

AIM: To investigate whether DNA vaccine encoding herpes simplex virus 1 (HSV-1) glycoprotein C (gC) and glycoprotein D (gD) will achieve better protective effect against herpes simplex keratitis (HSK) than DNA vaccine encoding gD alone. METHODS: DNA vaccine expressing gD or gC combined gD (gD.gC) were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293T cells by Western-blot. For immunization, mice w...

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.

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

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Li-Li Dong

2017-11-01

Full Text Available AIM: To investigate whether DNA vaccine encoding herpes simplex virus 1 (HSV-1 glycoprotein C (gC and glycoprotein D (gD will achieve better protective effect against herpes simplex keratitis (HSK than DNA vaccine encoding gD alone. METHODS: DNA vaccine expressing gD or gC combined gD (gD.gC were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS: Fusion protein gD.gC could be expressed successfully in cultured 293T cells. And, pRSC-gC.gD-IL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and sIgA production, enhanced cytotoxicities of splenocytes and nature killer cells (NK, when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION: gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future.

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

Science.gov (United States)

Dong, Li-Li; Tang, Ru; Zhai, Yu-Jia; Malla, Tejsu; Hu, Kai

2017-01-01

AIM To investigate whether DNA vaccine encoding herpes simplex virus 1 (HSV-1) glycoprotein C (gC) and glycoprotein D (gD) will achieve better protective effect against herpes simplex keratitis (HSK) than DNA vaccine encoding gD alone. METHODS DNA vaccine expressing gD or gC combined gD (gD.gC) were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS Fusion protein gD.gC could be expressed successfully in cultured 293T cells. And, pRSC-gC.gD-IL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and sIgA production, enhanced cytotoxicities of splenocytes and nature killer cells (NK), when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future. PMID:29181304

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

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Erica Lynn Bradshaw-Pierce

2013-03-01

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

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

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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.

Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

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Daniel J Hoover

Full Text Available The glycoprotein YKL-40 (CHI3L1 is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and

Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

International Nuclear Information System (INIS)

Changotra, Harish; Turk, Susan M.; Artigues, Antonio; Thakur, Nagendra; Gore, Mindy; Muggeridge, Martin I.; Hutt-Fletcher, Lindsey M.

2016-01-01

The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.

Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

Energy Technology Data Exchange (ETDEWEB)

Changotra, Harish; Turk, Susan M. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Artigues, Antonio [Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS (United States); Thakur, Nagendra; Gore, Mindy; Muggeridge, Martin I. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Hutt-Fletcher, Lindsey M., E-mail: lhuttf@lsuhsc.edu [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States)

2016-02-15

The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.

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

International Nuclear Information System (INIS)

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

2015-01-01

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

Ammonia transport in the kidney by Rhesus glycoproteins

Science.gov (United States)

Verlander, Jill W.

2014-01-01

Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport. PMID:24647713

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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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.

Comparison of glycoprotein expression between ovarian and colon adenocarcinomas

DEFF Research Database (Denmark)

Multhaupt, H A; Arenas-Elliott, C P; Warhol, M J

1999-01-01

, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both...... primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use...... in distinguishing between these 2 entities. CONCLUSION: A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot...

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

Science.gov (United States)

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

2013-01-01

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

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.

Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum

International Nuclear Information System (INIS)

Leberer, E.; Charuk, J.H.M.; MacLennan, D.H.; Green, N.M.

1989-01-01

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1,308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH 2 -terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca 2+ . The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45 C 2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca 2+ with the membrane. The authors propose that this lumenal Ca 2+ binding glycoprotein of the sarcoplasmic reticulum be designated sarcalumenin

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

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Chen HH

2015-08-01

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

THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

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A. V. Shulkin

2015-09-01

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

Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins

OpenAIRE

Buonocore, Linda; Blight, Keril J.; Rice, Charles M.; Rose, John K.

2002-01-01

We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein...

Engineered CHO cells for production of diverse, homogeneous glycoproteins

DEFF Research Database (Denmark)

Yang, Zhang; Wang, Shengjun; Halim, Adnan

2015-01-01

Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferas...

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

Science.gov (United States)

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

2017-01-01

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

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

Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

Energy Technology Data Exchange (ETDEWEB)

Yagi, Hirokazu [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Nakamura, Masatoshi [National Institute of Agrobiological Sciences, Genetic Resources Conservation Research Unit, Genetic Resources Center (Japan); Yokoyama, Jun [Taiyo Nippon Sanso Corporation, Tsukuba Laboratories (Japan); Zhang, Ying; Yamaguchi, Takumi [National Institutes of Natural Sciences, Institute for Molecular Science and Okazaki Institute for Integrative Bioscience (Japan); Kondo, Sachiko [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Kobayashi, Jun [Yamaguchi University, Department of Biological and Environmental Sciences, Faculty of Agriculture (Japan); Kato, Tatsuya; Park, Enoch Y. [Shizuoka University, Laboratory of Biotechnology, Research Institute of Green Science and Technology (Japan); Nakazawa, Shiori [Nagoya University, Sugashima Marine Biological Laboratory, Graduate School of Science (Japan); Hashii, Noritaka; Kawasaki, Nana [National Institute of Health Sciences, Division of Biological Chemistry and Biologicals (Japan); Kato, Koichi, E-mail: kkato@phar.nagoya-cu.ac.jp [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan)

2015-06-15

Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing {sup 15}N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a {sup 15}N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.

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

Science.gov (United States)

Crawford, Lindsey; Putnam, David

2014-08-20

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

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

International Nuclear Information System (INIS)

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

2002-01-01

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

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

Science.gov (United States)

Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

2017-02-01

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

Cell wall O-glycoproteins and N-glycoproteins: biosynthesis and some functional aspects.

Directory of Open Access Journals (Sweden)

Eric eNguema-Ona

2014-10-01

Full Text Available Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensin, the O-glycan chains of arabinogalactan proteins are highly heterogeneous consisting mostly of (i a short oligo-arabinoside chain of three to four residues, and (ii a larger -1,3-linked galactan backbone with -1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core 1,2-xylose, core 1,3-fucose residues and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins only extensin and arabinogalactan proteins are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review.

P-glycoprotein in autoimmune rheumatic diseases.

Science.gov (United States)

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

2015-07-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2002-08-01

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

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

International Nuclear Information System (INIS)

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

2002-01-01

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

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

International Nuclear Information System (INIS)

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

2017-01-01

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

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

Science.gov (United States)

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

2016-03-01

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

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

NARCIS (Netherlands)

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

1993-01-01

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

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

Directory of Open Access Journals (Sweden)

Alberto Lazarowski

2015-07-01

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

Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

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Anna Maisa

2009-06-01

Full Text Available Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

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

Science.gov (United States)

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

2014-01-01

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

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

Science.gov (United States)

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

2014-01-01

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

Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli.

OpenAIRE

Rose, J K; Shafferman, A

1981-01-01

Bacterial plasmids that directed expression of the vesicular stomatitis virus glycoprotein (G-protein) gene under control of the tryptophan operon regulatory region were constructed. A plasmid directing the synthesis of a G-protein-like protein (containing the NH2-terminal segment of seven amino acids encoded by the trpE gene fused to the complete G-protein sequence lacking only its NH2-terminal methionine) could be transformed into trpR+ (repressed) but not into trpR- (derepressed) cells. Th...

Glycoproteins and sialyl transferase of human B lymphoblastoid cell lines

International Nuclear Information System (INIS)

Lui, S.W.L.; Ng, M.H.

1980-01-01

We used two radiolabeling methods to study glycoproteins on the surface of lymphoblastoid cells. One of the methods affects tritiation of residues which are oxidized with galactose oxidase and the other causes tritiation of neuraminic acid residues. This approach was shown to allow a better resolution of cell surface glycoproteins than if either method were used alone. Glycoproteins of B 1 - 19 cells which harbor the Epstein-Barr virus genomes were compared with those of its parental cell line, BJAB, which does not harbor the viral genomes. These studies did not reveal a unique viral protein. A 28,000 mol. wt. glycoprotein was found to be the most prominent neuraminic acidlabeled product of B 1 - 19 cells and also of the two other cell lines, Raji and Ly38, which harbor the EBV genomes. A similar molecular weight species from BJAB cells identified by galactose oxidase labeling might be deficient in neuraminic acid residues as it was poorly labeled by the periodate oxidation method. The neuraminic acid content and level of sialyl transferase of BJAB cells were found to be lower than those of the other cell lines studied. (auth.)

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

Science.gov (United States)

Hirose, Masao

2009-04-01

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

Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

Energy Technology Data Exchange (ETDEWEB)

Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.; Langlois, A.J.; Lyerly, H.K.; Carson, H.; Krohn, K.; Ranki, A.; Gallo, R.C.; Bolognesi, D.P.; Putney, S.D.

1987-10-01

The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.

Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

International Nuclear Information System (INIS)

Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.

1987-01-01

The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120

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

Science.gov (United States)

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

2018-06-01

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

Early expression of pregnancy-specific glycoprotein 22 (PSG22) by trophoblast cells modulates angiogenesis in mice.

Science.gov (United States)

Blois, Sandra M; Tirado-González, Irene; Wu, Julie; Barrientos, Gabriela; Johnson, Briana; Warren, James; Freitag, Nancy; Klapp, Burghard F; Irmak, Ster; Ergun, Suleyman; Dveskler, Gabriela S

2012-06-01

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.

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

International Nuclear Information System (INIS)

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

1994-01-01

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

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

NARCIS (Netherlands)

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

2014-01-01

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

New baculovirus recombinants expressing Pseudorabies virus (PRV) glycoproteins protect mice against lethal challenge infection.

Science.gov (United States)

Grabowska, Agnieszka K; Lipińska, Andrea D; Rohde, Jörg; Szewczyk, Boguslaw; Bienkowska-Szewczyk, Krystyna; Rziha, Hanns-Joachim

2009-06-02

The present study demonstrates the protective potential of novel baculovirus recombinants, which express the glycoproteins gB, gC, or gD of Pseudorabies virus (PRV; Alphaherpesvirus of swine) and additionally contain the glycoprotein G of Vesicular Stomatitis Virus (VSV-G) in the virion (Bac-G-PRV). To evaluate the protective capacity, mixtures of equal amounts of the PRV gB-, gC-, and gD-expressing baculoviruses were used for immunization. Three intramuscular immunizations with that Bac-G-PRV mixture could protect mice against a lethal PRV challenge infection. To achieve complete protection high titers of Bac-G-PRV and three immunizations were necessary. This immunization with Bac-G-PRV resulted in the induction of high titers of PRV-specific serum antibodies of the IgG2a subclass and of interferon (IFN)-gamma, indicating a Th1-type immune response. Moreover, splenocytes of immunized mice exhibited natural killer cell activity accompanied by the production of IFN-alpha and IFN-gamma. Collectively, the presented data demonstrate for the first time that co-expression of VSV-G in baculovirus recombinant vaccines can improve the induction of a protective immune response against foreign antigens.

Evaluation of Zinc-alpha-2-Glycoprotein and Proteasome Subunit beta-Type 6 Expression in Prostate Cancer Using Tissue Microarray Technology.

LENUS (Irish Health Repository)

2010-07-23

Prostate cancer (CaP) is a significant cause of illness and death in males. Current detection strategies do not reliably detect the disease at an early stage and cannot distinguish aggressive versus nonaggressive CaP leading to potential overtreatment of the disease and associated morbidity. Zinc-alpha-2-glycoprotein (ZAG) and proteasome subunit beta-Type 6 (PSMB-6) were found to be up-regulated in the serum of CaP patients with higher grade tumors after 2-dimensional difference gel electrophoresis analysis. The aim of this study was to investigate if ZAG and PSMB-6 were also overexpressed in prostatic tumor tissue of CaP patients. Immunohistochemical analysis was performed on CaP tissue microarrays with samples from 199 patients. Confirmatory gene expression profiling for ZAG and PSMB-6 were performed on 4 cases using Laser Capture Microdissection and TaqMan real-time polymerase chain reaction. ZAG expression in CaP epithelial cells was inversely associated with Gleason grade (benign prostatic hyperplasia>G3>G4\\/G5). PSMB-6 was not expressed in either tumor or benign epithelium. However, strong PSMB-6 expression was noted in stromal and inflammatory cells. Our results indicate ZAG as a possible predictive marker of Gleason grade. The inverse association between grade and tissue expression with a rising serum protein level is similar to that seen with prostate-specific antigen. In addition, the results for both ZAG and PSMB-6 highlight the challenges in trying to associate the protein levels in serum with tissue expression.

California serogroup GC (G1) glycoprotein is the principal determinant of pH-dependent cell fusion and entry

International Nuclear Information System (INIS)

Plassmeyer, Matthew L.; Soldan, Samantha S.; Stachelek, Karen M.; Martin-Garcia, Julio; Gonzalez-Scarano, Francisco

2005-01-01

Members of the California serogroup of orthobunyaviruses, particularly La Crosse (LAC) and Tahyna (TAH) viruses, are significant human pathogens in areas where their mosquito vectors are endemic. Previous studies using wild-type LAC and TAH181/57, a highly neurovirulent strain with low neuroinvasiveness (Janssen, R., Gonzalez-Scarano, F., Nathanson, N., 1984. Mechanisms of bunyavirus virulence. Comparative pathogenesis of a virulent strain of La Crosse and an avirulent strain of Tahyna virus. Lab. Invest. 50 (4), 447-455), have demonstrated that the neuroinvasive phenotype maps to the M segment, the segment that encodes the two viral glycoproteins GN (G2) and GC (G1), as well as a non-structural protein NSm. To further define the role of GN and GC in fusion and entry, we prepared a panel of recombinant M segment constructs using LAC, TAH181/57, and V22F, a monoclonal-resistant variant of LAC with deficient fusion function. These M segment constructs were then tested in two surrogate assays for virus entry: a cell-to-cell fusion assay based on T7-luciferase expression, and a pseudotype transduction assay based on the incorporation of the bunyavirus glycoproteins on an MLV backbone. Both assays demonstrated that GC is the principal determinant of virus fusion and cell entry, and furthermore that the region delineated by amino acids 860-1442, corresponding to the membrane proximal two-thirds of GC, is key to these processes. These results, coupled with structural modeling suggesting homologies between the carboxy region of GC and Sindbis virus E1, suggest that the LAC GC functions as a type II fusion protein

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

Science.gov (United States)

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

2018-01-01

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

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.

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

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Ruben M Markosyan

2016-01-01

Full Text Available Ebola virus (EBOV is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.

Membrane glycoproteins of differentiating skeletal muscle cells

International Nuclear Information System (INIS)

Miller, K.R.; Remy, C.N.; Smith, P.B.

1987-01-01

The composition of N-linked glycoprotein oligosaccharides was studied in myoblasts and myotubes of the C2 muscle cell line. Oligosaccharides were radioactively labelled for 15 hr with [ 3 H] mannose and plasma membranes isolated. Ten glycopeptides were detected by SDS-PAGE and fluorography. The extent of labelling was 4-6 fold greater in myoblasts vs myotubes. A glycopeptide of Mr > 100,000 was found exclusively in myoblast membranes. Lectin chromatography revealed that the proportion of tri-, tetranntenary, biantennary and high mannose chains was similar throughout differentiation. The high mannose chain fraction was devoid of hybrid chains. The major high mannose chain contained nine mannose residues. The higher level of glycopeptide labelling in myoblasts vs myotubes corresponded to a 5-fold greater rate of protein synthesis. Pulse-chase experiments were used to follow the synthesis of the Dol-oligosaccharides. Myoblasts and myotubes labelled equivalently the glucosylated tetradecasaccharide but myoblasts labelled the smaller intermediates 3-4 greater than myotubes. Myoblasts also exhibited a 2-3 fold higher Dol-P dependent glycosyl transferase activity for chain elongation and Dol-sugar synthesis. Together these results show that the degree of protein synthesis and level of Dol-P are contributing factors in the higher capacity of myoblasts to produce N-glycoproteins compared to myotubes

Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells

International Nuclear Information System (INIS)

Ray, Ratna B.; Basu, Arnab; Steele, Robert; Beyene, Aster; McHowat, Jane; Meyer, Keith; Ghosh, Asish K.; Ray, Ranjit

2004-01-01

Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences

Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

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Yannan Qin

2014-11-01

Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

Science.gov (United States)

Marshall, Karen E; Hughson, Andrew; Vascellari, Sarah; Priola, Suzette A; Sakudo, Akikazu; Onodera, Takashi; Baron, Gerald S

2017-01-15

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP C ) influences PrP C misfolding into the disease-associated isoform, PrP res , as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP C away from rafts. Previous studies showed that nonraft transmembrane PrP C variants resist conversion to PrP res when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP C and GPI-anchored PrP res in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP C might convert to PrP res if seeded by an exogenous source of PrP res not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP C To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP C supported persistent PrP res propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP C is required for persistent PrP res propagation, implicating raft microdomains as a location for conversion. Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP C

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

DEFF Research Database (Denmark)

Saaby, Lasse; Brodin, Birger

2017-01-01

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

Morbillivirus glycoprotein expression induces ER stress, alters Ca2+ homeostasis and results in the release of vasostatin.

Directory of Open Access Journals (Sweden)

Jean-Marc Brunner

Full Text Available Although the pathology of Morbillivirus in the central nervous system (CNS is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV that we inoculated into two different cell systems: a monkey cell line (Vero and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H markedly accumulated in the endoplasmic reticulum (ER. This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT, another ER resident chaperon critically involved in the response to misfolded proteins and in Ca(2+ homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca(2+ homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses.

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells

International Nuclear Information System (INIS)

Wu, S.Y.; HungKuang Univ., Taichung; Lin, K.C.; Chiou, J.F.; Taipei Medical Univ. Hospital, Taipei; Taipei Medical Univ. Hospital, Taipei; Taipei Medical Univ. Hospital, Taipei; Jeng, S.C.; Cheng, W.H.; Chang, C.I.; Lin, W.C.; Wu, L.L.; Lee, H.L.; Chen, R.J.

2013-01-01

Background and purpose: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. Materials and methods: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. Results: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. Conclusion: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells. (orig.)

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Wu, S.Y. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Radiation-Oncology; HungKuang Univ., Taichung (China). Dept. of Biotechnology; Lin, K.C. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Oral and Maxillofacial Surgery; Chiou, J.F. [Taipei Medical Univ., Taipei (China). Dept. of Radiology; Taipei Medical Univ. Hospital, Taipei (China). Dept. of Radiation Oncology; Taipei Medical Univ. Hospital, Taipei (China). Dept. of Hospice and Palliative Center; Taipei Medical Univ. Hospital, Taipei (China). Cancer Center; Jeng, S.C. [Taipei Medical Univ. Hospital, Taipei (China). Dept. of Radiation Oncology; Cheng, W.H.; Chang, C.I. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Hemato-Ongology; Lin, W.C. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Div. of Thoracic Surgery; Wu, L.L. [National Taiwan Univ. Hospital, Taipei (China). Dept. of Ophthalmology; Lee, H.L. [Taipei Medical Univ., Wan Fang Hospital, Taipei (China). Dept. of Radiation-Oncology; Chen, R.J. [National Taiwan Univ. Hospital and National Taiwan Univ., Taipei (China). Dept. of Obstetrics and Gynecology

2013-08-15

Background and purpose: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. Materials and methods: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. Results: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. Conclusion: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells. (orig.)

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

Science.gov (United States)

Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

2015-01-02

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

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.

Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

Science.gov (United States)

Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

2010-11-01

Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2014-11-01

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

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.

Knockdown of p53 suppresses Nanog expression in embryonic stem cells

Energy Technology Data Exchange (ETDEWEB)

Abdelalim, Essam Mohamed, E-mail: emohamed@qf.org.qa [Qatar Biomedical Research Institute, Qatar Foundation, Doha 5825 (Qatar); Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

2014-01-10

Highlights: •We investigate the role of p53 in ESCs in the absence of DNA damage. •p53 knockdown suppresses ESC proliferation. •p53 knockdown downregulates Nanog expression. •p53 is essential for mouse ESC self-renewal. -- Abstract: Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.

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

Science.gov (United States)

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

2013-04-01

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

Study of the role of the covalently linked cell wall protein (Ccw14p) and yeast glycoprotein (Ygp1p) within biofilm formation in a flor yeast strain.

Science.gov (United States)

Moreno-García, J; Coi, A L; Zara, G; García-Martínez, T; Mauricio, J C; Budroni, M

2018-03-01

Flor yeasts are Saccharomyces cerevisiae strains noted by their ability to create a type of biofilm in the air-liquid interface of some wines, known as 'flor' or 'velum', for which certain proteins play an essential role. Following a proteomic study of a flor yeast strain, we deleted the CCW14 (covalently linked cell wall protein) and YGP1 (yeast glycoprotein) genes-codifying for two cell surface glycoproteins-in a haploid flor yeast strain and we reported that both influence the weight of the biofilm as well as cell adherence (CCW14).

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...

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.

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

International Nuclear Information System (INIS)

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

2001-01-01

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

Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

International Nuclear Information System (INIS)

Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

2007-01-01

The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions

Relation between expression p-glycoprotein and the findings of the centellography with Tc-99m MIBI in patients with advanced mammary cancer

International Nuclear Information System (INIS)

Delgado, L; Alonso, O; Gualco, G; Vargas, C; Ortega, V; Alonso, I; Suarez, L; Nunez, M; Roca, R; Sabini, G; Muse, IM

2004-01-01

We have previously shown that pre-treatment tumor uptake of 99m Tc-MIBI, a transport substrate of P-glycoprotein (Pgp), is correlated to clinical response to doxorubicin-based chemotherapy in advanced breast cancer (ABC) patients. The aim of this study was to investigate the possible association between Pgp expression, MIBI uptake and clinical response to chemotherapy in breast cancer lesions. Twenty-seven lesions from 26 patients with ABC were included in the study. Pre-chemotherapy Pgp expression was investigated by immunocytochemistry. MIBI scintigraphy was performed 2-8 days prior chemotherapy. Images were acquired 10 minutes (early phase) and 60 minutes (delayed phase) post injection of 740-1110 MBq of 99m Tc-MIBI. Tumor-to-normal background tissue uptake ratios were calculated in the early (T/Be) and delayed (T/Bd) phase of the study. Both ratios were significantly higher (p< 0.05) in Pgp negative (n=21) than in Pgp positive lesions (n=6). Furthermore, both ratios were higher in responder compared to non-responder lesions (T/Be 2.2 vs 1.4; T/Bd 1.8 vs 1.4; p< 0.05). All lesions with a T/Be ratio higher than 1.5 were classified as responders. No significant association was found between Pgp expression and response to chemotherapy. We concluded that MIBI scintigraphy may predict MDR1 phenotype and response to doxorubicin-based chemotherapy in ABC. Pgp expression would not be useful for predicting chemotherapy response (Au)

The amino-terminus of the hepatitis C virus (HCV p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types.

Directory of Open Access Journals (Sweden)

Solène Denolly

2017-12-01

Full Text Available Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP. HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.

Transient expression of P-type ATPases in tobacco epidermal cells

DEFF Research Database (Denmark)

Pedas, Lisbeth Rosager; Palmgren, Michael Broberg; Lopez Marques, Rosa Laura

2016-01-01

Transient expression in tobacco cells is a convenient method for several purposes such as analysis of protein-protein interactions and the subcellular localization of plant proteins. A suspension of Agrobacterium tumefaciens cells carrying the plasmid of interest is injected into the intracellula...... for example protein-protein interaction studies. In this chapter, we describe the procedure to transiently express P-type ATPases in tobacco epidermal cells, with focus on subcellular localization of the protein complexes formed by P4-ATPases and their β-subunits....

Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

International Nuclear Information System (INIS)

Kaleeba, Johnan A.R.; Berger, Edward A.

2006-01-01

The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of α3β1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Glycoprotein of the wall of sycamore tissue-culture cells.

Science.gov (United States)

Heath, M F; Northcote, D H

1971-12-01

1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the alpha-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the alpha-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.

Expression of p210 BCR/ABl increases hematopoietic progenitor cell radiosensitivity

International Nuclear Information System (INIS)

Santucci, M.A.; Anklesaria, P.; Das, I.J.; Sakakeeny, M.A.; FitzGerald, T.J.; Greenberger, J.S.; Laneuville, P.

1993-01-01

The cytogenetic finding of the Ph1+ chromosome and its molecular biologic marker bcr/abl gene rearrangement in cells from patients with chronic myeloid leukemia are associated with a proliferative advantage of the Ph1+ clone in vivo. Although the transition to the acute terminal phase or blastic crisis is often associated with additional cytogenetic abnormalities, the molecular events which correlate the initial cytogenetic lesion with the terminal phase are poorly understood. Defective cellular DNA repair capacity is often associated with chromosomal instability, increased mutation frequency, and biologic alterations. The authors tested whether the protein product of the bcr/abl translocation (p210) could alter DNA repair after gamma-irradiation of murine cell lines expressing the bcr/abl cDNA. The 32D cl 3 parent, 32D cl 3 pYN (containing the control vector plasmid) and each of two sources of 32D cl 3 cells expressing p210 cDNA (32D-PC1 cell line and 32D-LG7 subclone) showed a D 0 of 1.62, 1.57, 1.16, and 1.27 Gy, respectively. Thus, expression of the p210 product induced a significant increase in radiosensitivity at the clinically relevant radiation therapy dose-rate. The increased radiosensitivity of p210-expressing cells persisted if cells were held before plating in a density-inhibited state for 8 hr after gamma-irradiation, indicating little effect on the repair of potentially lethal gamma-irradiation damage. The IL-3 dependent parent 32D cl 3 cells demonstrated programmed cell death in the absence of growth factor or following gamma-irradiation to 200 cGy. Expression of p210 cDNA in the 32D-PC1 and 32D-LG7 subclones abrogated IL-3 requirement of these cell lines and inhibited gamma-irradiation induced programmed cell death. These data suggest a role for p210 in amplifying gamma-irradiation DNA damage or broadly inhibiting DNA repair, conditions that may stimulate further cytogenetic alterations in hematopoietic cells. 43 refs., 3 figs., 1 tab

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

Science.gov (United States)

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

2002-12-01

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

P2Y12 receptor upregulation in satellite glial cells is involved in neuropathic pain induced by HIV glycoprotein 120 and 2',3'-dideoxycytidine.

Science.gov (United States)

Yi, Zhihua; Xie, Lihui; Zhou, Congfa; Yuan, Huilong; Ouyang, Shuai; Fang, Zhi; Zhao, Shanhong; Jia, Tianyu; Zou, Lifang; Wang, Shouyu; Xue, Yun; Wu, Bing; Gao, Yun; Li, Guilin; Liu, Shuangmei; Xu, Hong; Xu, Changshui; Zhang, Chunping; Liang, Shangdong

2018-03-01

The direct neurotoxicity of HIV and neurotoxicity of combination antiretroviral therapy medications both contribute to the development of neuropathic pain. Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in mechanical and thermal hyperalgesia. The P2Y 12 receptor expressed in SGCs of the DRG is involved in pain transmission. In this study, we explored the role of the P2Y 12 receptor in neuropathic pain induced by HIV envelope glycoprotein 120 (gp120) combined with ddC (2',3'-dideoxycytidine). A rat model of gp120+ddC-induced neuropathic pain was used. Peripheral nerve exposure to HIV-gp120+ddC increased mechanical and thermal hyperalgesia in gp120+ddC-treated model rats. The gp120+ddC treatment increased expression of P2Y 12 receptor mRNA and protein in DRG SGCs. In primary cultured DRG SGCs treated with gp120+ddC, the levels of [Ca 2+ ] i activated by the P2Y 12 receptor agonist 2-(Methylthio) adenosine 5'-diphosphate trisodium salt (2-MeSADP) were significantly increased. P2Y 12 receptor shRNA treatment inhibited 2-MeSADP-induced [Ca 2+ ] i in primary cultured DRG SGCs treated with gp120+ddC. Intrathecal treatment with a shRNA against P2Y 12 receptor in DRG SGCs reduced the release of pro-inflammatory cytokines, decreased phosphorylation of p38 MAPK in the DRG of gp120+ddC-treated rats. Thus, downregulating the P2Y 12 receptor relieved mechanical and thermal hyperalgesia in gp120+ddC-treated rats.

YKL-40-Induced Inhibition of miR-590-3p Promotes Interleukin-18 Expression and Angiogenesis of Endothelial Progenitor Cells

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Te-Mao Li

2017-04-01

Full Text Available YKL-40, also known as human cartilage glycoprotein-39 or chitinase-3-like-1, is a pro-inflammatory protein that is highly expressed in rheumatoid arthritis (RA patients. Angiogenesis is a critical step in the pathogenesis of RA, promoting the infiltration of inflammatory cells into joints and providing oxygen and nutrients to RA pannus. In this study, we examined the effects of YKL-40 in the production of the pro-inflammatory cytokine interleukin-18 (IL-18, and the stimulation of angiogenesis and accumulation of osteoblasts. We observed that YKL-40 induces IL-18 production in osteoblasts and thereby stimulates angiogenesis of endothelial progenitor cells (EPCs. We found that this process occurs through the suppression of miR-590-3p via the focal adhesion kinase (FAK/PI3K/Akt signaling pathway. YKL-40 inhibition reduced angiogenesis in in vivo models of angiogenesis: the chick embryo chorioallantoic membrane (CAM and Matrigel plug models. We report that YKL-40 stimulates IL-18 expression in osteoblasts and facilitates EPC angiogenesis.

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

International Nuclear Information System (INIS)

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

2008-01-01

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

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)

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

Science.gov (United States)

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

MDR1 P-glycoprotein transports endogenous opioid peptides

NARCIS (Netherlands)

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

2001-01-01

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

Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

DEFF Research Database (Denmark)

Kristensen, Torsten; Schousboe, Inger; Boel, Espen

1991-01-01

Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

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

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Li JP

2016-11-01

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

Development of a recombinant poxvirus expressing bovine herpesvirus-1 glycoprotein D

International Nuclear Information System (INIS)

Ruiz Saenz, Julian; Osorio, Jorge E; Vera, Victor J.

2012-01-01

Bovine herpesvirus-1 is a DNA virus belonging to the family herpesviridae, which affects cattle, causing a wide spectrum of clinical manifestations and economic losses. The main immunogenic component is its envelope glycoprotein d (GD), which has been characterized and used as immunogen in different expression systems. The aim of this work was to generate a recombinant poxvirus (raccoonpox [RCN]) expressing a truncated version of BHV-1 GD to be used as a vaccine. to do this, it was amplified the gene for a truncated version of GD which subsequently was cloned in transfer plasmid PTK/IRES/TPA which has homology to sites of poxvirus thymidine kinase, an internal site of ribosome entry (IRES) and a secretory signal (TPA), generating the construct PTK/GD/IRES/TPA. to generate the recombinant RCN, we took BSC-1 cells and we infected with a wild type RCN (CDC/v71-i-85a) at a multiplicity of infection of 0.05, then cells were transfected with the construct PTK/GD/IRES/TPA, generating different viral populations with and without the gene of interest. To select recombinant viruses expressing the gene of interest, we performed a selection of recombinant thymidine kinase negative and positive for GD by three rounds of plaque purification on rat-2 cells monolayers which are thymidine kinase null and using bromodeoxyuridine. Recombinant viruses were recovered and confirmed by PCR and nucleotide sequencing and so called RCN-GD.

Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

International Nuclear Information System (INIS)

Kunz, Stefan; Edelmann, Kurt H.; Torre, Juan-Carlos de la; Gorney, Robert; Oldstone, Michael B.A.

2003-01-01

The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP

Prion propagation in cells expressing PrP glycosylation mutants.

Science.gov (United States)

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

Science.gov (United States)

Florentino, Pilar T. V.; Real, Fernando; Orikaza, Cristina M.; da Cunha, Julia P. C.; Vitorino, Francisca N. L.; Cordero, Esteban M.; Sobreira, Tiago J. P.; Mortara, Renato A.

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. PMID:29692765

Arbovirus vaccines: opportunities for the baculovirus-insect cell expression system

NARCIS (Netherlands)

Metz, S.W.H.; Pijlman, G.P.

2011-01-01

The baculovirus-insect cell expression system is a well-established technology for the production of heterologous viral (glyco)proteins in cultured cells, applicable for basic scientific research as well as for the development and production of vaccines and diagnostics. Arboviruses form an emerging

Glycoprotein component of plant cell walls

International Nuclear Information System (INIS)

Cooper, J.B.; Chen, J.A.; Varner, J.E.

1984-01-01

The primary wall surrounding most dicotyledonous plant cells contains a hydroxyproline-rich glycoprotein (HRGP) component named extensin. A small group of glycopeptides solubilized from isolated cell walls by proteolysis contained a repeated pentapeptide glycosylated by tri- and tetraarabinosides linked to hydroxyproline and, by galactose, linked to serine. Recently, two complementary approaches to this problem have provided results which greatly increase the understanding of wall extensin. In this paper the authors describe what is known about the structure of soluble extensin secreted into the walls of the carrot root cells

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

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Doublier Sophie

2012-01-01

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

Marburg Virus Glycoprotein GP2: pH-Dependent Stability of the Ectodomain α-Helical Bundle†

Science.gov (United States)

Harrison, Joseph S.; Koellhoffer, Jayne F.; Chandran, Kartik; Lai, Jonathan R.

2012-01-01

Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV is required for fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔGunf H2O, of 33.4 ± 2.5 kcal/mol and melting temperature, Tm, of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH with higher stability under lower pH conditions (Tm values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. Based on these results, we hypothesize that pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism to control conformational preferences such that the six-helix bundle ‘post-fusion’ state is preferred under conditions of appropriately matured endosomes. PMID:22369502

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.

Glycoprotein 90K Promotes E-Cadherin Degradation in a Cell Density-Dependent Manner via Dissociation of E-Cadherin–p120-Catenin Complex

Directory of Open Access Journals (Sweden)

So-Yeon Park

2017-12-01

Full Text Available Glycoprotein 90K (also known as LGALS3BP or Mac-2BP is a tumor-associated protein, and high 90K levels are associated with poor prognosis in some cancers. To clarify the role of 90K as an indicator for poor prognosis and metastasis in epithelial cancers, the present study investigated the effect of 90K on an adherens junctional protein, E-cadherin, which is frequently absent or downregulated in human epithelial cancers. Treatment of certain cancer cells with 90K significantly reduced E-cadherin levels in a cell-population-dependent manner, and these cells showed decreases in cell adhesion and increases in invasive cell motility. Mechanistically, 90K-induced E-cadherin downregulation occurred via ubiquitination-mediated proteasomal degradation. 90K interacted with the E-cadherin–p120-catenin complex and induced its dissociation, altering the phosphorylation status of p120-catenin, whereas it did not associate with β-catenin. In subconfluent cells, 90K decreased membrane-localized p120-catenin and the membrane fraction of the p120-catenin. Particularly, 90K-induced E-cadherin downregulation was diminished in p120-catenin knocked-down cells. Taken together, 90K upregulation promotes the dissociation of the E-cadherin–p120-catenin complex, leading to E-cadherin proteasomal degradation, and thereby destabilizing adherens junctions in less confluent tumor cells. Our results provide a potential mechanism to explain the poor prognosis of cancer patients with high serum 90K levels.

The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.

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Feldmann, H; Volchkov, V E; Volchkova, V A; Klenk, H D

1999-01-01

Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.

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

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

2013-03-01

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

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

NARCIS (Netherlands)

Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

1990-01-01

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

p16 expression in follicular dendritic cell sarcoma: a potential mimicker of human papillomavirus-related oropharyngeal squamous cell carcinoma.

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Zhang, Lingxin; Yang, Chen; Lewis, James S; El-Mofty, Samir K; Chernock, Rebecca D

2017-08-01

Follicular dendritic cell sarcoma is a rare mesenchymal neoplasm that most commonly occurs in cervical lymph nodes. It has histologic and clinical overlap with the much more common p16-positive human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx, which characteristically has nonkeratinizing morphology and often presents as an isolated neck mass. Not surprisingly, follicular dendritic cell sarcomas are commonly misdiagnosed as squamous cell carcinoma. Immunohistochemistry is helpful in separating the 2 entities. Follicular dendritic cell sarcoma expresses dendritic markers such as CD21 and CD23 and is almost always cytokeratin negative. However, in many cases of HPV-related oropharyngeal carcinoma, only p16 immunohistochemistry as a prognostic and surrogate marker for HPV is performed. p16 expression in follicular dendritic cell sarcoma has not been characterized. Here, we investigate the expression of p16 in follicular dendritic cell sarcoma and correlate it with retinoblastoma protein expression. A pilot study of dendritic marker expression in HPV-related oropharyngeal squamous cell carcinoma was also performed. We found that 4 of 8 sarcomas expressed p16 with strong and diffuse staining in 2 cases. In 2 of the 4 cases, p16 expression corresponded to loss of retinoblastoma protein expression. Dendritic marker expression (CD21 and CD23) was not found in HPV-related oropharyngeal squamous cell carcinomas. As such, positive p16 immunohistochemistry cannot be used as supportive evidence for the diagnosis of squamous cell carcinoma as strong and diffuse p16 expression may also occur in follicular dendritic cell sarcoma. Cytokeratins and dendritic markers are critical in separating the two tumor types. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple Drug Transport Pathways through Human P-Glycoprotein.

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

2015-07-21

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

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

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

2014-04-01

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

The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

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1995-04-13

vaccinia virus-T7 RNA polymerase s y stem for e xpression of target genes . Mol . Cell . BioI . 7 : 2538-2544 . Gagneten , S ., Gout , 0 ., Dubois-Dalcq...glycoprotein. These results showed f or the first time that two murine CEA- related genes can be co-expressed in some cell lines from inbred mice...49 Southern Hybridization ................ . ............ 50 Subcloning of PCR Products and Gene Cloning ........ 51 Growth

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

International Nuclear Information System (INIS)

Berger, W.

1993-08-01

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

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

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Pilar T. V. Florentino

2018-04-01

Full Text Available Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels, it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease.

Expression of peanut agglutinin-binding mucin-type glycoprotein in human esophageal squamous cell carcinoma as a marker

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Balakrishnan Ramathilakam

2003-11-01

Full Text Available Abstract Background The TF (Thomson – Friedenreich blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen by Peanut agglutinin (PNA and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. Results We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. Conclusion The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple

Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

International Nuclear Information System (INIS)

Filone, Claire Marie; Heise, Mark; Doms, Robert W.; Bertolotti-Ciarlet, Andrea

2006-01-01

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by β-galactosidase α-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion

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

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

1999-05-01

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

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

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

2010-04-01

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

N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

International Nuclear Information System (INIS)

Dey, Antu K.; David, Kathryn B.; Ray, Neelanjana; Ketas, Thomas J.; Klasse, Per J.; Doms, Robert W.; Moore, John P.

2008-01-01

The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B

Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins.

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Weir, Dawn L; Smith, Ina L; Bossart, Katharine N; Wang, Lin-Fa; Broder, Christopher C

2013-09-01

Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s). Published by Elsevier Inc.

Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP.

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Elkord, Eyad; Abd Al Samid, May; Chaudhary, Belal

2015-08-21

Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are expressed on CD4+FoxP3- T cells expressing Helios (FoxP3-Helios+) in the steady state. Following TCR activation, GARP/LAP are up-regulated on CD4+Helios+ T cells regardless of FoxP3 expression (FoxP3+/-Helios+). We show that CD4+GARP+/-LAP+ Tregs make IL-10 immunosuppressive cytokine but not IFN-γ effector cytokine. Further characterization of FoxP3/Helios subpopulations showed that FoxP3+Helios+ Tregs proliferate in vitro significantly less than FoxP3+Helios- Tregs upon TCR stimulation. Unlike FoxP3+Helios- Tregs, FoxP3+Helios+ Tregs secrete IL-10 but not IFN-γ or IL-2, confirming they are bona fide Tregs with immunosuppressive characteristics. Taken together, Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP, and FoxP3+Helios+ Tregs have more suppressive characteristics, compared with FoxP3+Helios- Tregs. Our work implies that therapeutic modalities for treating autoimmune and inflammatory diseases, allergies and graft rejection should be designed to induce and/or expand FoxP3+Helios+ Tregs, while therapies against cancers or infectious diseases should avoid such expansion/induction.

Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological, Immunohistochemical and Gene Expression Profiles

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Marta Anna Szychlinska

2016-03-01

Full Text Available Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1 are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA, whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren–Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01. By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01. Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease.

Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo

International Nuclear Information System (INIS)

Perryman, L.A.; Blair, J.M.; Kingsley, E.A.; Szymanska, B.; Ow, K.T.; Wen, V.W.; MacKenzie, K.L.; Vermeulen, P.B.; Jackson, P.; Russell, P.J.

2006-01-01

This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous 'take rate' in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation

Chemotherapy and Stem Cell Transplantation Increase p16INK4a Expression, a Biomarker of T-cell Aging

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William A. Wood

2016-09-01

Full Text Available The expression of markers of cellular senescence increases exponentially in multiple tissues with aging. Age-related physiological changes may contribute to adverse outcomes in cancer survivors. To investigate the impact of high dose chemotherapy and stem cell transplantation on senescence markers in vivo, we collected blood and clinical data from a cohort of 63 patients undergoing hematopoietic cell transplantation. The expression of p16INK4a, a well-established senescence marker, was determined in T-cells before and 6 months after transplant. RNA sequencing was performed on paired samples from 8 patients pre- and post-cancer therapy. In patients undergoing allogeneic transplant, higher pre-transplant p16INK4a expression was associated with a greater number of prior cycles of chemotherapy received (p = 0.003, prior autologous transplantation (p = 0.01 and prior exposure to alkylating agents (p = 0.01. Transplantation was associated with a marked increase in p16INK4a expression 6 months following transplantation. Patients receiving autologous transplant experienced a larger increase in p16INK4a expression (3.1-fold increase, p = 0.002 than allogeneic transplant recipients (1.9-fold increase, p = 0.0004. RNA sequencing of T-cells pre- and post- autologous transplant or cytotoxic chemotherapy demonstrated increased expression of transcripts associated with cellular senescence and physiological aging. Cytotoxic chemotherapy, especially alkylating agents, and stem cell transplantation strongly accelerate expression of a biomarker of molecular aging in T-cells.

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

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

2007-05-01

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

Histochemical and structural analysis of mucous glycoprotein secreted by the gill of Mytilus edulis

International Nuclear Information System (INIS)

Ahn, Hae-Young.

1988-01-01

Studies were carried out to characterized various mucous cells in the gill filament, to ascertain structural characteristics of the secreted mucous glycoproteins, and to determine the ability of the gill epithelium to incorporate [ 14 C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins. Using histochemical staining techniques, mucous cells containing neutral and acidic mucins were found in the lateral region, whereas mucous cells containing primarily neutral or sulfated mucins were found in the postlateral region. Serotonin, but not dopamine, stimulated the mucous secretion. In tissues pretreated with [ 14 C]glucosamine, the secreted glycoproteins contain incorporated radiolabel. Analysis by column chromatography using Bio-Gel P-2 and P-6 shows that the secretion contains two glycoprotein populations. Glycoprotein II has a molecular weight of 2.3 x 10 4 daltons. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of glycoprotein I, about 70% of the radiolabel was removed from the protein. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contains N-acetylglucosamine (GluNAc), N-acetylgalactosamine (GalNAc), and galactose, fucose and mannose. Amino acid analysis shows that the glycoproteins are rich in serine, threonine and proline

Cancer cell–derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo

Science.gov (United States)

Thomas, Grace M.; Panicot-Dubois, Laurence; Lacroix, Romaric; Dignat-George, Françoise; Lombardo, Dominique

2009-01-01

Recent publications have demonstrated the presence of tissue factor (TF)–bearing microparticles (MPs) in the blood of patients suffering from cancer. However, whether these MPs are involved in thrombosis remains unknown. We show that pancreatic and lung cancer cells produce MPs that express active TF and P-selectin glycoprotein ligand 1 (PSGL-1). Cancer cell–derived MPs aggregate platelets via a TF-dependent pathway. In vivo, cancer cell–derived MPs, but not their parent cells, infused into a living mouse accumulate at the site of injury and reduce tail bleeding time and the time to occlusion of venules and arterioles. This thrombotic state is also observed in mice developing tumors. In such mice, the amount of circulating platelet-, endothelial cell–, and cancer cell–derived MPs is increased. Endogenous cancer cell–derived MPs shed from the growing tumor are able to accumulate at the site of injury. Infusion of a blocking P-selectin antibody abolishes the thrombotic state observed after injection of MPs or in mice developing a tumor. Collectively, our results indicate that cancer cell–derived MPs bearing PSGL-1 and TF play a key role in thrombus formation in vivo. Targeting these MPs could be of clinical interest in the prevention of thrombosis and to limit formation of metastasis in cancer patients. PMID:19667060

p53 expression in biopsies from children with Langerhans cell histiocytosis

DEFF Research Database (Denmark)

Bank, Micha I; Lundegaard, Pia Rengtved; Carstensen, Henrik

2002-01-01

based on CD1a positivity. The slides were stained with p53 antibody and semiquantitatively evaluated using a grading system from 1 to 5 as an estimate for 0% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, and 80% to 100% p53-positive for pathologic Langerhans cells (pLC), respectively. RESULTS: The p53...... protein was expressed in various degrees in pLC in all lesions. The degree of p53 expression could not be correlated to either clinical manifestation or outcome. CONCLUSIONS: An increased expression of p53 in pLC indicates an altered DNA repair control with or without abnormal control of apoptosis....

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

Science.gov (United States)

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection

International Nuclear Information System (INIS)

Barretto, Naina; Hallak, Louay K.; Peeples, Mark E.

2003-01-01

Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly, if at all. We found that neuraminidase treatment of RSV-infected cells to remove sialic acid (SA) increases fusion dramatically and that the same treatment of transiently transfected cells expressing the three viral glycoproteins, or even cells expressing the fusion (F) protein alone, results in easily detectable fusion. Neuraminidase treatment of the effector cells, expressing the viral glycoproteins, enhanced fusion while treatment of the target cells did not. Likewise, infectivity was increased by treating virions with neuraminidase, but not by treating target cells. Reduction of charge repulsion by removal of the negatively charged SA is unlikely to explain this effect, since removal of negative charges from either membrane would reduce charge repulsion. Infection with neuraminidase-treated virus remained heparan-sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal. Interestingly, neuraminidase enhancement of RSV infectivity was less pronounced in a virus expressing both the G and the F glycoproteins, compared to virus expressing only the F glycoprotein, possibly suggesting that the G protein sterically hinders access of the neuraminidase to its fusion-enhancing target

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

International Nuclear Information System (INIS)

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

1998-01-01

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

Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth

International Nuclear Information System (INIS)

Quackenbush, E.; Clabby, M.; Gottesdiener, K.M.; Barbosa, J.; Jones, N.H.; Strominger, J.L.; Speck, S.; Leiden, J.M.

1987-01-01

Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a λgt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggest that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution

Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells

NARCIS (Netherlands)

Westra, DF; Glazenburg, KL; Harmsen, MC; Tiran, A; Scheffer, AJ; Welling, GW; The, TH; WellingWester, S

In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell

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

Science.gov (United States)

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

2016-02-19

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

Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression

Energy Technology Data Exchange (ETDEWEB)

Liu, Ming; Wang, Dan, E-mail: danwangwdd@163.com; Li, Ning

2016-04-22

The transcriptional cofactor Che-1 is an RNA polymerase II (Pol II) which is involved in tumorigenesis, such as breast cancer and multiple myeloma. Che-1 can also regulate mutant p53 expression, which plays roles in many types of cancer. In this study, we aimed to investigate the effects and specific mechanism of Che-1 in the regulation of osteosarcoma (OS) cell growth. We found that Che-1 is highly expressed in several kinds of OS cells compared with osteoblast hFOB1.19 cells. MTT and flow cytometry assays showed that Che-1 depletion by siRNA markedly suppressed MG-63 and U2OS cell proliferation and promoted apoptosis. The chromatin immunoprecipitation (ChIP) assay verified the presence of Che-1 on the p53 promoter in MG-63 and U2OS cells carrying mutant p53. Further studies showed that Che-1 depletion inhibited mutant p53 expression. Notably, our study showed that the loss of Che-1 inhibits proliferation and promotes apoptosis in MG-63 cells by decreasing the level of mutant p53. Therefore, these findings open the possibility that silencing of Che-1 will have therapeutic benefit in OS. - Highlights: • Che-1 is highly expressed in several kinds of OS cells. • Che-1 depletion suppressed MG-63 and U2OS cell growth. • Che-1 is existed in the p53 promoter in MG-63 and U2OS cells. • Che-1 depletion inhibited mutant p53 expression. • Che-1 depletion inhibits cell growth by decreasing the level of mutant p53.

Molecular insight into conformational transmission of human P-glycoprotein

International Nuclear Information System (INIS)

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

2013-01-01

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

Differential gene expression profile reveals deregulation of pregnancy specific β1 glycoprotein 9 early during colorectal carcinogenesis

Directory of Open Access Journals (Sweden)

Gallinger Steven

2005-06-01

Full Text Available Abstract Background APC (Adenomatous polyposis coli plays an important role in the pathogenesis of both familial and sporadic colorectal cancer. Patients carrying germline APC mutations develop multiple colonic adenomas at younger age and higher frequency than non-carrier cases which indicates that silencing of one APC allele may be sufficient to initiate the transformation process. Methods To elucidate the biological dysregulation underlying adenoma formation we examined global gene expression profiles of adenomas and corresponding normal mucosa from an FAP patient. Differential expression of the most significant gene identified in this study was further validated by mRNA in situ hybridization, reverse transcriptase PCR and Northern blotting in different sets of adenomas, tumours and cancer cell lines. Results Eighty four genes were differentially expressed between all adenomas and corresponding normal mucosa, while only seven genes showed differential expression within the adenomas. The first group included pregnancy specific β-1 glycoprotein 9 (PSG9 (p PSG9 is a member of the carcinoembryonic antigen (CEA/PSG family and is produced at high levels during pregnancy, mainly by syncytiotrophoblasts. Further analysis of sporadic and familial colorectal cancer confirmed that PSG9 is ectopically upregulated in vivo by cancer cells. In total, deregulation of PSG9 mRNA was detected in 78% (14/18 of FAP adenomas and 75% (45/60 of sporadic colorectal cancer cases tested. Conclusion Detection of PSG9 expression in adenomas, and at higher levels in FAP cases, indicates that germline APC mutations and defects in Wnt signalling modulate PSG9 expression. Since PSG9 is not found in the non-pregnant adult except in association with cancer, and it appears to be an early molecular event associated with colorectal cancer monitoring of its expression may be useful as a biomarker for the early detection of this disease.

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

DEFF Research Database (Denmark)

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

2007-01-01

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

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

International Nuclear Information System (INIS)

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

1996-01-01

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

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

OpenAIRE

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

2013-01-01

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

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

Science.gov (United States)

Efferth, Thomas; Volm, Manfred

2017-07-01

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

Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

NARCIS (Netherlands)

Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

The expression of GST isoenzymes and p53 in non-small cell lung cancer.

Directory of Open Access Journals (Sweden)

MĂźzeyyen Ozhavzali

2010-06-01

Full Text Available This study investigated the immunohistochemical staining characteristics of glutathione-S-transferase alpha, pi, mu, theta and p53 in non-small cell lung carcinoma and normal lung tissue from 50 patients. The relationships between expressions of the Glutathione-S-transferase isoenzymes and some clinicopathological features were also examined. Expression of glutathione-S-transferase pi, mu, alpha, theta and p53 was assessed by immunohistochemistry for primary lung carcinomas of 50 patients from the Sanitarium Education and Research Hospital, Ankara lung cancer collection. The relationships between expression of the glutathione-S-transferase isoenzymes, p53 in normal and tumor tissue by Student T test and the clinicopathological data were also examined by Spearman Rank tests. When the normal and tumor tissue of these cases were compared according to their staining intensity and percentage of positive staining, glutathione-S-transferase alpha, pi, mu, theta expressions in tumor cells was significantly higher than normal cells (p<0.05. There was no significant difference in the expression of p53 between normal and tumor cells (p>0.05. When the immunohistochemical results of glutathione-S-transferase isoenzymes and p53 were correlated with the clinical parameters, there were no significant associations between glutathione-S-transferases and p53 expressions and tumor stage, tumor grade and smoking status (p>0.05.

Low pH induces co-ordinate regulation of gene expression in oesophageal cells.

Science.gov (United States)

Duggan, Shane P; Gallagher, William M; Fox, Edward J P; Abdel-Latif, Mohammed M; Reynolds, John V; Kelleher, Dermot

2006-02-01

The development of gastro-oesophageal reflux disease (GORD) is known to be a causative risk factor in the evolution of adenocarcinoma of the oesophagus. The major component of this reflux is gastric acid. However, the impact of low pH on gene expression has not been extensively studied in oesophageal cells. This study utilizes a transcriptomic and bioinformatic approach to assess regulation of gene expression in response to low pH. In more detail, oesophageal adenocarcinoma cell lines were exposed to a range of pH environments. Affymetrix microarrays were used for gene-expression analysis and results were validated using cycle limitation and real-time RT-PCR analysis, as well as northern and western blotting. Comparative promoter transcription factor binding site (TFBS) analysis (MatInspector) of hierarchically clustered gene-expression data was employed to identify the elements which may co-ordinately regulate individual gene clusters. Initial experiments demonstrated maximal induction of EGR1 gene expression at pH 6.5. Subsequent array experimentation revealed significant induction of gene expression from such functional categories as DNA damage response (EGR1-4, ATF3) and cell-cycle control (GADD34, GADD45, p57). Changes in expression of EGR1, EGR3, ATF3, MKP-1, FOSB, CTGF and CYR61 were verified in separate experiments and in a variety of oesophageal cell lines. TFBS analysis of promoters identified transcription factors that may co-ordinately regulate gene-expression clusters, Cluster 1: Oct-1, AP4R; Cluster 2: NF-kB, EGRF; Cluster 3: IKRS, AP-1F. Low pH has the ability to induce genes and pathways which can provide an environment suitable for the progression of malignancy. Further functional analysis of the genes and clusters identified in this low pH study is likely to lead to new insights into the pathogenesis and therapeutics of GORD and oesophageal cancer.

Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport

Directory of Open Access Journals (Sweden)

Szilvia Veszelka

2018-05-01

Full Text Available Cell culture-based blood-brain barrier (BBB models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC, ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA. As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L, and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1 and influx transporters (GLUT-1, LAT-1 were present in all models at mRNA levels. The transcript of BCRP (ABCG2 was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which

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

OpenAIRE

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

2013-01-01

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

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.

(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

Directory of Open Access Journals (Sweden)

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.

(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

International Nuclear Information System (INIS)

Syvänen, Stina; Luurtsema, Gert; Molthoff, Carla FM; Windhorst, Albert D; Huisman, Marc C; Lammertsma, Adriaan A; Voskuyl, Rob A; Lange, Elizabeth C de

2011-01-01

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. (R)-[ 11 C]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)-[ 11 C]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)-[ 11 C]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). All data analysis approaches indicated only modest differences in brain distribution of (R)-[ 11 C]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. P-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate

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

Science.gov (United States)

Abdelfatah, Sara A A; Efferth, Thomas

2015-02-15

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

Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells

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Malgorzata Kloc

2012-10-01

Full Text Available The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
a key regulator of cell cycle, controls actin organization and negatively regulates cell motility via regulation of RhoA
expression. We studied the organization of actin and cytokeratin cytoskeleton and the expression of TCTP, p53,
cyclin A, RhoA and actin in HIO180 non-transformed ovarian epithelial cells, and OVCAR3 and SKOV3 (expressing
low level of inducible p53 ovarian epithelial cancer cells with different metastatic potential. Immunostaining
and ultrastructural analyses illustrated a dramatic difference in the organization of the cytokeratin and actin
filaments in non-transformed versus cancer cell lines. We also determined that there is an inverse relationship between
the level of TCTP/RhoA and actin/p53/cyclin A expression in ovarian cancer cell lines. This previously unidentified
negative relationship between TCTP/RhoA and actin/p53/cyclin A may suggest that this interaction is linked
with the high aggressiveness of ovarian cancers.The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
a key regulator of cell cycle, controls actin organization

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells

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Flora Cristina Lobo Penteado

2006-04-01

Full Text Available O retrovírus linfotrópico de células T humanas tipo 1 é o agente etiológico da leucemia das células T do adulto e da paraparesia espástica tropical/mielopatia associada ao HTLV-1. O genoma proviral é composto por 9.032 pares de bases, contendo genes estruturais e regulatórios. A glicoproteína transmembrana gp 21 é codificada pelo gene estrutural env. O desenvolvimento de metodologias para a expressão heteróloga de proteínas, assim como a obtenção de uma linhagem celular que expresse a gp 21 recombinante constitutivamente são os principais objetivos do trabalho. O fragmento codificante da gp 21 foi amplificado por Nested-PCR e clonado no vetor pCR2.1-TOPO. Posteriormente, foi realizada a subclonagem no vetor de expressão pcDNA3.1+. A transfecção da linhagem celular de mamíferos HEK 293 foi realizada de maneira transitória e permanente. A produção da gp 21 recombinante foi confirmada por citometria de fluxo e a linhagem celular produtora será utilizada em ensaios de imunogenicidade.The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent

International Nuclear Information System (INIS)

Bienz, D.; Clemetson, K.J.

1989-01-01

Glycoprotein Ia (GP Ia) is a relatively minor component of human blood platelets thought to be a receptor involved in collagen-induced platelet activation. However, some difficulties exist with the definition of this glycoprotein. The expression of GP Ia on resting (prostacyclin analogue-treated) and thrombin-activated platelets was compared by surface labeling with 125 I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/[ 3 H]NaBH 4 . Analysis on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that GP Ia is very poorly labeled in resting platelets. After activation a new spot (GP Ia*) appears with the same relative molecular mass as GP Ia under reducing conditions. GP Ia and Ia* can be clearly separated by two-dimensional nonreduced/reduced gel electrophoresis. Therefore, two glycoproteins which have been termed GP Ia exist in platelets with similar molecular weight and pI under reducing conditions. One of these (GP Ia*) is only surface-labeled when platelets are activated, indicating that it is only exposed on the surface of activated platelets. Supernatant from activated platelets contains this glycoprotein as well as other granule components. This glycoprotein is missing in platelets from two patients with collagen-response defects

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

DEFF Research Database (Denmark)

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A

2012-01-01

oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted......The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high...... YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3ß, PDX1, CD34, p63, nestin, PAX6) markers. Double...

The c-Myc target glycoprotein1balpha links cytokinesis failure to oncogenic signal transduction pathways in cultured human cells.

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Qian Wu

2010-05-01

Full Text Available An increase in chromosome number, or polyploidization, is associated with a variety of biological changes including breeding of cereal crops and flowers, terminal differentiation of specialized cells such as megakaryocytes, cellular stress and oncogenic transformation. Yet it remains unclear how cells tolerate the major changes in gene expression, chromatin organization and chromosome segregation that invariably accompany polyploidization. We show here that cancer cells can initiate increases in chromosome number by inhibiting cell division through activation of glycoprotein1b alpha (GpIbalpha, a component of the c-Myc signaling pathway. We are able to recapitulate cytokinesis failure in primary cells by overexpression of GpIbalpha in a p53-deficient background. GpIbalpha was found to localize to the cleavage furrow by microscopy analysis and, when overexpressed, to interfere with assembly of the cellular cortical contraction apparatus and normal division. These results indicate that cytokinesis failure and tetraploidy in cancer cells are directly linked to cellular hyperproliferation via c-Myc induced overexpression of GpIbalpha.

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

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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

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

Science.gov (United States)

Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

2002-09-15

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

EMA: a developmentally regulated cell-surface glycoprotein of CNS neurons that is concentrated at the leading edge of growth cones.

Science.gov (United States)

Baumrind, N L; Parkinson, D; Wayne, D B; Heuser, J E; Pearlman, A L

1992-08-01

To identify cell-surface molecules that mediate interactions between neurons and their environment during neural development, we used monoclonal antibody techniques to define a developmentally regulated antigen in the central nervous system of the mouse. The antibody we produced (2A1) immunolabels cells throughout the central nervous system; we analyzed its distribution in the developing cerebral cortex, where it is expressed on cells very soon after they complete mitosis and leave the periventricular proliferative zone. Expression continues into adult life. The antibody also labels the epithelium of the choroid plexus and the renal proximal tubules, but does not label neurons of the peripheral nervous system in the dorsal root ganglia. In dissociated cell culture of embryonic cerebral cortex, 2A1 labels the surface of neurons but not glia. Immunolabeling of neurons in tissue culture is particularly prominent on the edge of growth cones, including filopodia and the leading edge of lamellipodia, when observed with either immunofluorescence or freeze-etch immunoelectron microscopy. Immunopurification with 2A1 of a CHAPS-extracted membrane preparation from brains of neonatal mice produces a broad (32-36 kD) electrophoretic band and a less prominent 70 kD band that are sensitive to N-glycosidase but not endoglycosidase H. Thus the 2A1 antibody recognizes a developmentally regulated, neuronal cell surface glycoprotein (or glycoproteins) with complex N-linked oligosaccharide side chains. We have termed the glycoprotein antigen EMA because of its prominence on the edge membrane of growth cones. EMA is similar to the M6 antigen (Lagenaur et al: J. Neurobiol. 23:71-88, 1992) in apparent molecular weight, distribution in tissue sections, and immunoreactivity on Western blots, suggesting that the two antigens are similar or identical. Expression of EMA is a very early manifestation of neuronal differentiation; its distribution on growth cones suggests a role in mediating the

Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

Science.gov (United States)

Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

2016-06-01

Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

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Goldbach Rob W

2011-07-01

Full Text Available Abstract Background Chikungunya virus (CHIKV is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

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

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

Science.gov (United States)

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

2014-03-10

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

Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

International Nuclear Information System (INIS)

Manzotti, G; Mariani, S A; Corradini, F; Bussolari, R; Cesi, V; Vergalli, J; Ferrari-Amorotti, G; Fragliasso, V; Soliera, A R; Cattelani, S; Raschellà, G; Holyoake, T L; Calabretta, B

2012-01-01

The c-Myb gene encodes the p75 c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p c-Mybex9b , which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of p c-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p c-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75 c-Myb , p c-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p c-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p c-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34 + cells, without affecting the levels of p75 c-Myb . Together, these studies indicate that expression of the low-abundance p c-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

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Gale Smith

Full Text Available Respiratory Syncytial Virus (RSV is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

Glycan structures contain information for the spatial arrangement of glycoproteins in the plasma membrane.

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M Kristen Hall

Full Text Available Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells.

Up-Regulation of the Lymphatic Marker Podoplanin, a Mucin-Type Transmembrane Glycoprotein, in Human Squamous Cell Carcinomas and Germ Cell Tumors

Science.gov (United States)

Schacht, Vivien; Dadras, Soheil S.; Johnson, Louise A.; Jackson, David G.; Hong, Young-Kwon; Detmar, Michael

2005-01-01

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers. PMID:15743802

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

Science.gov (United States)

Saaby, Lasse; Brodin, Birger

2017-09-01

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

P-cadherin expression and survival rate in oral squamous cell carcinoma:an immunohistochemical study

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Laino Gregorio

2005-06-01

Full Text Available Abstract Background P-cadherin (P-cad is a transmembrane molecule involved in the cell-cell adhesion and similar to E-cadherin (E-cad, but less investigated in oncology, especially in in vivo studies. Aims of the present study were to assess the prevalence of P-cad expression in oral squamous cell carcinoma (OSCC and to verify whether P-cad can be considered a marker of prognosis in patients with OSCC. Methods In a retrospective study, a cohort of 67 OSCC patients was investigated for P-cad expression and its cellular localization by immunohistochemistry; some respective healthy margins of resection were similarly investigated as standard controls. After grouping for P-cad expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G, TNM, Staging, and overall survival rate. Univariate and multivariate analyses were performed. Results 37 cases (55.2% of OSCC showed membranous/cytoplasmic positivity for P-cad, whereas 30 (44.8 % were negative. Although with some differences in membranous vs cytoplasmic localization of P-cad in OSCC with different G, no statistical association was found between P-cad expression and any variables considered at baseline. In terms of prognostic significance, P-cad non expression was found to have an independent association with poorer overall survival rate than P-cad expressing group (P = 0.056; moreover, among P-cad +ve patients the best prognosis was for those OSCC with membranous (P Conclusion On the basis of these results, it is possible to suggest P-cad as an early marker of poor prognosis. The abnormal or lack of P-cad expression could constitute an hallmark of aggressive biological behavior in OSCC

Expressions of topoisomerase IIα and BCRP in metastatic cells are associated with overall survival in small cell lung cancer patients.

Science.gov (United States)

Rijavec, Matija; Silar, Mira; Triller, Nadja; Kern, Izidor; Cegovnik, Urška; Košnik, Mitja; Korošec, Peter

2011-09-01

The aim of this study was to investigate the mRNA expression levels of multidrug resistance-associated proteins in chemo-naïve metastatic lung cancer cells and to determine the correlation with response to chemotherapy and overall survival. Metastatic cells were obtained by transbronchial fine needle aspiration biopsy of enlarged mediastinal lymph nodes in 14 patients with small cell lung cancer (SCLC) and 7 patients with non-small cell lung cancer (NSCLC). After cytological confirmation of lung cancer type, total RNA was extracted from biopsy samples and reverse transcribed to cDNA, and real-time PCR for the genes of interest [P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP), lung resistance protein (LRP) and topoisomerase IIα (TOPIIα)], was performed. We observed significantly decreased expression of BCRP and significantly increased expression of TOPIIα in metastatic SCLC cells compared to NSCLC. Furthermore, in SCLC high topoisomerase IIα and low BCRP expression levels positively correlated with longer overall survival. Our results showed higher expression levels of BCRP as well as lower levels of topoisomerase IIα in chemo-naïve metastatic cells in NSCLC than in SCLC. These results correlate with previous observations that metastatic SCLC cells at the beginning of chemotherapy are potentially more sensitive to chemotherapeutic agents while in metastatic NSCLC cells resistance is usually inherent. We also showed that altered levels of topoisomerase IIα and BCRP in SCLC are important factors that contribute to resistance to chemotherapeutics that interfere with the enzyme and/or DNA and are highly associated with overall survival.

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

NARCIS (Netherlands)

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

2010-01-01

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

Transfected HEK293 Cells Expressing Functional Recombinant Intercellular Adhesion Molecule 1 (ICAM-1) - A Receptor Associated with Severe Plasmodium falciparum Malaria

DEFF Research Database (Denmark)

Bengtsson, Anja; Joergensen, Louise; Barbati, Zachary R

2013-01-01

Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes....... Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has...

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

Science.gov (United States)

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

2002-02-01

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

(R)-[{sup 11}C]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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Syvänen, Stina [Division of Pharmacology, LACDR, Leiden University, P.O. Box 9502, 2300 RA Leiden (Netherlands); Luurtsema, Gert [Department of Nuclear Medicine & Molecular Imaging, Groningen University Medical Center, P.O. Box 30.001 9700 RB Groningen (Netherlands); Molthoff, Carla FM; Windhorst, Albert D; Huisman, Marc C; Lammertsma, Adriaan A [Department of Nuclear Medicine & PET Research, VU University Medical Center, P.O. Box 7057, 1007 MB, Amsterdam (Netherlands); Voskuyl, Rob A [Division of Pharmacology, LACDR, Leiden University, P.O. Box 9502, 2300 RA Leiden (Netherlands); Epilepsy Institute of The Netherlands Foundation (SEIN), P.O. Box 21, 2100 AA, Heemstede (Netherlands); Lange, Elizabeth C de [Division of Pharmacology, LACDR, Leiden University, P.O. Box 9502, 2300 RA Leiden (Netherlands)

2011-01-03

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. (R)-[{sup 11}C]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)-[{sup 11}C]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)-[{sup 11}C]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). All data analysis approaches indicated only modest differences in brain distribution of (R)-[{sup 11}C]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. P-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate.

miR-208-3p promotes hepatocellular carcinoma cell proliferation and invasion through regulating ARID2 expression

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Yu, Peng; Wu, Dingguo; You, Yu; Sun, Jing; Lu, Lele; Tan, Jiaxing; Bie, Ping, E-mail: bieping2010@163.com

2015-08-15

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at post-transcriptional level. miRNA dysregulation plays a causal role in cancer progression. In this study, miR-208-3p was highly expressed and directly repressed ARID2 expression. As a result, ARID2 expression in hepatocellular carcinoma (HCC) was decreased. In vitro, miR-208-3p down-regulation and ARID2 over-expression elicited similar inhibitory effects on HCC cell proliferation and invasion. In vivo test results revealed that miR-208-3p down-regulation inhibited HCC tumorigenesis in Hep3B cells. Moreover, ARID2 was possibly a downstream element of transforming growth factor beta1 (TGFβ1)/miR-208-3p/ARID2 regulatory pathway. These findings suggested that miR-208-3p up-regulation is associated with HCC cell progression and may provide a new target for liver cancer treatment. - Highlights: • miR-208-3p was highly expressed and directly repressed the expression of ARID2 in HCC. • miR-208-3p contributed to HCC cell progression both in vitro and in vivo. • Over-expression of ARID2 inhibited the HCC cell proliferation and invasion. • Restoration of ARID2 partly reversed the the effect of miR-208-3p down-regulation on HCC cells. • Newly regulatory pathway: miR-208-3p mediated the repression of ARID2 by TGFβ1 in HCC cells.

p38 MAP kinase is required for Wnt3a-mediated osterix expression independently of Wnt-LRP5/6-GSK3β signaling axis in dental follicle cells

International Nuclear Information System (INIS)

Sakisaka, Yukihiko; Kanaya, Sousuke; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

2016-01-01

Wnt3a is a secreted glycoprotein that activates the glycogen synthase kinase-3β (GSK3β)/β-catenin signaling pathway through low-density-lipoprotein receptor-related protein (LRP)5/6 co-receptors. Wnt3a has been implicated in periodontal development and homeostasis, as well as in cementum formation. Recently, we have reported that Wnt3a increases alkaline phosphatase expression through the induction of osterix (Osx) expression in dental follicle cells, a precursor of cementoblasts. However, the molecular mechanism by which Wnt3a induces Osx expression is still unknown. In this study, we show that Wnt3a-induced Osx expression was inhibited in the presence of p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203580 and SB202190) at gene and protein levels, as assessed by real-time PCR and immunocytohistochemistry, respectively. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist binding to LRP5/6 co-receptors, did not influence Wnt3a-mediated p38 MAPK phosphorylation, suggesting that Wnt3a activates p38 MAPK through LRP5/6-independent signaling. On the other hand, pretreatment with p38 MAPK inhibitors had no effects on the phosphorylated status of GSK3β and β-catenin as well as β-catenin nuclear translocation, but inhibited Wnt3a-mediated β-catenin transcriptional activity. These findings suggest that p38 MAPK modulates canonical Wnt signaling at the β-catenin transcriptional level without any crosstalk with the Wnt3a-mediated LRP5/6-GSK3β signaling axis and subsequent β-catenin nuclear translocation. These findings expand our knowledge of the mechanisms controlling periodontal development and regeneration. - Highlights: • Wnt3a induces Osx expression via p38 MAPK signaling in dental follicle cells. • p38 MAPK has no crosstalk with Wnt3a-mediated LRP5/6 and GSK3β signaling. • p38 MAPK is required for Wnt signaling at the β-catenin transcriptional level.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Directory of Open Access Journals (Sweden)

A.R.M. Sabbatini

2011-02-01

Full Text Available Histidine-rich glycoprotein (HRG is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD. We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Two-dimensional analysis of metabolically and cell surface radiolabeled proteins of some human lymphoid and myeloid leukemia cell lines. II. Glycosylated and phosphorylated proteins

Energy Technology Data Exchange (ETDEWEB)

Chorvath, B; Duraj, J; Sedlak, J; Pleskova, I

1986-01-01

Cell surface glycoproteins, radiolabelled by the sodium metaperiodate/tritiated borohydride technique, and cell phosphoproteins, metabolically radiolabelled with /sup 32/P-orthophosphate were analyzed by two-dimensional electrophoretic analysis in some myeloid and lymphoid leukemia cell lines. Some markedly expressed major glycoproteins were predominant in some of the cell lines (such as 95k and 100k glycoproteins with marked charge heterogeneity in non-T, non-B acute lymphoblastic leukemia cell lines NALM 6 and NALM 16), but markedly quantitatively reduced in other examined cell lines, such as lymphoblastoid cell line UHKT 34/2. /sup 32/P-orthophosphate radiolabelled phosphoprotein two-dimensional patterns of the examined lymphoid leukemia cell lines were essentially similar, with some minor differences, in examined lymphoid and myeloid leukemia cell lines, such as marked expression of a series of large phosphoproteins in the molecular weight range 80-100k in lymphoid cell lines and almost complete absence of these phosphoproteins on the examined myeloid leukemia cell lines. Another configuration of acidic phosphoproteins (30-35k) exhibited individual cell line variability and differences between both individual myeloid leukemia cell lines and between the lymphoid and myeloid cell lines examined. (author) 2 figs., 15 refs.

Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

Science.gov (United States)

Rasheed, Muhammad Asif; Ansari, Abdur Rahman; Ihsan, Awais; Navid, Muhammad Tariq; Ur-Rehman, Shahid; Raza, Sohail

2018-03-01

Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.

Science.gov (United States)

Weir, Dawn L; Laing, Eric D; Smith, Ina L; Wang, Lin-Fa; Broder, Christopher C

2014-02-27

Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

MicroRNAs control transcription factor NF-kB (p65) expression in human ovarian cells.

Science.gov (United States)

Sirotkin, Alexander V; Alexa, Richard; Kišová, Gabriela; Harrath, Abdel Halim; Alwasel, Saleh; Ovcharenko, Dmitriy; Mlynček, Miloš

2015-05-01

MicroRNAs (miRNAs) are known to influence ovarian cell proliferation, apoptosis and hormone release, but it remains unknown whether miRNAs affect ovarian functions via transcription factors. We examined the effect of miRNAs on nuclear factor-κappaB (NF-kB) (p65) expression in human ovarian luteinized granulosa cells. We transfected cultured primary human ovarian luteinized granulosa cells with 80 different constructs encoding human pre-miRNAs and then evaluated NF-kB (p65) expression (percentage of cells containing p65) by immunocytochemistry. We found that 21 of the constructs stimulated NF-kB (p65) expression and 18 of the constructs inhibited NF-kB (p65) expression. This is the first direct demonstration that miRNAs affect NF-kB (p65) expression and the first genome-scale miRNA screen to identify upregulation and downregulation of NF-kB accumulation by miRNAs in the ovary. Novel miRNAs that affect the NF-kB signalling pathway could be useful for the control of NF-kB-dependent reproductive processes and the treatment of NF-kB-dependent reproductive disorders.

[Expression of Ki-67 and P53 protein in oral squamous cell carcinoma and its clinical significance].

Science.gov (United States)

He, Wei; Xiao, Yan; Chen, Wei-min

2015-04-01

To investigate the clinical and pathological features and its relationship with the expression of Ki-67 and p53 protein in oral squamous cell carcinoma. Immunohistochemical SP staining method was used to quantify the protein expression levels of Ki-67 and p53 protein in 10 cases of normal oral mucosa, 16 cases of oral leukoplakia (OLK) tissue, and 48 cases of oral squamous cell carcinoma. The relationship of the expression of Ki-67 and p53 protein to clinical and pathological data was analyzed, and SPSS17.0 software package was used for statistical analysis. The positive expression rate of Ki-67 protein in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma was 30%, 56.3% and 79.2%, respectively; The positive expression rate of p53 was 0%, 43.8%, and 70.8%, respectively; Ki-67 and p53 expression had significant difference among normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma (Poral squamous cell carcinoma (Poral squamous cell carcinoma tissues may play an important role in the development of oral squamous cell carcinoma.

Basal p53 expression is indispensable for mesenchymal stem cell integrity.

Science.gov (United States)

Boregowda, Siddaraju V; Krishnappa, Veena; Strivelli, Jacqueline; Haga, Christopher L; Booker, Cori N; Phinney, Donald G

2018-03-01

Marrow-resident mesenchymal stem cells (MSCs) serve as a functional component of the perivascular niche that regulates hematopoiesis. They also represent the main source of bone formed in adult bone marrow, and their bifurcation to osteoblast and adipocyte lineages plays a key role in skeletal homeostasis and aging. Although the tumor suppressor p53 also functions in bone organogenesis, homeostasis, and neoplasia, its role in MSCs remains poorly described. Herein, we examined the normal physiological role of p53 in primary MSCs cultured under physiologic oxygen levels. Using knockout mice and gene silencing we show that p53 inactivation downregulates expression of TWIST2, which normally restrains cellular differentiation to maintain wild-type MSCs in a multipotent state, depletes mitochondrial reactive oxygen species (ROS) levels, and suppresses ROS generation and PPARG gene and protein induction in response to adipogenic stimuli. Mechanistically, this loss of adipogenic potential skews MSCs toward an osteogenic fate, which is further potentiated by TWIST2 downregulation, resulting in highly augmented osteogenic differentiation. We also show that p53 - /- MSCs are defective in supporting hematopoiesis as measured in standard colony assays because of decreased secretion of various cytokines including CXCL12 and CSF1. Lastly, we show that transient exposure of wild-type MSCs to 21% oxygen upregulates p53 protein expression, resulting in increased mitochondrial ROS production and enhanced adipogenic differentiation at the expense of osteogenesis, and that treatment of cells with FGF2 mitigates these effects by inducing TWIST2. Together, these findings indicate that basal p53 levels are necessary to maintain MSC bi-potency, and oxygen-induced increases in p53 expression modulate cell fate and survival decisions. Because of the critical function of basal p53 in MSCs, our findings question the use of p53 null cell lines as MSC surrogates, and also implicate dysfunctional