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Sample records for inhibits angiogenesisin vitro

  1. Chrysin Inhibits Lymphangiogenesis in Vitro.

    PubMed

    Prangsaengtong, Orawin; Athikomkulchai, Sirivan; Xu, Jiuxiang; Koizumi, Keiichi; Inujima, Akiko; Shibahara, Naotoshi; Shimada, Yutaka; Tadtong, Sarin; Awale, Suresh

    2016-01-01

    The induction of lymphangiogenesis is an important process to promote cancer growth and cancer metastasis via the lymphatic system. Identifying the compounds that can prevent lymphangiogenesis for cancer therapy is urgently required. Chrysin, 5,7-dihydroxyflavone, a natural flavone extracted from Thai propolis, was used to investigate the effect on the lymphangiogenesis process of TR-LE, rat lymphatic endothelial cells. In this study, maximal nontoxic doses of chrysin on TR-LE cells were selected by performing a proliferation assay. The process of lymphangiogenesis in vitro was determined by cord formation assay, adhesion assay and migration assay. Chrysin at a nontoxic dose (25 μM) significantly inhibited cord formation, cell adhesion and migration of TR-LE cells when compared with the control group. We also found that chrysin significantly induced vascular endothelial growth factor C (VEGF-C) mRNA expression and nitric oxide (NO) production in TR-LE cells which was involved in decreasing the cord formation of TR-LE cells. In conclusion, we report for the first time that chrysin inhibited the process of lymphangiogenesis in an in vitro model. This finding may prove to be a natural compound for anti-lymphangiogenesis that could be developed for use in cancer therapy.

  2. Praziquantel inhibits Schistosoma mansoni attachment in vitro.

    PubMed

    da-Silva, S P; Noel, F

    1990-01-01

    Male adult Schistosoma mansoni worms were placed in a glass dish containing Tyrode solution and observed for 15 min after addition of praziquantel (0.01 to 1 microM). Praziquantel promoted a concentration- and time-dependent inhibition of sucker-mediated attachment of the worm. Attachment inhibition was correlated with shortening of the parasite. We propose that the rapid and total inhibition of worm attachment observed in vitro with 1 microM praziquantel indicates that therapeutic concentrations of this drug should promote a rapid hepatic shift, in vivo, which may facilitate host tissue reaction.

  3. Phytic acid inhibits lipid peroxidation in vitro.

    PubMed

    Zajdel, Alicja; Wilczok, Adam; Węglarz, Ludmiła; Dzierżewicz, Zofia

    2013-01-01

    Phytic acid (PA) has been recognized as a potent antioxidant and inhibitor of iron-catalyzed hydroxyl radical formation under in vitro and in vivo conditions. Therefore, the aim of the present study was to investigate, with the use of HPLC/MS/MS, whether PA is capable of inhibiting linoleic acid autoxidation and Fe(II)/ascorbate-induced peroxidation, as well as Fe(II)/ascorbate-induced lipid peroxidation in human colonic epithelial cells. PA at 100 μM and 500 μM effectively inhibited the decay of linoleic acid, both in the absence and presence of Fe(II)/ascorbate. The observed inhibitory effect of PA on Fe(II)/ascorbate-induced lipid peroxidation was lower (10-20%) compared to that of autoxidation. PA did not change linoleic acid hydroperoxides concentration levels after 24 hours of Fe(II)/ascorbate-induced peroxidation. In the absence of Fe(II)/ascorbate, PA at 100 μM and 500 μM significantly suppressed decomposition of linoleic acid hydroperoxides. Moreover, PA at the tested nontoxic concentrations (100 μM and 500 μM) significantly decreased 4-hydroxyalkenal levels in Caco-2 cells which structurally and functionally resemble the small intestinal epithelium. It is concluded that PA inhibits linoleic acid oxidation and reduces the formation of 4-hydroxyalkenals. Acting as an antioxidant it may help to prevent intestinal diseases induced by oxygen radicals and lipid peroxidation products.

  4. Phytic Acid Inhibits Lipid Peroxidation In Vitro

    PubMed Central

    Węglarz, Ludmiła; Dzierżewicz, Zofia

    2013-01-01

    Phytic acid (PA) has been recognized as a potent antioxidant and inhibitor of iron-catalyzed hydroxyl radical formation under in vitro and in vivo conditions. Therefore, the aim of the present study was to investigate, with the use of HPLC/MS/MS, whether PA is capable of inhibiting linoleic acid autoxidation and Fe(II)/ascorbate-induced peroxidation, as well as Fe(II)/ascorbate-induced lipid peroxidation in human colonic epithelial cells. PA at 100 μM and 500 μM effectively inhibited the decay of linoleic acid, both in the absence and presence of Fe(II)/ascorbate. The observed inhibitory effect of PA on Fe(II)/ascorbate-induced lipid peroxidation was lower (10–20%) compared to that of autoxidation. PA did not change linoleic acid hydroperoxides concentration levels after 24 hours of Fe(II)/ascorbate-induced peroxidation. In the absence of Fe(II)/ascorbate, PA at 100 μM and 500 μM significantly suppressed decomposition of linoleic acid hydroperoxides. Moreover, PA at the tested nontoxic concentrations (100 μM and 500 μM) significantly decreased 4-hydroxyalkenal levels in Caco-2 cells which structurally and functionally resemble the small intestinal epithelium. It is concluded that PA inhibits linoleic acid oxidation and reduces the formation of 4-hydroxyalkenals. Acting as an antioxidant it may help to prevent intestinal diseases induced by oxygen radicals and lipid peroxidation products. PMID:24260736

  5. Curcumin-loaded biodegradable polymeric micelles for colon cancer therapy in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Gou, Maling; Men, Ke; Shi, Huashan; Xiang, Mingli; Zhang, Juan; Song, Jia; Long, Jianlin; Wan, Yang; Luo, Feng; Zhao, Xia; Qian, Zhiyong

    2011-04-01

    Curcumin is an effective and safe anticancer agent, but its hydrophobicity inhibits its clinical application. Nanotechnology provides an effective method to improve the water solubility of hydrophobic drug. In this work, curcumin was encapsulated into monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles through a single-step nano-precipitation method, creating curcumin-loaded MPEG-PCL (Cur/MPEG-PCL) micelles. These Cur/MPEG-PCL micelles were monodisperse (PDI = 0.097 +/- 0.011) with a mean particle size of 27.3 +/- 1.3 nm, good re-solubility after freeze-drying, an encapsulation efficiency of 99.16 +/- 1.02%, and drug loading of 12.95 +/- 0.15%. Moreover, these micelles were prepared by a simple and reproducible procedure, making them potentially suitable for scale-up. Curcumin was molecularly dispersed in the PCL core of MPEG-PCL micelles, and could be slow-released in vitro. Encapsulation of curcumin in MPEG-PCL micelles improved the t1/2 and AUC of curcuminin vivo. As well as free curcumin, Cur/MPEG-PCL micelles efficiently inhibited the angiogenesis on transgenic zebrafish model. In an alginate-encapsulated cancer cell assay, intravenous application of Cur/MPEG-PCL micelles more efficiently inhibited the tumor cell-induced angiogenesisin vivo than that of free curcumin. MPEG-PCL micelle-encapsulated curcumin maintained the cytotoxicity of curcumin on C-26 colon carcinoma cellsin vitro. Intravenous application of Cur/MPEG-PCL micelle (25 mg kg-1curcumin) inhibited the growth of subcutaneous C-26 colon carcinoma in vivo (p < 0.01), and induced a stronger anticancer effect than that of free curcumin (p < 0.05). In conclusion, Cur/MPEG-PCL micelles are an excellent intravenously injectable aqueous formulation of curcumin; this formulation can inhibit the growth of colon carcinoma through inhibiting angiogenesis and directly killing cancer cells.

  6. Glycerol inhibition of ruminal lipolysis in vitro

    USDA-ARS?s Scientific Manuscript database

    Supplemental glycerol inhibits rumen lipolysis, a prerequisite for rumen biohydrogenation, which is responsible for the saturation of dietary fatty acids consumed by ruminant animals. Feeding excess glycerol, however, adversely affects dry matter digestibility. To more clearly define the effect of...

  7. Resveratrol inhibits periodontal pathogens in vitro.

    PubMed

    O'Connor, D J; Wong, R W K; Rabie, A B M

    2011-11-01

    The gram-negative anaerobic bacteria A. actinomycetemcomitans (Aa) and P. gingivalis (Pg) are key components in the aetiology of periodontal disease, and associated hard-tissue destruction. Resveratrol is a phytoalexin, produced naturally by several plants when under attack by bacterial or fungal pathogens. It is found in many foods including mulberries, peanuts and the skin of labrusca and muscadine grapes. The objective of this study was to evaluate the effect of resveratrol on the in vitro growth of periodontal pathogens Aa and Pg. For comparison, resveratrol's effect on a variety of other oral microorganisms was also evaluated. Resveratrol demonstrates a poor solubility in water, thus different concentrations of resveratrol in the solvent dimethyl sulphoxide (DMSO) were added to calibrated suspensions of Aa and Pg. As a control, a parallel series of dilutions containing the vehicle DMSO alone was made to measure the effect of the solvent. Minimum inhibitory concentrations of the periodontal pathogens were calculated. All suspensions were incubated for 1, 3, 6 and 24 h in an anaerobic chamber at 37 °C. At each time interval, selected dilutions from each culture broth were plated on blood agar plates. Colonies appearing on blood agar plates were visually counted at 3 days for Aa, and at 5 days for Pg. The periodontal bacteria showed a significant decrease (p < 0.05) in viable counts after 1 h, whilst no colony forming units could be observed after 24 h. The results suggest that resveratrol possesses significant antimicrobial properties on periodontal pathogens in vitro. Copyright © 2011 John Wiley & Sons, Ltd.

  8. Geranylgeranylacetone inhibits ovarian cancer progression in vitro and in vivo

    SciTech Connect

    Hashimoto, Kae; Morishige, Ken-ichirou . E-mail: mken@gyne.med.osaka-u.ac.jp; Sawada, Kenjiro; Ogata, Seiji; Tahara, Masahiro; Shimizu, Shoko; Sakata, Masahiro; Tasaka, Keiichi; Kimura, Tadashi

    2007-04-27

    Geranylgeranylacetone (GGA), an isoprenoid compound, is an anti-ulcer drug developed in Japan. In our previous study, GGA was shown to inhibit ovarian cancer invasion by attenuating Rho activation [K. Hashimoto, K. Morishige, K. Sawada, M. Tahara, S. Shimizu, M. Sakata, K. Tasaka, Y. Murata, Geranylgeranylacetone inhibits lysophosphatidic acid-induced invasion of human ovarian carcinoma cells in vitro. Cancer 103 (2005) 1529-1536.]. In the present study, GGA treatment inhibited ovarian cancer progression in vitro and suppressed the tumor growth and ascites in the in vivo ovarian cancer model. In vitro analysis, treatment of cancer cells by GGA resulted in the inhibition of cancer cell proliferation, the inactivation of Ras, and the suppression of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK). In conclusion, this is the first report that GGA inhibited ovarian cancer progression and the anti-tumor effect by GGA is, at least in part, derived not only from the suppression of Rho activation but also Ras-MAPK activation.

  9. Geranylgeranylacetone inhibits ovarian cancer progression in vitro and in vivo.

    PubMed

    Hashimoto, Kae; Morishige, Ken-ichirou; Sawada, Kenjiro; Ogata, Seiji; Tahara, Masahiro; Shimizu, Shoko; Sakata, Masahiro; Tasaka, Keiichi; Kimura, Tadashi

    2007-04-27

    Geranylgeranylacetone (GGA), an isoprenoid compound, is an anti-ulcer drug developed in Japan. In our previous study, GGA was shown to inhibit ovarian cancer invasion by attenuating Rho activation [K. Hashimoto, K. Morishige, K. Sawada, M. Tahara, S. Shimizu, M. Sakata, K. Tasaka, Y. Murata, Geranylgeranylacetone inhibits lysophosphatidic acid-induced invasion of human ovarian carcinoma cells in vitro. Cancer 103 (2005) 1529-1536.]. In the present study, GGA treatment inhibited ovarian cancer progression in vitro and suppressed the tumor growth and ascites in the in vivo ovarian cancer model. In vitro analysis, treatment of cancer cells by GGA resulted in the inhibition of cancer cell proliferation, the inactivation of Ras, and the suppression of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK). In conclusion, this is the first report that GGA inhibited ovarian cancer progression and the anti-tumor effect by GGA is, at least in part, derived not only from the suppression of Rho activation but also Ras-MAPK activation.

  10. Clodronate inhibits angiogenesis in vitro and in vivo.

    PubMed

    Ribatti, D; Maruotti, N; Nico, B; Longo, V; Mangieri, D; Vacca, A; Cantatore, F P

    2008-05-01

    The effects of amino-bisphosphonate clodronate on endothelial cell functions involved in angiogenesis, namely proliferation and morphogenesis on matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo. This was performed by using the chick embryo chorioallantoic membrane (CAM) assay. In vitro, clodronate inhibited the endothelial cell proliferation in a dose-dependent fashion, peaking at 30 microM. At the same concentration, clodronate inhibited the fibroblast growth factor-2 (FGF-2)-induced capillary-like tube formation in the morphogenesis assay on matrigel. In vivo, when tested with the CAM assay, clodronate again displayed the capability to inhibit FGF-2-induced angiogenesis. Overall, these results suggest that antiangiogenesis by clodronate can be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases and cancer.

  11. Aluminum Trichloride Inhibits the Rat Osteoblasts Mineralization In Vitro.

    PubMed

    Song, Miao; Huo, Hui; Cao, Zheng; Han, Yanfei; Gao, Li

    2017-01-01

    Aluminum (Al) is an accumulative toxic metal. Excessive Al accumulation inhibits osteoblasts mineralization and induces osteoporosis. However, the inhibition mechanism of Al on the mineralization is not fully understood. Thus, in this study, the rat osteoblasts were cultured and exposed to 0 mmol L(-1) (control group, CG) and 0.52 mmol L(-1) aluminum trichloride (AlCl3, treatment group, TG) for 7, 14, and 21 days, respectively. We found that mineralized matrix nodules, the activity of bone alkaline phosphatase, the concentration of extracellular calcium, the mRNA expression of type-I collagen, the mRNA and protein expressions of osteopontin, osteocalcin, and bone sialoprotein were all decreased, while the concentration of extracellular phosphorus was increased in TG compared with CG with time prolonged. Taken together, these results indicated that AlCl3 inhibited osteoblasts mineralization in vitro.

  12. Nitric oxide inhibition of coxsackievirus replication in vitro.

    PubMed Central

    Zaragoza, C; Ocampo, C J; Saura, M; McMillan, A; Lowenstein, C J

    1997-01-01

    Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (CVB3) replication in vitro by determining the effect of NO upon a single replicative cycle of CVB3 grown in HeLa cells. Transfection of inducible NO synthase cDNA into HeLa cells reduces the number of viral particles produced during a single cycle of growth. Similarly, a noncytotoxic concentration of the NO donor S-nitroso-amino-penicillamine reduces the number of viral particles in a dose-dependent manner. To explore the mechanisms by which NO exerts its antiviral effect, we assayed the attachment, replication, and translation steps of the CVB3 life cycle. NO does not affect the attachment of CVB3 to HeLa cells. However, NO inhibits CVB3 RNA synthesis, as shown by a [3H]uridine incorporation assay, reverse transcription-PCR, and Northern analysis. In addition, NO inhibits CVB3 protein synthesis, as shown by [35S]methionine protein labeling and Western blot analysis of infected cells. Thus, NO inhibits CVB3 replication in part by inhibiting viral RNA synthesis by an unknown mechanism. PMID:9312175

  13. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2015-12-08

    During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

  14. Inhibition of AIDS virus replication by acemannan in vitro.

    PubMed

    Kahlon, J B; Kemp, M C; Carpenter, R H; McAnalley, B H; McDaniel, H R; Shannon, W M

    1991-09-01

    Acemannan (ACE-M), a beta-(1,4)-linked acetylated mannan, was evaluated for in vitro activity against human immunodeficiency virus type 1 (HIV-1). Castanospermine (CAS), deoxymannojirimycin (DMN), swainsonine (SWS), azidothymidine (AZT), and dideoxythymidine (DDC) were tested in parallel as control compounds. In vitro antiviral efficacy of ACE-M was evaluated in a variety of cell lines including human peripheral mononuclear, CEM-SS1 and MT-2(2) cells. The virus strain, number of infectious units per cell, and target cell line were important factors in determining the degree of inhibition of viral cytopathic effect in the presence of ACE-M and other control compounds tested. Maximum inhibitory effect was observed in CEM-SS cells infected with the RFII strain of HIV-1. This inhibitory effect was determined to be concentration-dependent. Assay design included primary screening to measure cell viabilities of infected target cells in the presence and absence of test compounds. When tested on HIV-1/RFII-infected CEM-SS cells, the 50% inhibitory effect of CAS (IC50 = 28), an inhibitor of alpha-glucosidase I, was determined to be similar to that observed for ACE-M (IC50 = 45). However, DMN and SWS, inhibitors of mannosidase I and II, tested in parallel to CAS and ACE-M, exhibited no IC50 values. Antiviral potential of ACE-M as an inhibitor of syncytia formation was also explored using CEM-SS cells. Suppression of syncytia formation was observed at an ACE-M concentration of 31.25 micrograms/ml, and complete inhibition was observed at 62.5 micrograms/ml. In addition, HIV-1 RNA levels were studied to establish the antiviral potential of ACE-M in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Trichostatin a inhibits corneal haze in vitro and in vivo.

    PubMed

    Sharma, Ajay; Mehan, Maneesh M; Sinha, Sunilima; Cowden, John W; Mohan, Rajiv R

    2009-06-01

    Trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to suppress TGF-beta-induced fibrogenesis in many nonocular tissues. The authors evaluated TSA cytotoxicity and its antifibrogenic activity on TGF-beta-driven fibrosis in the cornea with the use of in vitro and in vivo models. Human corneal fibroblasts (HSFs) were used for in vitro studies, and New Zealand White rabbits were used for in vivo studies. Haze in the rabbit cornea was produced with photorefractive keratectomy (PRK) using excimer laser. Trypan blue exclusion and MTT assays evaluated TSA cytotoxicity to the cornea. Density of haze in the rabbit eye was graded with slit lamp biomicroscopy. Real-time PCR, immunoblotting, or immunocytochemistry was used to measure alpha-smooth muscle actin (SMA), fibronectin, and collagen type IV mRNA or protein levels. TUNEL assay was used to detect cell death. TSA concentrations of 250 nM or less were noncytotoxic and did not alter normal HSF morphology or proliferation. TGF-beta1 treatment of HSF significantly increased mRNA and protein levels of SMA (9-fold), fibronectin (2.5-fold), and collagen type IV (2-fold). TSA treatment showed 60% to 75% decreases in TGF-beta1-induced SMA and fibronectin mRNA levels and 1.5- to 3.0-fold decreases in protein levels but had no effect on collagen type IV mRNA or protein levels in vitro. Two-minute topical treatment of TSA on rabbit corneas subjected to -9 D PRK significantly decreased corneal haze in vivo. TSA inhibits TGF-beta1-induced accumulation of extracellular matrix and myofibroblast formation in the human cornea in vitro and markedly decreases haze in rabbit cornea in vivo.

  16. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    SciTech Connect

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site.

  17. Inhibition of biofilms of Pseudomonas aeruginosa by Medihoney in vitro.

    PubMed

    Cooper, R; Jenkins, L; Hooper, S

    2014-03-01

    Pseudomonas aeruginosa has been linked to chronic wound infections, where its ability to form biofilms and to tolerate antimicrobial agents helps to facilitate its persistence. This study aimed to investigate the susceptibility of biofilms of Pseudomonas aeruginosa to Medihoney in vitro. Biofilms were cultivated in microtitre plates with and without a range of concentrations of Medihoney, and effects on biofilm were monitored by optical density (at 650nm), biomass (by staining with crystal violet), metabolic activity (using an esterase assay) and viability (by determining total cell counts). Structural effects on established biofilms were examined by scanning electron microscopy and epifluorescence following staining by LIVE/DEAD® BacLight, which also showed effects on vitality. The lowest concentration of Medihoney found to prevent biofilm formation was 17%(w/v), whereas on average 35.5%(w/v) of Medihoney was required to inhibit established biofilms. Susceptibility did not vary with length of biofilm establishment between 24 and 72 hours. Extensive structural changes in established biofilms were seen in the sample with less than or equal to 30%(w/v) Medihoney using scanning electron microscopy and loss of viability was found in test samples with less than or equal to 20%(w/v) Medihoney concentration using fluorescent staining, together with loss of biofilm structure. Using a range of methods to evaluate biofilm integrity, this study demonstrates that Medihoney inhibits Pseudomonas aeruginosa biofilms in vitro at concentrations that are attainable in clinical use. Whether Medihoney has the potential to disrupt Pseudomonas aeruginosa biofilms in cutaneous wounds must now be tested in patients. This study was sponsored by Derma Sciences Inc, NJ. An unrestricted grant was provided and the sponsors were not involved in the design of the experiments or the preparation of this manuscript.

  18. Experimental investigation of HGF inhibiting glial scar in vitro.

    PubMed

    Liu, Cheng; Wu, Zu-ze; Shu, Cui-li; Li, Ding-feng; Zeng, Yan-jun; Cui, Qiu; Jiang, Wei-hao

    2011-03-01

    To study the inhibitory effect of Hepatocyte growth factor (HGF) on the responsive hyperplasia of damaged astrocytes in vitro. We prepared damaged model of astrocytes to simulate the responsive hyperplasia of damaged astrocytes in vivo by culturing astrocytes in vitro; After the first day of Ad-HGF transfection, astrocytes were scratched, then after the first, the third, and the fifth day of scratch, we detect the expression amount of astrocytes specific glial fibrillary acidic protein (GFAP) and the ratio of S-phase cells with flow cytometry, both of which can reflect the proliferation status of damaged astrocytes; After HGF was added in scratched astrocytes, the activity of SPK and MAPK (P42/44) were detected by autoradiography and immunoblotting test; After adding different concentrations of HGF protein in astrocytes cultured in different serum concentrations and adding diverse concentrations of HGF protein, SPK and SPK inhibitor DMS in scratched astrocytes, we detect cell proliferation with 3H-TDR incorporation. The first day after Ad-HGF transfected astrocytes were scratched, the amount of GFAP secreted by astrocytes were decreased significantly (P < 0.05), and the cells in S phase were declined obviously. HGF has bidirectional regulation on SPK of scratched astrocytes: increases the SPK activity when HGF in low dose, while inhibits when in high dose. In addition, DMS can block the signal passage; HGF had no effects on MAPK (P42/44) of damaged astrocytes cells. In conclusion, after the transfection of Ad-HGF, it can inhibit the responsive hyperplasia of damaged astrocytes by the means of blocking SPK passage.

  19. Apigenin inhibits African swine fever virus infection in vitro.

    PubMed

    Hakobyan, Astghik; Arabyan, Erik; Avetisyan, Aida; Abroyan, Liana; Hakobyan, Lina; Zakaryan, Hovakim

    2016-12-01

    African swine fever virus (ASFV) is one of the most devastating diseases of domestic pigs for which no effective vaccines are available. Flavonoids, natural products isolated from plants, have been reported to have significant in vitro and in vivo antiviral activity against different viruses. Here, we tested the antiviral effect of five flavonoids on the replication of ASFV in Vero cells. Our results showed a potent, dose-dependent anti-ASFV effect of apigenin in vitro. Time-of-addition experiments revealed that apigenin was highly effective at the early stages of infection. Apigenin reduced the ASFV yield by more than 99.99 % when it was added at 1 hpi. The antiviral activity of apigenin was further investigated by evaluation of ASFV protein synthesis and viral factories. This flavonoid inhibited ASFV-specific protein synthesis and viral factory formation. ASFV-infected cells continuously treated with apigenin did not display a cytopathic effect. Further studies addressing the use of apigenin in vivo are needed.

  20. In vitro inhibition of mitochondrial respiratory rate by antidepressants.

    PubMed

    Hroudová, Jana; Fišar, Zdeněk

    2012-09-18

    Mitochondria represent a possible drug target with unexplored therapeutic and toxicological potential. The possibility was suggested that antidepressants, mood stabilizers and other drugs may show some therapeutic and/or toxic effects through their action on mitochondrial functions. There are no sufficient data about the effect of these drugs on mitochondrial respiration in the brain. We investigated the in vitro effects of amitriptyline, fluoxetine, tianeptine, ketamine, lithium, valproate, olanzapine, chlorpromazine and propranolol on mitochondrial respiration in crude mitochondrial fractions of pig brains. Respiration was energized using substrates of complex I or complex II and dose dependent drug-induced changes in mitochondrial respiratory rate were measured by high-resolution respirometry. Antidepressants, but not mood stabilizers, ketamine and propranolol were found to inhibit mitochondrial respiratory rate. The effective dose of antidepressants reaching half the maximal respiratory rate was in the range of 0.07-0.46 mmol/L. Partial inhibition was found for all inhibitors. Differences between individual drugs with similar physicochemical properties indicate selectivity of drug-induced changes in mitochondrial respiratory rate. Our findings suggest that mood stabilizers do not interfere with brain mitochondrial respiration, whereas direct mitochondrial targeting is involved in mechanisms of action of pharmacologically different antidepressants. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. Quercetin inhibits rhinovirus replication in vitro and in vivo

    PubMed Central

    Ganesan, Shyamala; Faris, Andrea N.; Comstock, Adam T.; Wang, Qiong; Nanua, Suparna; Hershenson, Marc B.; Sajjan, Uma S.

    2012-01-01

    Summary Rhinovirus (RV), which is responsible for the majority of common colds, also causes exacerbations in patients with asthma and chronic obstructive pulmonary disease. So far, there are no drugs available for treatment of rhinovirus infection. We examined the effect of quercetin, a plant flavanol on RV infection in vitro and in vivo. Pretreatment of airway epithelial cells with quercetin decreased Akt phosphosphorylation, viral endocytosis and IL-8 responses. Addition of quercetin 6 h after RV infection (after viral endocytosis) reduced viral load, IL-8 and IFN responses in airway epithelial cells. This was associated with decreased levels of negative and positive strand viral RNA, and RV capsid protein, abrogation of RV-induced eIF4GI cleavage and increased phosphorylation of eIF2α. In mice infected with RV, quercetin treatment decreased viral replication as well as expression of chemokines and cytokines. Quercetin treatment also attenuated RV-induced airway cholinergic hyperresponsiveness. Together, our results suggest that quercetin inhibits RV endocytosis and replication in airway epithelial cells at multiple stages of the RV life cycle. Quercetin also decreases expression of pro-inflammatory cytokines and improves lung function in RV-infected mice. Based on these observations, further studies examining the potential benefits of quercetin in the prevention and treatment of RV infection are warranted. PMID:22465313

  2. Further evidence that naphthoquinone inhibits Toxoplasma gondii growth in vitro.

    PubMed

    da Silva, Luciana Lemos Rangel; Portes, Juliana de Araujo; de Araújo, Marlon Heggdorne; Silva, Jéssica Lays Sant'ana; Rennó, Magdalena Nascimento; Netto, Chaquip Daher; da Silva, Alcides José Monteiro; Costa, Paulo Roberto Ribeiro; De Souza, Wanderley; Seabra, Sergio Henrique; DaMatta, Renato Augusto

    2015-12-01

    Toxoplasmosis is a widely disseminated disease caused by Toxoplasma gondii, an intracellular protozoan parasite. Standard treatment causes many side effects, such as depletion of bone marrow cells, skin rashes and gastrointestinal implications. Therefore, it is necessary to find chemotherapeutic alternatives for the treatment of this disease. It was shown that a naphthoquinone derivative compound is active against T. gondii, RH strain, with an IC50 around 2.5 μM. Here, three different naphthoquinone derivative compounds with activity against leukemia cells and breast carcinoma cell were tested against T. gondii (RH strain) infected LLC-MK2 cell line. All the compounds were able to inhibit parasite growth in vitro, but one of them showed an IC50 activity below 1 μM after 48 h of treatment. The compounds showed low toxicity to the host cell. In addition, these compounds were able to induce tachyzoite-bradyzoite conversion confirmed by morphological changes, Dolichus biflorus lectin cyst wall labeling and characterization of amylopectin granules in the parasites by electron microscopy analysis using the Thierry technique. Furthermore, the compounds induced alterations on the ultrastructure of the parasite. Taken together, our results point to the naphthoquinone derivative (LQB 151) as a potential compound for the development of new drugs for the treatment of toxoplasmosis.

  3. Overexpression of KAI1 inhibits retinoblastoma metastasis in vitro

    PubMed Central

    Yan, Hui; Ji, Xunda; Li, Jing; Zhang, Lei; Zhao, Peiquan

    2017-01-01

    The present study aimed to investigate the expression of cluster of differentiation 82 (KAI1), a gene involved in the suppression of tumor metastasis, in human retinoblastoma (RB) tissue and to study the effect of KAI1 expression on RB cell migration and invasion. KAI1 expression was examined in 26 patients with non-invasive and invasive retinoblastoma using reverse transcription-quantitative polymerase chain reaction and western blot analysis. A lentiviral vector containing KAI1 cDNA was used to transfect the two RB cell lines, HXO-Rb44-Gl and Y79. Following successful transfection, the migratory and invasive capacity of the two RB cell lines was evaluated using a Transwell® migration assay. KAI1 expression was observed to be downregulated in invasive RB compared to non-invasive RB. The migratory and invasive capacities of KAI1 transfected cell lines were significantly decreased compared to those of the control cells. KAI1 may be involved in retinoblastoma metastasis, and increased expression of KAI1 significantly inhibits the metastatic ability of RB cells in vitro. PMID:28356965

  4. Artemisinins Inhibit Trypanosoma cruzi and Trypanosoma brucei rhodesiense In Vitro Growth▿

    PubMed Central

    Mishina, Yuliya V.; Krishna, Sanjeev; Haynes, Richard K.; Meade, John C.

    2007-01-01

    Artemisinin compounds inhibit in vitro growth of cultured Trypanosoma cruzi and Trypanosoma brucei rhodesiense at concentrations in the low micromolar range. Artemisinin also inhibits calcium-dependent ATPase activity in T. cruzi membranes, suggesting a mode of action via membrane pumps. Artemisinins merit further investigation as chemotherapeutic options for these pathogens. PMID:17339374

  5. Statin Drugs Markedly Inhibit Testosterone Production by Rat Leydig Cells In Vitro: Implications for Men

    EPA Science Inventory

    Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. During drug development it was shown that statins inhibit production of cholesterol in the testis. We evaluated testosterone production in vitro, using highly purified rat ...

  6. Statin Drugs Markedly Inhibit Testosterone Production by Rat Leydig Cells In Vitro: Implications for Men

    EPA Science Inventory

    Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. During drug development it was shown that statins inhibit production of cholesterol in the testis. We evaluated testosterone production in vitro, using highly purified rat ...

  7. Lactate dehydrogenase activity is inhibited by methylmalonate in vitro.

    PubMed

    Saad, Laura O; Mirandola, Sandra R; Maciel, Evelise N; Castilho, Roger F

    2006-04-01

    Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K (i)=3.02+/-0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.

  8. In Vitro Acetylcholinesterase Inhibition by Type A Botulinum Toxin

    PubMed Central

    Marshall, R.; Quinn, L. Y.

    1967-01-01

    Type A botulinum toxin was studied for its ability to inhibit the action of acetyl-cholinesterase. The chromogenic substrate, indophenyl acetate, was used for assay of enzyme activity. Inhibition of enzyme function was detected through use of both 6.6 × 10−6 mg (20 ld50) and 6.6 × 10−10 mg (2 × 10−3ld50) of type A botulinal toxin. Control assays were performed by use of both homologous antitoxin and heterologous antitoxins (types B and E). Enzyme inhibition was effectively prevented by use of homologous antitoxin only. The inhibition noted was specific and reproducible for given substrate, enzyme, and toxin concentrations. PMID:4860916

  9. Inhibition of rat liver microsomal fatty acid chain elongation by gemfibrozil in vitro.

    PubMed

    Sánchez, R M; Viñals, M; Alegret, M; Vázquez, M; Adzet, T; Merlos, M; Laguna, J C

    1992-03-23

    Gemfibrozil, a hypolipidemic drug mainly used in the treatment of hypertriglyceridemic states, strongly inhibits the rat hepatic microsomal fatty acid chain elongation system in vitro. The inhibition is independent on the reducing cofactor used in the assay. Furthermore, gemfibrozil seems to act by inhibiting the rate-limiting step of the elongation process, the condensing reaction, without discriminating among the proposed three different condensing enzymes, devoted to condensation of saturated, mono-unsaturated and polyunsaturated acyl-CoA substrates.

  10. Inhibition of in vitro cholesterol synthesis by fatty acids.

    PubMed

    Kuroda, M; Endo, A

    1976-01-18

    Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.

  11. Synthetic polyphosphate inhibits endogenous coagulation and platelet aggregation in vitro

    PubMed Central

    Yang, Xiaoyang; Wan, Mengjie; Liang, Ting; Peng, Minyuan; Chen, Fangping

    2017-01-01

    Platelet-derived polyphosphate has previously been indicated to induce coagulation. However, industrially synthesized polyphosphate has been found to have different effects from those of the platelet-derived form. The present study investigated whether synthetic sodium polyphosphate inhibits coagulation using routine coagulation tests and thromboelastography. Synthetic polyphosphate was found to inhibit adenosine diphosphate-, epinephrine-, arachidonic acid-, ristocetin-, thrombin-, oxytocin- and pituitrin-induced platelet aggregation. The effects of synthetic polyphosphate in clotting inhibition were revealed by the analysis of clotting factor activity and platelet aggregation tests. Synthetic polyphosphate may inhibit platelet aggregation by reducing platelet calcium levels, as indicated by the results of flow cytometric analysis and high-throughput fluorescent screening. Furthermore, analysis of thromboxane (TX)B2 by ELISA indicated that synthetic polyphosphate reduces platelet aggregation by inhibiting the TXA2 signaling pathway. In conclusion, synthetic polyphosphate inhibits clotting factor activity and endogenous coagulation by reducing the levels of calcium ions and TXA2 to curb platelet aggregation. PMID:28123708

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

    SciTech Connect

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

    2013-11-01

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

  13. In vitro growth inhibition of mastitis pathogens by bovine teat skin normal flora.

    PubMed

    Woodward, W D; Besser, T E; Ward, A C; Corbeil, L B

    1987-01-01

    One factor contributing to differences in the susceptibility of cows to mastitis may be differences in the teat skin normal flora, which could inhibit or enhance the growth of pathogenic bacteria. Using in vitro cross-streaking methods, we found that 25% of the isolates of teat normal flora of non-lactating heifers inhibited the growth of selected mastitis pathogens, but enhancers were not detected. Gram-positive pathogens were inhibited to a greater extent than Gram-negative pathogens. Inhibition was not a characteristic of specific genera or species of normal flora, but rather a property of certain variants within a species. This phenomenon of inhibition of mastitis pathogens in vitro by normal flora may be useful as an in vivo biological control method to reduce the incidence of mastitis.

  14. Inhibition of catalase activity in vitro by diesel exhaust particles.

    PubMed

    Mori, Y; Murakami, S; Sagae, T; Hayashi, H; Sakata, M; Sagai, M; Kumagai, Y

    1996-02-09

    The effect of diesel exhaust particles (DEP) on the activity of catalase, an intracellular antioxidant, was investigated because H2O2 is a cytotoxic oxidant, and catalase released from alveolar cells is an important antioxidant in the epithelial lining fluid in the lung. DEP inhibited the activity of bovine liver catalase dose-dependently, to 25-30% of its original value. The inhibition of catalase by DEP was observed only in the presence of anions such as Cl-,Br-, or thiocyanate. Other anions, such as CH3COO- or SO4-, and cations such as K+, Na+, Mg2+, or Fe2+, did not affect the activity of catalase, even in the presence of DEP extract. Catalase from guinea pig alveolar cells and catalase from red blood cells were also inhibited by DEP extracts, as was catalase from bovine liver. These results suggest that DEP taken up in the lung and located on alveolar spaces might cause cell injury by inhibiting the activity of catalase in epithelial lining fluid, enhancing the toxicity of H2O2 generated from cells in addition to that of O2- generated by the chemical reaction of DEP with oxygen.

  15. Inhibition of catalase activity in vitro by diesel exhaust particles

    SciTech Connect

    Mori, Yoki; Murakami, Sumika; Sagae, Toshiyuki

    1996-02-09

    The effect of diesel exhaust particles (DEP) on the activity of catalase, an intracellular anti-oxidant, was investigated because H{sub 2}O{sub 2} is a cytotoxic oxidant, and catalase released from alveolar cells is an important antioxidant in the epithelial lining fluid in the lung. DEP inhibited the activity of bovine liver catalase dose-dependently, to 25-30% of its original value. The inhibition of catalase by DEP was observed only in the presence of anions such as Cl{sup {minus}}, Br{sup {minus}}, or thiocyanate. Other anions, such as CH{sub 3}COO{sup {minus}} or SO{sub 4}{sup {minus}}, and cations such as K{sup +}, Na{sup +}, Mg{sup 2+}, or Fe{sup 2+}, did not affect the activity of catalase, even in the presence of DEP extract. Catalase from guinea pig alveolar cells and catalase from red blood cells were also inhibited by DEP extracts, as was catalase from bovine liver. These results suggest that DEP taken up in the lung and located on alveolar spaces might cause cell injury by inhibiting the activity of catalase in epithelial lining fluid, enhancing the toxicity of H{sub 2}O{sub 2} generated from cells in addition to that of O{sub 2}{sup {minus}} generated by the chemical reaction of DEP with oxygen. 10 refs., 6 figs.

  16. Glucose-induced inhibition of in vitro bone mineralization.

    PubMed

    Balint, E; Szabo, P; Marshall, C F; Sprague, S M

    2001-01-01

    Patients with diabetes tend to have an increased incidence of osteopenia that may be related to hyperglycemia. However, little is known about how glucose may alter bone formation and osteoblast maturation. To determine whether glucose affects osteoblastic calcium deposition, MC3T3-E1 cells were incubated in media containing either a normal (5.5 mmol/L) or high glucose concentration (15 mmol/L) or mannitol (15 mmol/L), and bone nodule formation was examined. Net calcium flux was measured thrice weekly and cumulative calcium uptake was determined. Compared with control incubations, glucose significantly inhibited daily and cumulative calcium uptake into the nodules. At the time of matrix maturation, cultures undergo a rapid phase of increased calcium deposition; this was significantly inhibited by the presence of glucose. Total calcium uptake, determined by acid digestion, was also significantly inhibited by glucose. Area and number of nodules were quantitated at the end of the incubation period (day 30) by staining with Alizarin Red S calcium stain. Compared with both control and mannitol-treated cultures, the number of nodules was increased by incubation with glucose. Furthermore, both the average total nodular area and calcified nodular area of large nodules were increased by glucose. Cellular proliferation as well as the release of markers of osteoblast activity (osteocalcin and alkaline phosphatase) were determined at the end of the experimental period (day 30). Cellular proliferation and alkaline phosphatase activity was significantly increased in the presence of glucose, however, the release of osteocalcin into culture media was similar in all three groups. In conclusion, the present study shows that elevated glucose concentration present throughout the development of murine osteoblasts stimulates cellular proliferation while inhibiting calcium uptake. The result of glucose inhibition of calcium uptake suggests that bone could be structurally altered in

  17. Phorbol diesters inhibit enzymatic hydrolysis of diacylglycerols in vitro.

    PubMed Central

    Chabbott, H; Cabot, M C

    1986-01-01

    The effect of phorbol 12-myristate 13-acetate (PMA) on diacylglycerol lipase activity was examined in rat serum, tissue, and cellular preparations by using di[14C]oleoylglycerol, [3H]palmitoylacetylglycerol, and membrane-resident phospholipase C-generated diacylglycerols as substrates. These experiments were conducted to address whether phorbol esters can mimic diacylglycerols in interacting with enzymes other than protein kinase C. Serum hydrolysis of palmitoylacetylglycerol, assayed by the formation of [3H]palmitic acid, was inhibited by PMA, 4-O-methyl-PMA, or phorbol 12,13-dibutyrate (in order of decreasing potency). The hydrolysis of palmitoylacetylglycerol was inhibited more than 40% by the addition of PMA at a 1:1 molar ratio with substrate. The inhibition resembled the competitive type, with a Ki of approximately 2.7 microM. PMA in the 10-60 microM range also inhibited hydrolysis of palmitoylacetylglycerol by lipases from rat brain microsomes and by homogenates of C3H/10T1/2 mouse fibroblasts. PMA was likewise inhibitory when assayed in an intramembrane enzyme-substrate milieu in which diacylglycerols were generated, in situ, by treatment of [3H]palmitate-labeled cell homogenates with phospholipase C. Collectively, these data demonstrate that PMA, which is now thought to act by mimicry of diacylglycerols, can inhibit the action of diacylglycerol lipase. It is possible that such a mechanism is linked to the multiplicity of responses elicited by phorbol diesters and that other agents may function by means of enzyme interactions (post-phospholipase C) to influence the levels of the cellular diacylglycerol mediators. PMID:3458169

  18. 4-methylumbelliferone inhibits angiogenesis in vitro and in vivo.

    PubMed

    García-Vilas, Javier A; Quesada, Ana R; Medina, Miguel Ángel

    2013-05-01

    4-Methylumbelliferone (4-MU) is a hyaluronic acid biosynthesis inhibitor with antitumoral and antimetastatic effects. The objective of the present study was to determine the potential of 4-MU as an antiangiogenic compound. To fulfill this aim, cultured endothelial cells were used to perform an array of in vitro assays, as well as two different in vivo angiogenesis assays. This study demonstrates that, in fact, 4-MU behaves as a new inhibitor of both in vitro and in vivo angiogenesis. In vitro, 4-MU affects several key steps of angiogenesis, including endothelial cell proliferation, adhesion, tube formation, and extracellular matrix remodeling. Half-maximal inhibitory concentrations (IC50) values in the proliferation assay were 0.65 ± 0.04 and 0.37 ± 0.03 mM for HMEC and RF-24 endothelial cells, respectively. 4-MU (2 mM) treatment for 24 h induced apoptosis in 13% of HMEC and 5% of RF-24 cells. The number of adherent endothelial cells decreased by >20% after 24 h of treatment with 1 mM 4-MU. Minimal inhibitory concentrations in the tube formation assay were 2 and 0.5 mM 4-MU for HMEC and RF-24, respectively. Matrix metalloproteinase-2 expression was differentially altered upon 4-MU treatment in both tested endothelial cell lines. Taken together, the results suggest that 4-MU may have potential as a new candidate multitargeted bioactive compound for antiangiogenic therapy.

  19. DNase inhibits Gardnerella vaginalis biofilms in vitro and in vivo.

    PubMed

    Hymes, Saul R; Randis, Tara M; Sun, Thomas Yang; Ratner, Adam J

    2013-05-15

    Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities.

  20. Inhibition of adenovirus replication in vitro by trifluridine.

    PubMed

    Lennette, D A; Eiferman, R A

    1978-09-01

    At present, there is no effective chemotherapeutic agent available for the treatment of adenoviral keratoconjunctivitis. Recent evidence suggests that trifluridine (3FT) may effectively inhibit the replication of some adenovirus serotypes known to cause keratoconjunctivitis. The ability of 3FT to inhibit two reference strains of adenoviruses, type 8 and type 19, was examined using cell cultures. Two second-passage isolates of adenoviruses, identified as serotype 13, were also tested. Compared with untreated, virusinfected cell cultures, drug-treated cell cultures developed a lesser degree of cytopathic effect following infection with all three serotypes. Virus production was reduced in the drug-treated cell cultures: approximately tenfold for type 8, more than 1,000-fold for type 19, and 5,000-fold for the type 13 isolates.

  1. DNase Inhibits Gardnerella vaginalis Biofilms In Vitro and In Vivo

    PubMed Central

    Hymes, Saul R.; Randis, Tara M.; Sun, Thomas Yang; Ratner, Adam J.

    2013-01-01

    Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities. PMID:23431033

  2. Vitamin E inhibits retinal pigment epithelium cell proliferation in vitro.

    PubMed

    Mojon, D; Boscoboinik, D; Haas, A; Bohnke, M; Azzi, A

    1994-01-01

    Retinal pigment epithelium (RPE) cells migrating through the damaged retina play an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We found that alpha-tocopherol (vitamin E) inhibits proliferation of human RPE in culture without exerting cytotoxic effects. Maximal inhibition was achieved with 100 microM alpha-tocopherol. Our result could explain the observation that vitamin E supplements have an adverse effect on light-damaged retina and on the course of retinitis pigmentosa. Since it has been shown that supplemental oral administrations of vitamin E can raise the RPE concentration of alpha-tocopherol well above 100 microM and supplementation is not associated with any clinical relevant adverse effect, we believe that vitamin E could be beneficial in the treatment of PVR.

  3. Bafetinib inhibits functional responses of human eosinophils in vitro.

    PubMed

    Milara, Javier; Martinez-Losa, Maleles; Sanz, Celia; Almudéver, Patricia; Peiró, Teresa; Serrano, Adela; Morcillo, Esteban Jesus; Zaragozá, Cristóbal; Cortijo, Julio

    2013-09-05

    Eosinophils play a prominent role in the process of allergic inflammation. Non-receptor associated Lyn tyrosine kinases generate key initial signals in eosinophils. Bafetinib, a specific Abl/Lyn tyrosine kinase inhibitor has shown a potent antiproliferative activity in leukemic cells, but its effects on eosinophils have not been reported. Therefore, we studied the effects of bafetinib on functional and mechanistic responses of isolated human eosinophils. Bafetinib was more potent than non-specific tyrosin kinase comparators genistein and tyrphostin inhibiting superoxide anion triggered by N-formyl-Met-Leu-Phe (fMLF; 100 nM) (-log IC50=7.25 ± 0.04 M; 6.1 ± 0.04 M; and 6.55 ± 0.03 M, respectively). Bafetinib, genistein and tyrphostin did not modify the [Ca(2+)]i responses to fMLF. Bafetinib inhibited the release of EPO induced by fMLF with higher potency than genistein and tyrphostin (-log IC50=7.24 ± 0.09 M; 5.36 ± 0.28 M; and 5.37 ± 0.19 M, respectively), and nearly suppressed LTC4, ECP and chemotaxis. Bafetinib, genistein and tyrphostin did not change constitutive apoptosis. However bafetinib inhibited the ability of granulocyte-monocyte colony-stimulating factor to prevent apoptosis. The activation of Lyn tyrosine kinase, p-ERK1/2 and p-38 induced by fMLF was suppressed by bafetinib and attenuated by genistein and tyrphostin. In conclusion, bafetinib inhibits oxidative burst and generation of inflammatory mediators, and reverses the eosinophil survival. Therefore, future anti-allergic therapies based on bafetinib, could help to suppress excessive inflammatory response of eosinophils at inflammatory sites.

  4. Lactoperoxidase inhibits glucosyltransferases from Streptococcus mutans in vitro.

    PubMed

    Korpela, A; Yu, X; Loimaranta, V; Lenander-Lumikari, M; Vacca-Smith, A; Wunder, D; Bowen, W H; Tenovuo, J

    2002-01-01

    This study examines the possible effect of the antimicrobial peroxidase system on the activity of streptococcal glucosyltransferases B, C and D (GtfB, GtfC and GtfD), either in solution (GtfB and GtfC) or when adsorbed to hydroxyapatite (GtfC and GtfD) at pH 6.5. The lactoperoxidase (LP) system (LP, H(2)O(2), SCN(-)) had no effect on the activity of dissolved GtfC, but the activity of dissolved GtfB was enhanced. The LP system, however, strongly inhibited the activities of both GtfC and GtfD in their adsorbed form. LP enzyme, without its substrates, inhibited all three Gtf enzymes: GtfB and GtfC in concentrations between 10 and 100 microg/ml in liquid phase and adsorbed GtfC and GtfD in concentrations between 25 and 50 microg/ml. This inhibition was in part abolished in liquid phase, but not in solid phase, if the substrates of LP were added. This study shows that the lactoperoxidase system can exert inhibitory activity against streptococcal Gtfs without generating oxidizing agents. Copyright 2002 S. Karger AG, Basel

  5. In Vitro and In Vivo Phlebovirus Inhibition by Ribavirin

    DTIC Science & Technology

    1988-03-01

    inhibitori in vitro to Adames and Balliet strains of Punta Tooi virus 411TV). a Phlebot’irltN related to Rift V’alle% fexer and sandfiv lever viruses . By...Panama in 1966. This virus , \\\\ as plaque purified twice as’ Alenquer. (’andirti. and Punta Toro viruses 114). 14. 23. 251. described above, its...cells. reductions of 2.7 log,,, unit,, in the Adames virus strain experiment, a group of 2M0 female mice (age. 3 weeks: yield and 1.0 log... units in the

  6. In vitro inhibition of Eimeria tenella invasion by indigenous chicken Lactobacillus species.

    PubMed

    Tierney, J; Gowing, H; Van Sinderen, D; Flynn, S; Stanley, L; McHardy, N; Hallahan, S; Mulcahy, G

    2004-07-14

    The aim of this study was to determine the effects of indigenous chicken Lactobacillus species isolates from different parts of the gastrointestinal tract on Eimeria tenella invasion in vitro and to characterise the nature of inhibition, if any. The effects of competitive exclusion, steric interference and bacterial extracellular factors on E. tenella invasion were examined in an MDBK cell model. Several Lactobacillus species were initially isolated from chickens and identified by biochemical characteristics and 16S-rRNA. All Lactobacillus species isolates tested, significantly inhibited E. tenella invasion. Steric interference did not affect parasite invasion. Extracellular metabolic factors secreted by Lactobacillus species isolates into the surrounding media were shown to inhibit parasite invasion and these factors appeared to be heat stable. These results show that the natural microflora of poultry can provide a source of E. tenella-inhibiting Lactobacillus species in vitro, and thus may contribute to the control of Eimeria infection.

  7. Ethanol inhibits human bone cell proliferation and function in vitro

    SciTech Connect

    Friday, K.E.; Howard, G.A. )

    1991-06-01

    The direct effects of ethanol on human bone cell proliferation and function were studied in vitro. Normal human osteoblasts from trabecular bone chips were prepared by collagenase digestion. Exposure of these osteoblasts to ethanol in concentrations of 0.05% to 1% for 22 hours induced a dose-dependent reduction in bone cell DNA synthesis as assessed by incorporation of 3H-thymidine. After 72 hours of ethanol exposure in concentrations of 0.01% to 1%, protein synthesis as measured by 3H-proline incorporation into trichbroacetic acid (TCA)-precipitable material was reduced in a dose-dependent manner. Human bone cell protein concentrations and alkaline phosphatase total activity were significantly reduced after exposure to 1% ethanol for 72 hours, but not with lower concentrations of ethanol. This reduction in osteoblast proliferation and activity may partially explain the development of osteopenia in humans consuming excessive amounts of ethanol.

  8. Inhibition of glycation of albumin and hemoglobin by acetylation in vitro and in vivo.

    PubMed

    Rendell, M; Nierenberg, J; Brannan, C; Valentine, J L; Stephen, P M; Dodds, S; Mercer, P; Smith, P K; Walder, J

    1986-10-01

    Aspirin (acetylsalicylic acid or ASA) is known to inhibit glycosylation (glycation) of albumin in vitro. The mechanism has been presumed to be acetylation, but this has never been validated. The new affinity aminophenylboronic acid procedure for determination of glycosylated albumin was used to demonstrate inhibition of glycosylation by aspirin. ASA, but not salicylic acid, inhibited glycation. The inhibition of glycation by equimolar acetic anhydride was greater than that by ASA. Pretreatment of albumin with ASA in the absence of glucose demonstrated that inhibition was extremely rapid, occurring in a matter of minutes. However, the inhibition by ASA could not be prevented by massive acceleration of glycation induced by borohydride reduction. Glycation of hemoglobin was also inhibited by ASA, but the dose requirement was considerably higher. Various analogues of ASA were evaluated for inhibition of glycation. Only acetyl-5-ethylsalicylic acid was more effective than ASA in inhibiting albumin glycation. None of these agents was more potent than ASA in inhibiting glycation of hemoglobin. ASA was fed to diabetic rats in a long-term experiment. Glycohemoglobin and glycoalbumin levels were decreased by ASA administration. We conclude that ASA inhibits glycation by a very rapid acetylation process. This process is apparently quite selective in terms of the protein involved, presumably because of the local environment of affected lysine groups. The phenomenon can be produced in vivo by administration of ASA.

  9. Strontium Promotes Cementoblasts Differentiation through Inhibiting Sclerostin Expression In Vitro

    PubMed Central

    Bao, Xingfu; Liu, Xianjun; Zhang, Yi; Cui, Yue; Yao, Jindan

    2014-01-01

    Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose. PMID:25003114

  10. Strontium promotes cementoblasts differentiation through inhibiting sclerostin expression in vitro.

    PubMed

    Bao, Xingfu; Liu, Xianjun; Zhang, Yi; Cui, Yue; Yao, Jindan; Hu, Min

    2014-01-01

    Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose.

  11. In vitro inhibition of oocyte nuclear maturation in the bovine.

    PubMed

    Sirard, M A; First, N L

    1988-09-01

    Bovine follicular oocytes (N = 5991) were exposed to an analog of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cyclic AMP (db-cAMP) (2.5, 5, and 10 mM), the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 0.2 mM), or the purine, hypoxanthine (0.5, 1.0, 2.0 mM), and the nucleoside, adenosine (0.05, 0.1, 0.2 mM), for 6 or 21 h to assess their effects on oocyte nuclear maturation. Potential effects of bovine follicular fluid (BFF) were also evaluated after different preculture washing procedures. In a separate experiment, denuded oocytes were used to study the effect of cumulus removal on meiotic inhibition. Db-cAMP decreased the frequency of germinal vesicle breakdown (GVBD) at 6 h (88% for control and 51%, 45%, and 32% for 2.5, 5, and 10 mM concentrations, respectively). IBMX had a comparable effect with only 41% of the oocytes resuming meiosis. Hypoxanthine and adenosine alone or in combinations also decreased the number of oocytes undergoing GVBD at 6 h. Only 22% GVBD occurred when the combined highest concentration of both substances was used compared to 88% in controls. If oocytes were incubated in 50% BFF after a wash in control medium during processing, 56% would resume meiosis at 6 h vs. 35% if the washing procedure included inhibitors (db-cAMP + IBMX). Total BFF (100%) during washing and maturation prevented 72% of the oocytes from resuming meiosis. Db-cAMP and IMBX combined or BFF also inhibited meiotic resumption of denuded oocytes. At 21 h, the inhibitory effects were less pronounced, with most oocytes only delayed in completing the first reduction division.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro

    PubMed Central

    1995-01-01

    The amyloid precursor protein (APP) is a transmembrane protein expressed in several cell types. In the nervous system, APP is expressed by glial and neuronal cells, and several lines of evidence suggest that it plays a role in normal and pathological phenomena. To address the question of the actual function of APP in normal developing neurons, we undertook a study aimed at blocking APP expression using antisense oligonucleotides. Oligonucleotide internalization was achieved by linking them to a vector peptide that translocates through biological membranes. This original technique, which is very efficient and gives direct access to the cell cytosol and nucleus, allowed us to work with extracellular oligonucleotide concentrations between 40 and 200 nM. Internalization of antisense oligonucleotides overlapping the origin of translation resulted in a marked but transient decrease in APP neosynthesis that was not observed with the vector peptide alone, or with sense oligonucleotides. Although transient, the decrease in APP neosynthesis was sufficient to provoke a distinct decrease in axon and dendrite outgrowth by embryonic cortical neurons developing in vitro. The latter decrease was not accompanied by changes in the spreading of the cell bodies. A single exposure to coupled antisense oligonucleotides at the onset of the culture was sufficient to produce significant morphological effects 6, 18, and 24 h later, but by 42 h, there were no remaining significant morphologic changes. This report thus demonstrates that amyloid precursor protein plays an important function in the morphological differentiation of cortical neurons in primary culture. PMID:7876315

  13. Growth hormone inhibits apoptosis in in vitro produced bovine embryos.

    PubMed

    Kölle, Sabine; Stojkovic, Miodrag; Boie, Gudrun; Wolf, Eckhard; Sinowatz, Fred

    2002-02-01

    Growth hormone (GH) has recently been shown to exert distinct effects on the differentiation and metabolism of early embryos. Up to now, however, it is not clear whether GH is able to modulate apoptosis during early embryogenesis. Differential cell staining of 8-day-old bovine embryos cultured with 100 ng bovine recombinant GH (rbGH) per ml medium (synthetic oviduct fluid-polyvinylalcohol) demonstrated that GH significantly increased the number of inner cell mass (ICM) and trophectoderm cells in bovine expanded blastocysts. As shown by terminal deoxynucleotidyl transferase mediated dUTP labeling (TUNEL) supplementation of bGH decreased the percentage of 8-day-old embryos showing at least one apoptotic cell from 58 to 21%. The percentage of apoptotic cells in one blastocyst was significantly (P < 0.01) reduced from 4.6 to 1.1% by GH treatment. Incubation of the embryos with 150 mM vanillylnonanamide induced apoptosis in all embryos. Whereas in control embryos 14% of the embryonic cells were TUNEL-positive, the percentage of apoptotic cells declined to 2.7% in the GH treated embryos. Expression of immunoreactive bcl-2 in blastocysts was not affected by GH treatment. Synthesis of the bax protein which is known to promote apoptosis was reduced in embryos cultured with GH. Our results suggest that GH acts as survival factor during in vitro culture and reduces apoptosis by altering the bax to bcl-2 ratio during early embryogenesis.

  14. Pseudorabies virus infection inhibits autophagy in permissive cells in vitro

    PubMed Central

    Sun, Mingxia; Hou, Linlin; Tang, Yan-dong; Liu, Yonggang; Wang, Shujie; Wang, Jingfei; Shen, Nan; An, Tongqing; Tian, Zhijun; Cai, Xuehui

    2017-01-01

    A large number of studies have demonstrated that autophagy is involved in the infection processes of different pathogens. Autophagy is now recognized as an essential component of innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral mechanism. Pseudorabies virus (PRV) is a swine herpesvirus with a broad host range that causes devastating disease in infected pigs. In this study, we described the interaction between PRV and autophagy for the first time. PRV infection had a dual effect on the cell autophagy response; during the early period of infection, PRV virions induced autophagy without viral replication, and with viral protein expression, PRV reduced the basal level of autophagy in several permissive cells. We observed that inhibit the level of autophagy could increase the titer of infectious PRV. We also found that the conserved alphaherpesvirus US3 tegument protein may reduce the level of autophagy via activation of the AKT/mTOR pathways in PRV infected cells. These findings suggest that autophagy likely contributes to clearance of PRV, and that the virus has evolved strategies to antagonize this pathway. PMID:28059118

  15. Ionene polymers for selectively inhibiting the vitro growth of malignant cells

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor)

    1977-01-01

    Ionene polymers of the structure ##STR1## WHERE X AND Y ARE INTEGERS FROM 3 TO 16, Z.sup.- is an anion such as a halogen and n is an integer from 50 to 150 are found to bind negatively charged mammalian cells such as malignant cells and can be utilized to selectively inhibit the growth of malignant cells in vitro.

  16. IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN ADULT AND NEONATAL RAT TESTIS

    EPA Science Inventory

    IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN THE ADULT AND NEONATAL TESTIS
    Chad R. Blystone1, 2, David J. Dix2, and John C. Rockett2
    1Department of Environmental and Molecular Toxicology, Box 7633, NC State University, Raleigh, NC 27695, USA and 2U.S. Envi...

  17. ’In VitroInhibition of Microsomal Calcium Atpase from Eggshell Gland of Mallard Duck.

    DTIC Science & Technology

    1976-12-01

    shell gland homogenate. Previous studies have shown DDT-induced eggshell thinning to be due to decreased calcium content of eggshells . Since calcium...ATPase has been shown to be associated with calcium transport in eggshell gland, the inhibition of this enzyme in vitro offers a possible explanation for DDT-induced eggshell thinning.

  18. Disulfiram inhibits the in vitro growth of methicillin-resistant staphylococcus aureus.

    PubMed Central

    Phillips, M; Malloy, G; Nedunchezian, D; Lukrec, A; Howard, R G

    1991-01-01

    Several antibiotics have disulfiram-like effects; we evaluated disulfiram for its antibiotic-like effects. Disulfiram inhibited the in vitro growth of methicillin-resistant Staphylococcus aureus, with an MIC of 1.33 micrograms/ml, but was not effective against members of the family Enterobacteriaceae or Pseudomonas species. PMID:2069390

  19. IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN ADULT AND NEONATAL RAT TESTIS

    EPA Science Inventory

    IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN THE ADULT AND NEONATAL TESTIS
    Chad R. Blystone1, 2, David J. Dix2, and John C. Rockett2
    1Department of Environmental and Molecular Toxicology, Box 7633, NC State University, Raleigh, NC 27695, USA and 2U.S. Envi...

  20. Roscovitine inhibits extrusion of second polar body and induces apoptosis in rat eggs cultured in vitro.

    PubMed

    Tripathi, Anima; Chaube, Shail K

    2015-10-01

    Inhibition of cyclin-dependent kinases (Cdks) may result in meiotic cell cycle arrest and apoptosis in rat eggs in vitro. We aimed to find out whether roscovitine, a Cdk inhibitor, inhibits extrusion of second polar body (II PB) and induced egg apoptosis in vitro. The metaphase-II (M-II) arrested eggs were collected from oviduct and exposed to various concentrations of roscovitine for 3h in vitro. The morphological changes, phosphorylation status of Cdk1, cyclin B1 level, hydrogen peroxide (H2O2), p53, Bax, Bcl2 and cytochrome c expressions, caspase-3 activity and DNA fragmentation were analyzed. We showed that the lower concentrations of roscovitine significantly reduced Thr-161 phosphorylated Cdk1 level and inhibited extrusion of II PB. The higher concentrations of roscovitine significantly reduced Thr-161 phosphorylated Cdk1 level but total Cdk as well as cyclin B1 levels remained high. Higher concentrations of roscovitine increased H2O2 level and expressions of p53, Bax and cytochrome c in treated eggs. The increased proapoptotic factors induced capsase-3 activity and thereby DNA fragmentation that finally resulted in cytoplasmic fragmentation, a morphological apoptotic feature. Our data suggest that roscovitine inhibited II PB extrusion possibly by reducing Thr-161 phosphorylated Cdk1 level and induced apoptosis through mitochondria-caspase-mediated apoptotic pathway in rat eggs cultured in vitro. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  1. Cinnamon extract inhibits tau aggregation associated with Alzheimer’s Disease in vitro

    USDA-ARS?s Scientific Manuscript database

    An aqueous extract of Ceylon cinnamon (C. zeylanicum) was found to inhibit tau aggregation and filament formation, hallmarks of Alzheimer’s disease (AD) in vitro using brain cells taken from patients who died with AD. The extract also promoted complete disassembly of recombinant tau filaments, and ...

  2. Arsenic(III) species inhibit oxidative protein folding in vitro.

    PubMed

    Ramadan, Danny; Rancy, Pumtiwitt C; Nagarkar, Radhika P; Schneider, Joel P; Thorpe, Colin

    2009-01-20

    The success of arsenic trioxide in the treatment of acute promyelocytic leukemia has renewed interest in the cellular targets of As(III) species. The effects of arsenicals are usually attributed to their ability to bind vicinal thiols or thiol selenols in prefolded proteins thereby compromising cellular function. The present studies suggest an additional, more pleiotropic, contribution to the biological effects of arsenicals. As(III) species, by avid coordination to the cysteine residues of unfolded reduced proteins, can compromise protein folding pathways. Three representative As(III) compounds (arsenite, monomethylarsenous acid (MMA), and an aryl arsenical (PSAO)) have been tested with three reduced secreted proteins (lysozyme, ribonuclease A, and riboflavin binding protein (RfBP)). Using absorbance, fluorescence, and pre-steady-state methods, we show that arsenicals bind tightly to low micromolar concentrations of these unfolded proteins with stoichiometries of 1 As(III) per 2 thiols for MMA and PSAO and 1 As(III) for every 3 thiols with arsenite. Arsenicals, at 10 microM, strongly disrupt the oxidative folding of RfBP even in the presence of 5 mM reduced glutathione, a competing ligand for As(III) species. MMA catalyzes the formation of amyloid-like monodisperse fibrils using reduced RNase. These in vitro data show that As(III) species can slow, or even derail, protein folding pathways. In vivo, the propensity of As(III) species to bind to unfolded cysteine-containing proteins may contribute to oxidative and protein folding stresses that are prominent features of the cellular response to arsenic exposure.

  3. In vitro inhibition of CYP3A4 by herbal remedies frequently used by cancer patients.

    PubMed

    Engdal, Silje; Nilsen, Odd Georg

    2009-07-01

    The herbal remedies Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic, frequently used by cancer patients, were investigated for their in vitro inhibition potential of cytochrome P-450 3A4 (CYP3A4) metabolism. To our knowledge, only garlic and green tea had available data on the possible inhibition of CYP3A4 metabolism. Metabolic studies were performed with human c-DNA baculovirus expressed CYP3A4. Testosterone was used as a substrate and ketoconazole as a positive quantitative inhibition control. The formation of 6-beta-OH-testosterone was quantified by a validated HPLC methodology. Green tea was the most potent inhibitor of CYP3A4 metabolism (IC(50): 73 microg/mL), followed by Agaricus, mistletoe and noni juice (1324, 3594, >10 000 microg/mL, respectively). All IC(50) values were high compared with those determined for crude extracts of other herbal remedies. The IC(50)/IC(25) ratios for the inhibiting herbal remedies ranged from 2.15 to 2.67, indicating similar inhibition profiles of the herbal inhibitors of CYP3A4. Garlic and Natto K2 were classified as non-inhibitors. Although Agaricus, noni juice, mistletoe and green tea inhibited CYP3A4 metabolism in vitro, clinically relevant systemic or intestinal interactions with CYP3A4 were considered unlikely, except for a probable inhibition of intestinal CYP3A4 by the green tea product. Copyright 2009 John Wiley & Sons, Ltd.

  4. Quiescin-sulfhydryl oxidase inhibits prion formation in vitro

    PubMed Central

    Yuan, Jue; Zeng, Liang; Dang, Johnny; Camacho Martinez, Manuel; Wang, Zerui; Mikol, Jacqueline; Lehmann, Sylvain; Bu, Shizhong; Steyaert, Jan; Cui, Li; Petersen, Robert B.; Kong, Qingzhong; Wang, Gong-Xiang; Wohlkonig, Alexandre; Zou, Wen-Quan

    2016-01-01

    Prions are infectious proteins that cause a group of fatal transmissible diseases in animals and humans. The scrapie isoform (PrPSc) of the cellular prion protein (PrPC) is the only known component of the prion. Several lines of evidence have suggested that the formation and molecular features of PrPSc are associated with an abnormal unfolding/refolding process. Quiescin-sulfhydryl oxidase (QSOX) plays a role in protein folding by introducing disulfides into unfolded reduced proteins. Here we report that QSOX inhibits human prion propagation in protein misfolding cyclic amplification reactions and murine prion propagation in scrapie-infected neuroblastoma cells. Moreover, QSOX preferentially binds PrPSc from prion-infected human or animal brains, but not PrPC from uninfected brains. Surface plasmon resonance of the recombinant mouse PrP (moPrP) demonstrates that the affinity of QSOX for monomer is significantly lower than that for octamer (312 nM vs 1.7 nM). QSOX exhibits much lower affinity for N-terminally truncated moPrP (PrP89-230) than for the full-length moPrP (PrP23-231) (312 nM vs 2 nM), suggesting that the N-terminal region of PrP is critical for the interaction of PrP with QSOX. Our study indicates that QSOX may play a role in prion formation, which may open new therapeutic avenues for treating prion diseases. PMID:27959866

  5. Inhibition of dentin demineralization by fluoride in vitro.

    PubMed

    ten Cate, J M; Damen, J J; Buijs, M J

    1998-01-01

    Compared with the knowledge accumulated on enamel-fluoride interactions, relatively little data is available regarding fluoride effects on dentin. This applies to both laboratory and clinical studies into the efficacy of fluoride schemes for the prevention of root surface caries. This study aimed to determine the effects of fluoride and pH on the demineralization of dentin, such as to provide information necessary to develop preventive programmes. Bovine dentin blocks were subjected to undersaturated calcium- and phosphate-containing solutions in the pH range 4.0-6.0 with fluoride added at concentrations between 0.5 and 10 ppm. Non-fluoride solutions served as controls. Mineral loss was assessed chemically and by transversal microradiography. Comparisons were made with similar studies on enamel demineralization. The results showed that demineralization of dentin depends on both pH and fluoride concentration in the demineralizing solution. Inhibition of demineralization that could be relevant from a clinical point of view was found at fluoride values 5-10 times the corresponding values for enamel. Also rapid depletion of fluoride from the solutions was observed, indicating the high uptake capacity of dentin for fluoride. Lesion depth depended on pH of the solution while the fluoride levels were associated with the surface layer, both in mineral content and depth. For dentin we propose a demineralization mechanism where acid penetrates rapidly into the tissue, presumably through the tubules, after which the released calcium and phosphate is partly trapped by the inward diffusing fluoride. This leads to the formation of a surface layer, which may even be hypermineralized compared to sound dentin.

  6. Inhibition of human low-density lipoprotein oxidation in vitro by ginger extracts.

    PubMed

    Gunathilake, K D Prasanna P; Rupasinghe, H P Vasantha

    2014-04-01

    Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in atherosclerotic plaque formation. Currently, there is a renewed interest in ginger because of its antioxidants and cardioprotective properties. The effects of ethanol, methanol, ethyl acetate, and hexane solvent extracts of ginger and pure major ginger constituents on Cu(2+)-induced oxidation of human LDL in vitro were examined. The LDL oxidation inhibition by ethanol, methanol, ethyl acetate, and hexane extracts of ginger was 71%, 76%, 67%, and 67%, respectively, at their optimum extraction conditions. Inhibition of LDL oxidation by water extracts of ginger, which was prepared by ultrasonic-assisted extraction conditions of 52°C for 15 min, was about 43%. Phenolic bioactives of ginger-6-gingerols, 8-gingerols, 10-gingerols, and 6-shogaol-seem to be strong inhibitors of Cu(+2)-induced LDL oxidation. Overall, ginger extracts, including the water extract possess the antioxidant activities to inhibit human LDL oxidation in vitro.

  7. Failure to Inhibit In Vitro or In Vivo Acetycholinesterase with Botulinum Toxin Type A

    PubMed Central

    Simpson, Lance L.; Morimoto, Hiromi

    1969-01-01

    An attempt has been made to replicate an earlier finding that type A botulinum toxin can inhibit the in vitro activity of acetylcholinesterase. Two methods of enzyme assay were employed, but with neither technique were we able to reproduce the finding of in vitro enzyme inhibition. In fact, an examination of the data from the previous report leads us to question the possibility of the observations that were given. Furthermore, an investigation was carried out to determine if botulinum toxin can exert an inhibiting effect on acetylcholinesterase that is situated in the biological tissue. The answer again is negative. The experimental observations, coupled with several mathematical computations, do not support the notion that botulinum toxin is an acetylcholinesterase inhibitor. PMID:5773011

  8. [In vitro inhibition of celastrol on spermatozoa fertilization ability of guinea pig].

    PubMed

    Yuan, Y Y; Gu, Z P; Shi, Q X; Qin, G W; Xu, R S; Cao, L

    1995-01-01

    The effects of celastrol (Cel), isolated from Tripterygium wilfordii, on guinea pig sperm forward motility (FM), capacitation (Cap), the acrosome reaction (AR) and sperm penetration assay (SPA) were assessed in vitro. Cel (5 micrograms.ml-1) was found to inhibit these spermatozoal functions, and the inhibitions were proportional to the concentrations of Cel used. The potency of inhibition of Cel on the fertilizing ability in guinea pig spermatozoa in vitro seems to follow the order: Cap > FM > SPA > AR. The inhibitory effect appeared to be reversible after washing away Cel if the duration of exposure of spermatozoa to Cel was shorter than 3 h. In a comparative study, the inhibitory effects of Cel on guinea pig sperm FM and AR were significantly stronger than those of gossypol acetic acid.

  9. Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

    PubMed Central

    Mahalingam, Sharada; Gao, Liying; Gonnering, Marni; Helferich, William; Flaws, Jodi A.

    2016-01-01

    Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. PMID:26876617

  10. Selective inhibition of human acetylcholinesterase by xanthine derivatives: in vitro inhibition and molecular modeling investigations.

    PubMed

    Mohamed, Tarek; Osman, Wesseem; Tin, Gary; Rao, Praveen P N

    2013-08-01

    The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC₅₀=6.40 μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC₅₀=7.25 μM)inhibition of hAChE with no inhibition of hBuChE (IC₅₀>50 μM) relative to the reference agent donepezil (hBuChE IC₅₀=13.60 μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia.

  11. Synergistic inhibition of glycinergic transmission in vitro and in vivo by flavonoids and strychnine.

    PubMed

    Raafat, Karim; Breitinger, Ulrike; Mahran, Laila; Ayoub, Nahla; Breitinger, Hans-Georg

    2010-11-01

    The inhibitory glycine receptor (GlyR) is a key mediator of synaptic signaling in spinal cord, brain stem, and higher central nervous system regions. The flavonoids quercetin and genistein have been identified previously as promising GlyR antagonists in vitro, but their detailed mechanism of action was not known. Here, inhibition of recombinant human α1 GlyRs in HEK 293 cells by genistein, quercetin, and strychnine was studied using whole-cell recording techniques. The interaction of several inhibitors applied alone or in combination was analyzed using a minimum mechanism of receptor activation and inhibition. Receptor inhibition in vivo was studied in a mouse model of strychnine toxicity. Genistein, quercetin, and strychnine were noncompetitive GlyR inhibitors. The inhibitory potency of one flavonoid (either genistein or quercetin) was not affected by simultaneous application of the other, suggesting that both flavonoids target the same site on the receptor. In combination with strychnine, flavonoid inhibition was augmented, indicating that strychnine binds to a position on the receptor physically distant from the flavonoid site. Potentiation of strychnine inhibition by flavonoids was also observed in vivo, where harmless doses of flavonoids enhanced strychnine toxicity in mice. Thus, in vitro and in vivo studies demonstrated a true synergism between flavonoids and strychnine for GlyR inhibition. The mechanism-based approach used here allows a rapid analysis of the effects of single drugs versus drug combinations.

  12. Inhibition of mycobacterial growth in vitro following primary but not secondary vaccination with Mycobacterium bovis BCG.

    PubMed

    Fletcher, Helen A; Tanner, Rachel; Wallis, Robert S; Meyer, Joel; Manjaly, Zita-Rose; Harris, Stephanie; Satti, Iman; Silver, Richard F; Hoft, Dan; Kampmann, Beate; Walker, K Barry; Dockrell, Hazel M; Fruth, Uli; Barker, Lew; Brennan, Michael J; McShane, Helen

    2013-11-01

    Despite the widespread use of the Mycobacterium bovis BCG vaccine, there are more than 9 million new cases of tuberculosis (TB) every year, and there is an urgent need for better TB vaccines. TB vaccine candidates are selected for evaluation based in part on the detection of an antigen-specific gamma interferon (IFN-γ) response. The measurement of mycobacterial growth in blood specimens obtained from subjects immunized with investigational TB vaccines may be a better in vitro correlate of in vivo vaccine efficacy. We performed a clinical study with 30 United Kingdom adults who were followed for 6 months to evaluate the abilities of both a whole-blood- and a novel peripheral blood mononuclear cell (PBMC)-based mycobacterial growth inhibition assay to measure a response to primary vaccination and revaccination with BCG. Using cryopreserved PBMCs, we observed a significant improvement in mycobacterial growth inhibition following primary vaccination but no improvement in growth inhibition following revaccination with BCG (P < 0.05). Mycobacterial growth inhibition following primary BCG vaccination was not correlated with purified protein derivative (PPD) antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. We demonstrate that a mycobacterial growth inhibition assay can detect improved capacity to control growth following primary immunization, but not revaccination, with BCG. This is the first study to demonstrate that an in vitro growth inhibition assay can identify a difference in vaccine responses by comparing both primary and secondary BCG vaccinations, suggesting that in vitro growth inhibition assays may serve as better surrogates of clinical efficacy than the assays currently used for the assessment of candidate TB vaccines.

  13. Momilactone B Inhibits Ketosis In Vitro by Regulating the ANGPTL3-LPL Pathway and Inhibiting HMGCS2.

    PubMed

    Kang, Dong Young; S P, Nipin; Darvin, Pramod; Joung, Youn Hee; Byun, Hyo Joo; Do, Chang Hee; Park, Kyung Do; Park, Mi Na; Cho, Kwang Hyun; Yang, Young Mok

    2016-11-22

    Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.

  14. Selective inhibition of in vitro synthesis of cancer DNA by alkaloids of beta-carboline class.

    PubMed

    Beljanski, M; Beljanski, M S

    1982-01-01

    The high template in vitro activity of native DNA from cancerous mammalian and plant tissues, compared to DNA from healthy tissues, enabled us to select substances which selectively inhibit cancer DNA synthesis. Among them, alstonine, serpentine, sempervirine and flavopereirine, all alkaloids which belong to the Beta-carboline class, distinguish cancer DNA from healthy tissue DNA inhibit DNA in vitro synthesis when native DNA from different cancerous tissues or cells is used as template. They have practically no effect on DNA from healthy tissues. The inhibitory effect of alkaloids is due to their capacity to form an 'alkaloid-cancer DNA' complex which has been characterized by use of the Sephadex column. Evidence is presented showing that these alkaloids inhibit the initiation of DNA synthesis but not chain elongation. The stimulating action caused by carcinogens during cancer DNA in vitro synthesis may be prevented and reversed by alkaloids. Furthermore, the stimulating action of steroids during in vitro synthesis of hormone target tissue DNA might be neutralized by alkaloids. However, at relatively high doses, steroids reversibly compete with alkaloids for binding sites on breast cancer DNA. This is not observed with DNA from nonhormone target tissues.

  15. Green tea extract and aged garlic extract inhibit anion transport and sickle cell dehydration in vitro.

    PubMed

    Ohnishi, S T; Ohnishi, T; Ogunmola, G B

    2001-01-01

    Both green tea extract (GTE or tea polyphenols) and aged garlic extract (AGE) effectively inhibited in vitro dehydration of sickle red blood cells induced by K-Cl cotransport or red cell storage. For K-Cl cotransport induced by 500 mM urea, 0.3 mg/ml EGCg (epigallocatechin gallate; a major component in GTE) almost completely inhibited dehydration, and 6 mg/ml AGE inhibited dehydration to 30% of the control level. Both vitamins E and C had no effect at the level of 2 mM. Different tea extracts had different degrees of inhibition, but the inhibitory activity increased when the number of hydroxyl groups in the compounds increased. With storage of sickle cells at 4 degrees C for 6 days, the cells started to undergo spontaneous dehydration when incubated at 37 degrees C. Neither inhibitors for Ca-induced K efflux nor K-Cl cotransport could inhibit cell dehydration of stored sickle cells, but both GTE and AGE effectively inhibited it. Chloride efflux measurements using a chloride electrode demonstrated that both GTE and AGE inhibited anion transport in red blood cells. The inhibitory mechanism of these compounds may be related to anion transport inhibition, although involvement of their antioxidant activities can not yet be ruled out. Copyright 2001 Academic Press.

  16. A practical in vitro growth inhibition assay for the evaluation of TB vaccines.

    PubMed

    Kolibab, Kristopher; Parra, Marcela; Yang, Amy L; Perera, Liyanage P; Derrick, Steven C; Morris, Sheldon L

    2009-12-11

    New vaccines and novel immunization strategies are needed to improve the control of the global tuberculosis epidemic. To facilitate vaccine development, we have been creating in vitro mycobacterial intra-macrophage growth inhibition assays. Here we describe the development of an in vitro assay designed for BSL-2 laboratories which measures the capacity of vaccine-induced immune splenocytes to control the growth of isoniazid-resistant Mycobacterium bovis BCG (INH(r) BCG). The use of the INH(r) BCG as the infecting organism allows the discrimination of BCG bacilli used in murine vaccinations from BCG used in the in vitro assay. In this study, we showed that protective immune responses evoked by four different types of Mycobacterium tuberculosis vaccines [BCG, an ESAT6/Antigen 85B fusion protein formulated in DDA/MPL adjuvant, a DNA vaccine expressing the same fusion protein, and a TB Modified Vaccinia Ankara construct expressing four TB antigens (MVA-4TB)] were detected. Importantly, the levels of vaccine-induced protective immunity seen in the in vitro assay correlated with the results from in vivo protection studies in the mouse model of pulmonary tuberculosis. Furthermore, the growth inhibition data for the INH(r) BCG assay was similar to the previously reported results for a M. tuberculosis infection assay. The cytokine expression profiles at day 7 of the INH(r) BCG growth inhibition studies were also similar but not identical to the cytokine patterns detected in earlier M. tuberculosis co-culture assays. Overall, we have shown that a BSL-2 compatible in vitro growth inhibition assay using INH(r) BCG as the intra-macrophage target organism should be useful in developing and evaluating new TB immunization strategies.

  17. Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

    PubMed

    Baureithel, K H; Büter, K B; Engesser, A; Burkard, W; Schaffner, W

    1997-06-01

    Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

  18. In vitro inhibition of Helicobacter pylori growth and adherence to gastric mucosal cells by Pycnogenol.

    PubMed

    Rohdewald, Peter; Beil, Winfried

    2008-05-01

    The emergence of antibiotic resistant H. pylori strains has necessitated the identification of alternative additive therapies for the treatment of this infection. The study tested whether a specific pine bark extract (Pycnogenol is effective in inhibiting the growth and adherence of H. pylori in vitro. Inhibition of H. pylori growth by Pycnogenol was tested in liquid medium as well as in an in vitro model by using sessile bacteria attached to AGS cells. Adherence was determined by co-incubation of gastric cells with Pycnogenol and H. pylori in vitro. Pycnogenol inhibited H. pylori growth in suspension with an MIC(50) of 12.5 microg/mL. Growth of H. pylori in infected cells was reduced to 10% of the control value by 125 microg/mL Pycnogenol. Adherence of H. pylori to gastric cells was reduced by 70% after 3 h incubation with 125 microg/mL Pycnogenol. The results show a significant, yet limited inhibition of growth and adherence of H. pylori to gastric cells by Pycnogenol. In vivo studies have to demonstrate the clinical relevance of these findings.

  19. Mechanism of inhibition defines CETP activity: a mathematical model for CETP in vitro.

    PubMed

    Potter, Laura K; Sprecher, Dennis L; Walker, Max C; Tobin, Frank L

    2009-11-01

    Because cholesteryl ester transfer protein (CETP) inhibition is a potential HDL-raising therapy, interest has been raised in the mechanisms and consequences of CETP activity. To explore these mechanisms and the dynamics of CETP in vitro, a mechanistic mathematical model was developed based upon the shuttle mechanism for lipid transfer. Model parameters were estimated from eight published experimental datasets, and the resulting model captures observed dynamics of CETP in vitro. Simulations suggest the shuttle mechanism yields behaviors consistent with experimental observations. Three key findings predicted from model simulations are: 1) net CE transfer activity from HDL to VLDL and LDL can be significantly altered by changing the balance of homoexchange versus heteroexchange of neutral lipids via CETP; 2) lipemia-induced increases in CETP activity are more likely caused by increases in lipoprotein particle size than particle number; and 3) the inhibition mechanisms of the CETP inhibitors torcetrapib and JTT-705 are significantly more potent than a classic competitive inhibition mechanism with the irreversible binding mechanism having the most robust response. In summary, the model provides a plausible representation of CETP activity in vitro, corroborates strong evidence for the shuttle hypothesis, and provides new insights into the consequences of CETP activity and inhibition on lipoproteins.

  20. ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expression in vitro

    PubMed Central

    Jia, Xu-feng; Ye, Fei; Wang, Yan-bo; Feng, Da-xiong

    2016-01-01

    Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway. PMID:27482229

  1. Inhibition of in Vitro Pollen Tube Growth by Isolated S-Glycoproteins of Nicotiana alata.

    PubMed Central

    Jahnen, W.; Lush, W. M.; Clarke, A. E.

    1989-01-01

    Pollen from three S-genotypes of Nicotiana alata was grown in vitro in the presence of S-glycoproteins isolated from styles of the same three genotypes. Pollen germination was not affected by the presence of the S-glycoproteins, but pollen tube growth of all genotypes was inhibited. S2 pollen was preferentially inhibited by the S2-glycoprotein and S3 pollen by the S3-glycoprotein. The S6-glycoprotein preferentially inhibited growth of both S2 and S6 pollen over S3 pollen. Heat treatment dramatically increased the inhibitory activity of the S-glycoproteins as inhibitors both of pollen germination and tube growth; after heat treatment, S-allele specificity of pollen tube inhibition was not detected. PMID:12359898

  2. Molecular basis of alpha-tocopherol inhibition of smooth muscle cell proliferation in vitro.

    PubMed

    Boscoboinik, D; Chatelain, E; Bartoli, G M; Azzi, A

    1992-01-01

    The molecular events responsible for the inhibition of cell proliferation by alpha-tocopherol have been investigated. Smooth muscle cells in vitro have been shown to be specifically inhibited by alpha-tocopherol with a concomitant inhibition of protein kinase C activity. beta-Tocopherol was inactive, despite its similar radical scavenging activity. The point of inhibition of alpha-tocopherol relative to the cell cycle was localized in the late G1 phase. A second effect of alpha-tocopherol observed with smooth muscle cells was the stimulation of protein kinase C biosynthesis in both the S and G2 phases of the cell cycle. The implications of these findings for the onset of arteriosclerosis are discussed.

  3. Vitamin E inhibits proliferation of human Tenon's capsule fibroblasts in vitro.

    PubMed

    Haas, A L; Boscoboinik, D; Mojon, D S; Böhnke, M; Azzi, A

    1996-01-01

    Failure of glaucoma surgery is mostly due to fibrocellular scar formation, derived from Tenon's capsule fibroblasts. In high-risk cases, postoperative Tenon's capsule fibroblast proliferation is inhibited by mitomycin C or 5-fluorouracil. Toxicity to other ocular cell types and the risk of ocular hypotony limits the use of these agents. We have found that d-alpha-tocopherol (vitamin E) was able to inhibit proliferation of in vitro human Tenon's capsule fibroblasts obtained from seven different donors. At 48 h, inhibition of cell proliferation was 30-78% (mean 60%) for 50 microM d-alpha-tocopherol and 46-97% (mean 77%) for 100 microM d-alpha-tocopherol. This inhibition was statistically significant. No cytotoxic effects were observed.

  4. The inhibition of DNA synthesis in chronic lymphocytic leukaemia cells by chlorambucil in vitro.

    PubMed Central

    Bentley, D. P.; Blackmore, J. A.

    1992-01-01

    The inhibition of 3H-thymidine incorporation into the DNA of mitogen-stimulated lymphocytes from patients with chronic lymphocytic leukaemia by chlorambucil was measured in vitro and the results related to clinical drug resistance. The assay proved to be both sensitive and specific showing a clear separation of those patients with responsive disease from those with disease resistant to treatment. There was evidence of primary drug resistance in untreated patients. In almost all patients who received treatment this led to increasing resistance to chlorambucil in vitro. The assay is predictive of clinical responsiveness and provides a potential means whereby new therapeutic agents and treatment modifiers may be investigated. PMID:1739612

  5. In vitro inhibition of human papillomavirus following use of a carrageenan-containing vaginal gel.

    PubMed

    Novetsky, Akiva P; Keller, Marla J; Gradissimo, Ana; Chen, Zigui; Morgan, Stephanie L; Xue, Xiaonan; Strickler, Howard D; Fernández-Romero, José A; Burk, Robert; Einstein, Mark H

    2016-11-01

    To assess in vitro efficacy of Divine 9, a carrageenan-based vaginal lubricant that is being studied as a microbicide to inhibit HPV16 pseudovirus (PsV) infection. Sexually active US women between 19 and 35years without prior HPV vaccination or cervical intraepithelial neoplasia were instructed to use Divine 9 vaginally with an applicator either before sex only or before and after intercourse. Women who applied a single dose of gel returned for cervicovaginal lavage (CVL) collection 1, 4 or 8-12h after intercourse versus those who applied gel before and after intercourse returned 1, 4 or 8-12h after the second gel dose. Carrageenan concentrations were assessed using an ELISA assay and the inhibitory activity was assessed using a PsV-based neutralization assay against HPV16 infection. Carrageenan concentrations and the percentage of PsV16 inhibition were compared using the Wilcoxon rank sum test. Thirteen women were enrolled and thirty specimens from different time-points were assessed. 87% of CVL samples had detectable carrageenans with levels decreasing over time from intercourse. 93% of CVL samples had detectable PsV16 inhibition with median inhibition of 97.5%. PsV16 inhibition decreased over time, but remained high, with median inhibition of 98.1%, 97.4% and 83.4% at 1, 4 and 8-12h, respectively. Higher carrageenan concentrations were associated with higher levels of PsV16 inhibition (rho=0.69). This is the first report of a human study investigating in vitro HPV inhibition of a carrageenan-based vaginal lubricant with CVL collected after sexual intercourse. We demonstrate excellent efficacy in preventing PsV16 infection. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Glucosamine prevents in vitro collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation

    PubMed Central

    Tiku, Moti L; Narla, Haritha; Jain, Mohit; Yalamanchili, Praveen

    2007-01-01

    Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix

  7. The Effect of Cyclooxygenase Inhibition on Tendon-Bone Healing in an In Vitro Coculture Model

    PubMed Central

    Schwarting, Tim; Pretzsch, Sebastian; Debus, Florian; Ruchholtz, Steffen; Lechler, Philipp

    2015-01-01

    The effects of cyclooxygenase (COX) inhibition following the reconstruction of the anterior cruciate ligament remain unclear. We examined the effects of selective COX-2 and nonselective COX inhibition on bone-tendon integration in an in vitro model. We measured the dose-dependent effects of ibuprofen and parecoxib on the viability of lipopolysaccharide- (LPS-) stimulated and unstimulated mouse MC3T3-E1 and 3T3 cells, the influence on gene expression at the osteoblast, interface, and fibroblast regions measured by quantitative PCR, and cellular outgrowth assessed on histological sections. Ibuprofen led to a dose-dependent suppression of MC3T3 cell viability, while parecoxib reduced the viability of 3T3 cultures. Exposure to ibuprofen significantly suppressed expression of Alpl (P < 0.01), Bglap (P < 0.001), and Runx2 (P < 0.01), and although parecoxib reduced expression of Alpl (P < 0.001), Fmod (P < 0.001), and Runx2 (P < 0.01), the expression of Bglap was increased (P < 0.01). Microscopic analysis showed a reduction in cellular outgrowth in LPS-stimulated cultures following exposure to ibuprofen and parecoxib. Nonselective COX inhibition and the specific inhibition of COX-2 led to region-specific reductions in markers of calcification and cell viability. We suggest further in vitro and in vivo studies examining the biologic and biomechanical effects of selective and nonselective COX inhibition. PMID:26063979

  8. Phloroglucinol Inhibits the in vitro Differentiation Potential of CD34 Positive Cells into Endothelial Progenitor Cells

    PubMed Central

    Kwon, Yi-Hong; Lee, Jun-Hee; Jung, Seok-Yun; Kim, Jae-Won; Lee, Sang-Hun; Lee, Dong-Hyung; Lee, Kyu-Sup; Lee, Boo-Yong; Kwon, Sang-Mo

    2012-01-01

    Inhibiting the bioactivities of circulating endothelial progenitor cells (EPCs) results in significant inhibition of neovessel formation during tumor angiogenesis. To investigate the potential effect of phloroglucinol as an EPC inhibitor, we performed several in vitro functional assays using CD34+ cells isolated from human umbilical cord blood (HUCB). Although a high treatment dose of phloroglucinol did not show any cell toxicity, it specifically induced the cell death of EPCs under serum free conditions through apoptosis. In the EPC colony-forming assay (EPC-CFA), we observed a significant decreased in the small EPC-CFUs for the phloroglucinol group, implying that phloroglucinol inhibited the early stage of EPC commitment. In addition, in the in vitro expansion assay using CD34+ cells, treatment with phloroglucinol was shown to inhibit endothelial lineage commitment, as demonstrated by the decrease in endothelial surface markers of EPCs including CD34+, CD34+/CD133+, CD34+/CD31+ and CD34+/CXCR4+. This is the first report to demonstrate that phloroglucinol can inhibit the functional bioactivities of EPCs, indicating that phloroglucinol may be used as an EPC inhibitor in the development of biosafe anti-tumor drugs that target tumor angiogenesis. PMID:24116289

  9. Apple peel bioactive rich extracts effectively inhibit in vitro human LDL cholesterol oxidation.

    PubMed

    Thilakarathna, Surangi H; Rupasinghe, H P Vasantha; Needs, Paul W

    2013-05-01

    Apple peels are rich in antioxidant bioactives and hence can possess the ability to inhibit human low density lipoprotein cholesterol (LDL-C) oxidation. LDL-C oxidation is known to initiate atherosclerotic plaque formation. Unique quercetin-rich (QAE) and triterpene-rich (TAE) apple peel extracts, their constituent compounds and three in vivo quercetin metabolites were investigated for in vitro LDL-C oxidation inhibition. Both extracts effectively inhibited Cu(2+)-induced LDL-C oxidation. IC(50) of QAE and TAE for LDL-C oxidation products were 0.06-8.29 mg/L and 29.58-95.49 mg/L, respectively. Quercetin compounds, chlorogenic acid and phloridzin could contribute more to the effectiveness of QAE at physiological concentrations. The three in vivo quercetin metabolites; quercetin-3'-sulfate, quercetin-3-glucuronic acid and isorhamnetin-3-glucuronic acid were effective at physiological concentrations and therefore, QAE can be effective in LDL-C oxidation inhibition under physiological conditions. Constituent TAE compounds did not perform well under Cu(2+)-induction. Overall, both extracts effectively inhibited LDL-C oxidation in vitro. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. In vitro inhibition of cytochrome P450 3A4 by Aronia melanocarpa constituents.

    PubMed

    Bräunlich, Marie; Christensen, Hege; Johannesen, Siri; Slimestad, Rune; Wangensteen, Helle; Malterud, Karl E; Barsett, Hilde

    2013-01-01

    Extracts, subfractions, isolated anthocyanins and procyanidins, and two phenolic acids from aronia [Aronia melanocarpa] were investigated for their CYP3A4 inhibitory effects, using midazolam as the probe substrate and recombinant insect cell microsomes expressing CYP3A4 as the enzyme source. Procyanidin B5 was a considerably stronger CYP3A4 inhibitor in vitro than the isomeric procyanidin B2 and comparable to bergamottin, a known CYP3A4 inhibitor from grapefruit juice. The inhibitory activity of proanthocyanidin-containing fractions was correlated to the degree of polymerization. Among the anthocyanins, cyanidin 3-arabinoside showed stronger CYP3A4 inhibition than cyanidin 3-galactoside and cyanidin 3-glucoside. Thus, the ability to inhibit CYP3A4 in vitro seems to be influenced by the sugar unit linked to the anthocyanidin. Georg Thieme Verlag KG Stuttgart · New York.

  11. Ets2 knockdown inhibits tumorigenesis in esophageal squamous cell carcinoma in vivo and in vitro

    PubMed Central

    Zhu, Liqiang; Zhang, Yanting; Ma, Shanshan; Zhang, Kun; Yang, Bo; Guan, Fangxia

    2016-01-01

    Increased expression of Ets2 is reported upregulated in esophageal squamous cell carcinoma tissue. However, the function of Ets2 in carcinogenesis of ESCC is poorly understood. Here, the rise of Ets2 was confirmed in ESCC cells and Ets2 depletion by RNA interference promotes cell apoptosis, inhibits cell proliferation, attenuates cell invasion and induces cell cycle G0/G1 arrest in vitro. Moreover, in vivo, Xenograft mouse model studies showed Ets2 knockdown inhibits tumor formation and metastasis significantly. Furthermore, Ets2 depletion inactivates the mTOR/p70S6K signaling pathway both in vitro and in vivo. Taken together, these findings strongly suggest that a critical role of Ets2 in human ESCC pathogenesis via the inactivation of the mTOR/p70S6K signaling pathway. PMID:27556183

  12. In Vitro Inhibition of Plasmodium falciparum Rosette Formation by Curdlan Sulfate▿

    PubMed Central

    Kyriacou, Helen M.; Steen, Katie E.; Raza, Ahmed; Arman, Monica; Warimwe, George; Bull, Peter C.; Havlik, Ivan; Rowe, J. Alexandra

    2007-01-01

    Spontaneous binding of infected erythrocytes to uninfected erythrocytes to form rosettes is a property of some strains of Plasmodium falciparum that is linked to severe complications of malaria. Curdlan sulfate (CRDS) is a sulfated glycoconjugate compound that is chemically similar to known rosette-inhibiting drugs such as heparin. CRDS has previously been shown to have antimalarial activity in vitro and is safe for clinical use. Here we show that CRDS at therapeutic levels (10 to 100 μg/ml) significantly reduces rosette formation in vitro in seven P. falciparum laboratory strains and in a group of 18 African clinical isolates. The strong ability to inhibit rosetting suggests that CRDS has the potential to reduce the severe complications and mortality rates from P. falciparum malaria among African children. Our data support further clinical trials of CRDS. PMID:17283200

  13. Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro

    PubMed Central

    Sipola, Annina; Nelo, Katri; Hautala, Timo; Ilvesaro, Joanna; Tuukkanen, Juha

    2006-01-01

    Background Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. Methods Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits. Results Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. Conclusion Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption. PMID:16839420

  14. Serum factors from infected baboons inhibit oviposition and cause unpairing of Schistosoma mansoni in vitro.

    PubMed

    Bosshardt, S C; Damian, R T

    1986-08-01

    A reliable in vitro fecundity assay for Schistosoma mansoni was established. The main features that reduced variability in in vitro oviposition were pre-selection and randomization of worm pairs producing moderate numbers of eggs in initial 2-day culture, and short pre-incubation in serumless medium prior to addition of test sera to the cultures. In 4 of 6 total experiments testing the effects of serum from chronically infected baboons, significant (P less than or equal to 0.025) fecundity reduction ranging from 29 to 82% was found. Chronically infected baboon serum also caused consistently higher unpairing than normal serum. These results demonstrate the existence of serum factors which inhibit egg production and maintenance of the paired status of Schistosoma mansoni in vitro.

  15. Salidroside inhibits the growth of human breast cancer in vitro and in vivo.

    PubMed

    Zhao, Gang; Shi, Aiping; Fan, Zhimin; Du, Ye

    2015-05-01

    Salidroside has been identified as one of the most potent compounds isolated from the plant Rhodiola rosea, and was found to have several important biological properties, including antioxidant and anti-inflammatory activity; however, its anticancer effects are poorly understood. Thus, the present study focused on evaluating the effects of purified salidroside on the growth of human breast cancer in vitro and in vivo, and on further investigating its possible molecular mechanisms. The human breast cancer cell line, MCF-7, was incubated with various concentrations of salidroside, and cell proliferation, colony formation, cell cycle distribution, apoptosis, migration and invasion were assayed by several in vitro approaches. As a result, it was found that salidroside treatment significantly inhibited cell proliferation, colony formation, migration and invasion, as well as induced cell apoptosis and cell cycle arrest at the G0/G1 phase in vitro. In addition, we also evaluated the effect of salidroside on tumor growth in a nude mouse model, and found that salidroside treatment significantly suppressed tumor growth in vivo. We also further disclosed that salidroside treatment significantly inhibited the intracellular reactive oxygen species (ROS) formation and MAPK pathway activation, which may contribute to the inhibition of tumor growth of breast cancer and reduction of oxidative stress. In conclusion, these findings suggest that salidroside may be a promising candidate target for the prevention and treatment of human breast cancer.

  16. Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats.

    PubMed

    Gadi, Dounia; Bnouham, Mohamed; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Legrand, Chantal; Lafeve, Françoise Fauvel; Mekhfi, Hassane

    2009-08-17

    Many cardiovascular diseases are associated with an increase in blood platelet activity. In Morocco, parsley (Petroselinum crispum, Apiaceae) is one of the medicinal herbs used to treat cardiovascular diseases such as arterial hypertension. In this study, crude aqueous extract (CAE) of parsley was evaluated for its anti-platelet activity in experimental animals on platelet aggregation in vitro and ex vivo; and on bleeding time in vivo. The in vitro aggregation was monitored after pre-incubation of platelets with CAE. The bleeding time and ex vivo aggregation were performed after oral treatment. CAE inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine. The oral administration of CAE (3g/kg) inhibited significantly (p<0.001) platelet aggregation ex vivo and prolonged bleeding time (p<0.001) without changes in the platelet amount. The prolongation of bleeding time by CAE may be attributed to the observed inhibition of platelet aggregation. These effects could be related in part to the polyphenolic compounds present in the extract. These results support the hypothesis that the dietary intake of parsley may be benefit in the normalization of platelet hyperactivation, in the nutritional prevention of cardiovascular diseases and are potentially interesting in the development of new prevention strategies.

  17. IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN THE ADULT AND NEONATAL RAT TESTIS THROUGH THE INHIBITION OF CYP17 ACTIVITY

    EPA Science Inventory

    IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN THE ADULT AND NEONATAL RAT TESTIS THROUGH THE INHIBITION OF CYP17 ACTIVITY

    Chad R. Blystone1, David J. Dix2, and John C. Rockett2
    1Department of Environmental and Molecular Toxicology, NC State University, R...

  18. IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN THE ADULT AND NEONATAL RAT TESTIS THROUGH THE INHIBITION OF CYP17 ACTIVITY

    EPA Science Inventory

    IN VITRO CONAZOLE EXPOSURE INHIBITS TESTOSTERONE PRODUCTION IN THE ADULT AND NEONATAL RAT TESTIS THROUGH THE INHIBITION OF CYP17 ACTIVITY

    Chad R. Blystone1, David J. Dix2, and John C. Rockett2
    1Department of Environmental and Molecular Toxicology, NC State University, R...

  19. PAC-1 activates procaspase-3 in vitro through relief of zinc-mediated inhibition.

    PubMed

    Peterson, Quinn P; Goode, David R; West, Diana C; Ramsey, Kara N; Lee, Joy J Y; Hergenrother, Paul J

    2009-04-24

    The direct induction of apoptosis has emerged as a powerful anticancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key "executioner" caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procaspase-3 and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small-molecule-mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds.

  20. [Perspectives of inhibition of multidrug resistance during cancer chemotherapy, in vitro and in vivo experiments].

    PubMed

    Engi, Helga

    2009-03-29

    The development of pharmacological agents able to counteract the mechanisms of multidrug resistance in oncology has remained a major goal for the past ten years. Our purpose was to find multidrug resistance reversal agents less toxic than verapamil among various synthetic compounds: cinnamylidene ketones; 1,4-dihydropyridines; phenothiazines; heat shock 90 inhibitor peptides; betti base derivative of tylosin and among some naturally occurring plant derived jatrophane and lathyrane-type diterpenes. The first part of this thesis presents the inhibition of multidrug resistance through inhibition of the P-glycoprotein efflux pump in various cell lines. In general, the newly identified multidrug resistance modifiers were able to enhance the antiproliferative activity of selected anticancer drugs in a synergistic or additive way in in vitro experiments. The in vitro activity of betti base derivative of tylosin was confirmed by further in vivo efficacy studies in DBA/2 mice. As an alternative way of antitumor effect, apoptosis inductions of resistance modifiers were studied. The substituted dihydropyridine 13 was the most promising apoptosis inducer on mouse lymphoma cells. Human cytomegalovirus was used in a modified in vitro model for characterizing lathyrane compounds with antipromotion effect on human lung cancer cells. All the six macrocyclic lathyrane-type diterpenoids reduced the promotion in vitro , except latilagascene D, decreased IE-antigen expression of cytomegalovirus to prevent progression of tumor malignancy.

  1. Inhibition of cholesterol synthesis by squalene synthase inhibitors does not induce myotoxicity in vitro.

    PubMed

    Flint, O P; Masters, B A; Gregg, R E; Durham, S K

    1997-07-01

    The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previous in vitro study, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-induced in vitro myotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis ([3H]leucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis ([14C]acetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-induced in vitro myotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that

  2. A new small molecule inhibits streptococcus mutans biofilms in vitro and in vivo

    PubMed Central

    Pan, Wenting; Fan, Mingwen; Wu, Hui; Melander, Christian; Liu, Chang

    2015-01-01

    Aims The aim of this study is to identify new small molecules that can inhibit Streptococcus mutans biofilms by in-vitro and in-vivo model. Methods and Results We evaluated the effect of a small molecule 2-amino-imidazole/triazole conjugate (2-AI/T) on the formation of S. mutans biofilms by culturing in 96-well plates. Toxicity was assessed through cell culture and intragastrically administering to mice. The anti-biofilm and anti-caries effects were investigated in vivo. The inhibitive mechanism was detected by isobaric tag for relative and absolute quantitation (itraq) and RT-QPCR. In vitro and in vivo study revealed that 2-AI/T significantly inhibited biofilm formation of S. mutans and is more so than inhibiting planktonic cells without toxicity. The ribosome and histidine metabolism pathways of S. mutans were significantly regulated by this compound. Conclusions These results suggest that the 2-AI/T conjugate is a potent inhibitor that can be potentially developed into a new drug to treat and prevent dental caries. Significance and Impact of the Study This is the first study to use small molecule from marine natural products, to protect from dental cariesin vivo. It has potential broad range application in clinical caries prevention, or as a bioactive ingredient for food applications. PMID:26294263

  3. In vitro evidence of baicalein's inhibition of the metabolism of zidovudine (AZT).

    PubMed

    Wang, Yu-Cun; Yang, Hai-Yan; Kong, Ling-Ting; Yu, Feng-Xia

    2014-03-01

    Herb-drug interaction (HDI) has been regarded as a key factor limiting the clinical application of herbs and drugs. Potential baicalein-zidovudine (AZT) interaction was predicted in the present study. In vitro evaluation of baicalein's inhibition towards human liver microsomes (HLMs)-catalyzed metabolism of zidovudine (AZT) was performed. Dixon and Lineweaver-Burk plots were used to determine the inhibition kinetic type, and second plot with the slopes from Lineweaver-Burk plot versus the concentrations of baicalein was employed to calculate the inhibition parameter (Ki). In combination with the in vivo concentration of baicalein, in vitro-in vivo extrapolation (IVIVE) was carried out to predict in vivo baicalein-AZT interaction. Competitive inhibition of baicalein towards AZT metabolism was demonstrated, and the Ki value was calculated to be 101.2 µM. The value of AUCi/AUC was calculated to be 2. Potential baicalein-AZT interaction was indicated in the present study, indicating the need for monitoring when AZT is co-administrated with baicalein or baicalein-containing herbs.

  4. Potassium humate inhibits complement activation and the production of inflammatory cytokines in vitro

    SciTech Connect

    van Rensburg, C.E.J.; Naude, P.J.

    2009-08-15

    The effects of brown coal derived potassium humate on lymphocyte proliferation, cytokine production and complement activation were investigated in vitro. Potassium humate increased lymphocyte proliferation of phytohaemaglutinin A (PHA) and pokeweed mitogen (PWM) stimulated mononuclear lymphocytes (MNL) in vitro from concentrations of 20 to 80 {mu} g/ml, in a dose dependant manner. On the other hand potassium humate, at 40 {mu} g/ml, significantly inhibited the release of TNF-alpha, IL-1 beta, IL-6 and IL-10 by PHA stimulated MNL. Regarding complement activation it was found that potassium humate inhibits the activation of both the alternative and classical pathways without affecting the stability of the red blood cell membranes. These results indicate that the anti-inflammatory potential of potassium humate could be partially due to the inhibition of pro-inflammatory cytokines responsible for the initiation of these reactions as well as inhibition of complement activation. The increased lymphocyte proliferation observed, might be due to increased IL-2 production as previously been documented.

  5. DTD, an anti-inflammatory ditriazine, inhibits angiogenesis in vitro and in vivo.

    PubMed

    Martínez-Poveda, Beatriz; Muñoz-Chápuli, Ramón; Riguera, Ricardo; Fernández, Antonio; Medina, Miguel Angel; Quesada, Ana R

    2008-08-01

    The ditriazine derivative DTD (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3,2-d]-1,2,3-ditriazine) has been previously reported to reduce the degree of granulomatous inflammation and vascular density in a murine air pouch granuloma model. The aim of this study was to test whether DTD affects angiogenesis. Our results show that DTD inhibits in vivo angiogenesis in the chorioallantoic membrane (CAM) assay at doses equal or lower than 0.3 nmol/egg. Different in vitro assays were used to study the potential effects of this compound on key steps of angiogenesis, namely, a colorimetric assay of cell proliferation/viability, a morphogenesis on Matrigel assay, zymographic assays for gelatinases and nuclear morphology and cell cycle analysis for apoptosis induction. Our data indicate that DTD inhibits proliferation but does not induce apoptosis in endothelial cells in vitro. DTD suppresses the endothelial capillary-like chord formation at concentrations lower than those required to inhibit proliferation. DTD treatment inhibits the matrix metalloproteinase-2 production in endothelial and fibrosarcoma cells, but does not affect the cyclooxygenase-2 expression in endothelial cells, as assessed by western blot analysis. Taken together, results here presented indicate that DTD exhibits an anti-angiogenic activity that is independent of inflammatory processes and make it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.

  6. Amygdalin-mediated inhibition of non-small cell lung cancer cell invasion in vitro.

    PubMed

    Qian, Liyu; Xie, Bo; Wang, Yaguo; Qian, Jun

    2015-01-01

    Lung cancer is a common malignant tumor claiming the highest fatality worldwide for a long period of time. Unfortunately, most of the current treatment methods are still based on the characteristics of cancer cells in the primary lesion and the prognosis is often much poorer in patients with metastatic cancers. Amygdalin, a natural product of glycosides and lots of evidence shows that amygdalin can inhibit the proliferation of some kinds of cancer cells. In this study, we first obtained the highly metastatic NSCLC cell lines H1299/M and PA/M and further treated these cells with amygdalin. We found that the in vitro proliferability of H1299/M and PA/M was inhibited, but such inhibition required higher concentration of amygdalin. When lower concentration of amygdalin was used for the experiments, we observed that the in vitro invasive and migration capacities of H1299/M and PA/M were significantly inhibited. These results strongly suggested that amygdalin was likely to have anti-metastatic NSCLC effect. This study offers information of the role of amygdalin that may be useful as a therapeutic target in lung tumors.

  7. In vitro inhibition of superoxide anion production and superoxide dismutase activity by zinc in human spermatozoa.

    PubMed

    Gavella, M; Lipovac, V; Vucić, M; Sverko, V

    1999-08-01

    The in vitro effect of zinc on superoxide anion (O2-) generation and on SOD-like activity in spermatozoa of infertile men was investigated. The formation of superoxide anion was stimulated by NADPH and the level of superoxide anion was measured by the reduction of ferricytochrome c. Both Percoll-isolated (n = 14) and washed spermatozoa (n = 14) exposed to 1 mmol/L zinc (60 min, 37 degrees C), released less (p < 0.002 and p < 0.04, respectively) superoxide anions than did zinc-untreated spermatozoa. These results implicate a possible role for zinc as a scavenger of excessive superoxide anions produced by defective spermatozoa in semen after ejaculation. Additionally, zinc was found to dose-dependently inhibit superoxide dismutase (SOD)-like activity of spermatozoa in vitro. The inhibition of SOD-like activity by an equal concentration of zinc (1 mmol/L) was less pronounced in oligospermic (p < 0.002; n = 16) and asthenozoospermic (p < 0.0005; n = 20) than in normozoospermic samples (p < 0.0001; n = 20). This differential ability of zinc to inhibit SOD-like activity may be relevant to the physiological function of spermatozoa in fertilization. The evidence that zinc may elicit an inhibition of both superoxide anion production and SOD-like activity in human spermatozoa, indicate the existence of novel, zinc-related mechanism(s) involved in the oxidative events occurring after ejaculation, with a possible modulatory effect on germ cell function.

  8. Syntheses and in vitro evaluations of uncharged reactivators for human acetylcholinesterase inhibited by organophosphorus nerve agents.

    PubMed

    Renou, Julien; Mercey, Guillaume; Verdelet, Tristan; Păunescu, Emilia; Gillon, Emilie; Arboléas, Mélanie; Loiodice, Mélanie; Kliachyna, Maria; Baati, Rachid; Nachon, Florian; Jean, Ludovic; Renard, Pierre-Yves

    2013-03-25

    Organophosphorus nerve agents (OPNAs) are highly toxic compounds that represent a threat to both military and civilian populations. They cause an irreversible inhibition of acetylcholinesterase (AChE), by the formation of a covalent P-O bond with the catalytic serine. Among the present treatment of nerve agents poisoning, pyridinium and bis-pyridinium aldoximes are used to reactivate this inhibited enzyme but these compounds do not readily cross the blood brain barrier (BBB) due to their permanent cationic charge and thus cannot efficiently reactivate cholinesterases in the central nervous system (CNS). In this study, a series of seven new uncharged oximes reactivators have been synthesized and their in vitro ability to reactivate VX and tabun-inhibited human acetylcholinesterase (hAChE) has been evaluated. The dissociation constant K(D) of inhibited enzyme-oxime complex, the reactivity rate constant kr and the second order reactivation rate constant k(r2) have been determined and have been compared to reference oximes HI-6, Obidoxime and 2-Pralidoxime (2-PAM). Regarding the reactivation of VX-inhibited hAChE, all compounds show a better reactivation potency than those of 2-PAM, nevertheless they are less efficient than obidoxime and HI-6. Moreover, one of seven described compounds presents an ability to reactivate tabun-inhibited hAChE equivalent to those of 2-PAM. Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

  9. In vitro inhibition and induction of human liver cytochrome p450 enzymes by milnacipran.

    PubMed

    Paris, Brandy L; Ogilvie, Brian W; Scheinkoenig, Julie A; Ndikum-Moffor, Florence; Gibson, Remi; Parkinson, Andrew

    2009-10-01

    Milnacipran (Savella) inhibits both norepinephrine and serotonin reuptake and is distinguished by a nearly 3-fold greater potency in inhibiting norepinephrine reuptake in vitro compared with serotonin. We evaluated the ability of milnacipran to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6 (IC(50) >or= 100 microM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC(50) = 7 microM) and CYP2B6 (IC(50) = 15 microM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1'-hydroxylation IC(50) approximately 30 microM; testosterone 6beta-hydroxylation IC(50) approximately 100 microM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC(50) = 37 and 38 microM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of P450 activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19, and 3A4/5. To evaluate P450 induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for 3 days with milnacipran (3, 10, and 30 microM), after which microsomal P450 activities were measured. Whereas positive controls (omeprazole, phenobarbital, and rifampin) caused anticipated P450 induction, milnacipran had minimal effect on CYP1A2, 2C8, 2C9, or 2C19 activity. The highest concentration of milnacipran (30 microM; >10 times plasma C(max)) produced 2.6- and 2.2-fold increases in CYP2B6 and CYP3A4/5 activity (making it 26 and 34% as effective as phenobarbital and rifampin, respectively). Given these results, milnacipran is not expected to cause clinically significant P450 inhibition or induction.

  10. G-protein ligands inhibit in vitro reactions of vacuole inheritance

    PubMed Central

    1994-01-01

    During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469- 1479). We now report a simple and reliable assay to quantify vacuole-to- vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2- 1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP- hydrolyzing G proteins may play a key role in the in vitro fusion events. PMID:8027189

  11. EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin.

    PubMed

    Sakariassen, Kjell S; Femia, Eti A; Daray, Federico M; Podda, Gian M; Razzari, Cristina; Pugliano, Mariateresa; Errasti, Andrea E; Armesto, Arnaldo R; Nowak, Wanda; Alberts, Pēteris; Meyer, Jean-Philippe; Sorensen, Alexandra S; Cattaneo, Marco; Rothlin, Rodolfo P

    2012-11-01

    This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. Fifty-two type-2 diabetics with CAD on chronic aspirin (100 mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100 nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). Arachidonic acid (1 mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63 ± 4 AU), which was significantly reduced by in vitro exposure to EV-077 (38 ± 3 AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Inhibition of Streptococcus mutans Growth and Biofilm Formation by Probiotics in vitro.

    PubMed

    Schwendicke, Falk; Korte, Franziska; Dörfer, Christof E; Kneist, Susanne; Fawzy El-Sayed, Karim; Paris, Sebastian

    2017-01-01

    To exert anticaries effects, probiotics are described to inhibit growth and biofilm formation of cariogenic bacteria such as Streptococcus mutans (SM). We screened 8 probiotics and assessed how SM growth or biofilm formation inhibition affects cariogenicity of probiotic-SM mixed-species biofilms in vitro. Growth inhibition was assessed by cocultivating probiotics and 2 SM strains (ATCC 20532/25175) on agar. Probiotics were either precultured before SM cultivation (exclusion), or SM precultured prior to probiotic cultivation (displacement). Inhibition of SM culture growth was assessed visually. Inhibition of SM biofilm formation on bovine enamel was assessed using a continuous-flow short-term biofilm model, again in exclusion or displacement mode. The cariogenicity of mixed-species biofilms of SM with the most promising growth and biofilm formation inhibiting probiotic strains was assessed using an artificial mouth model, and enamel mineral loss (ΔZ) was measured microradiographically. We found limited differences in SM growth inhibition in exclusion versus displacement mode, and in inhibition of SM 20532 versus 25175. Results were therefore pooled. Lactobacillus acidophilus LA-5 inhibited significantly more SM culture growth than most other probiotics. L. casei LC-11 inhibited SM biofilm formation similarly to other alternatives but showed the highest retention of probiotics in the biofilms (p < 0.05). Mineral loss from SM monospecies biofilms (ΔZ = 9,772, 25th/75th percentiles: 6,277/13,558 vol% × µm) was significantly lower than from mixed-species SM × LA-5 biofilms (ΔZ = 24,578, 25th/75th percentiles: 19,081/28,768 vol% × µm; p < 0.01) but significantly higher than from SM × LC-11 biofilms (ΔZ = 4,835, 25th/75th percentiles: 263/7,865 vol% × µm; p < 0.05). Probiotics inhibiting SM culture growth do not necessarily reduce the cariogenicity of SM-probiotic biofilms. Nevertheless, SM biofilm formation inhibition may be relevant in the reduction of

  13. In vitro CYP3A4 metabolism: inhibition by Echinacea purpurea and choice of substrate for the evaluation of herbal inhibition.

    PubMed

    Hansen, Torstein Schrøder; Nilsen, Odd Georg

    2008-11-01

    The in vitro CYP3A4 inhibition profiles of Echinacea purpurea, St. John's wort and ketoconazole were evaluated by three different substrates: 7-benzyloxy-trifluoromethylcoumarin (BFC), 7-benzyloxyquinoline (BQ) and testosterone. St. John's wort and ketoconazole produced similar inhibition profiles regardless of substrate. For E. purpurea, testosterone metabolism showed a much lower CYP3A4 inhibition (IC(50) 5394 microg/ml) compared to the fluorescent substrates BFC and BQ (IC(50) 354 and 452 mg/ml, respectively). It is suggested that the substrate/assay-dependent effects may arise from a complex nature of E. purpurea constituents, involving different CYP3A4 substrate binding sites. The choice of substrate might thus be essential for evaluation of the inhibition of CYP3A4 metabolism for some herbs. A weak inhibition potential of E. purpurea towards CYP3A4-mediated metabolism in vitro was confirmed by the use of three different substrates.

  14. Sulforaphane, a natural component of broccoli, inhibits vestibular schwannoma growth in vitro and in vivo

    PubMed Central

    Kim, Bo Gyung; Fujita, Takeshi; Stankovic, Konstantina M.; Welling, D. Bradley; Moon, In Seok; Choi, Jae Young; Yun, Jieun; Kang, Jong Soon; Lee, Jong Dae

    2016-01-01

    Vestibular schwannoma (VS) is an intracranial tumor that causes significant morbidity, including hearing loss, tinnitus, dizziness, and possibly even death from brainstem compression. However, FDA-approved pharmacologic treatments for VS do not exist. Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli, with potent chemoprotective effects in several cell types. Our objective was to determine whether SFN is effective against VS in vitro and in vivo. Human primary VS cells, HEI-193 schwannoma cells, and SC4 Nf2−/− Schwann cells were used to investigate the inhibitory effects of SFN in vitro. Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation, and cell viability and metabolic activity was calculated by MTT assay. Apoptosis was measured by flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and Western blot for cleaved caspases. A mouse model with a murine schwannoma allograft was also used to examine the antitumor activity of SFN. SFN exhibited significant antiproliferative activity in schwannoma cells in vitro, via the inhibition of HDAC activity and the activation of ERK. SFN treatment induced apoptosis and cell cycle arrest at the G2/M phase. SFN also significantly inhibited schwannoma growth in vivo. Our preclinical studies motivate a future prospective clinical study of SFN for the treatment of VS. PMID:27805058

  15. Noscapine inhibits human hepatocellular carcinoma growth through inducing apoptosis in vitro and in vivo.

    PubMed

    Xu, G; Niu, Z; Dong, J; Zhao, Y; Zhang, Y; Li, X

    2016-01-01

    Noscapine, a phthalideisoquinoline alkaloid derived from opium, has been demonstrated as a promising anti-tumor compound against various cancers. However, the anti-cancer activity of noscapine in hepatocellular carcinoma has not been defined. In this study, we investigate the inhibitive effects of noscapine on human hepatocellular carcinoma (HCC) using both in vitro and in vivo models. In vitro proliferation assay showed that noscapine suppressed HepG2 and Huh7 cells in dose- and time-dependent manners. With a mouse xenograft model, noscapine showed notable inhibition on HCC tumor growth in vivo without suppression of body weight. Moreover, apoptotic induction and regulation of related signalings by noscapine were examined by nuclear DNA staining, TUNEL, and western blotting assays. Results showed that noscapine induced apoptosis in HCC cells both in vitro and in vivo. Further studies indicated that noscapine induced antive-capsase-3, cleavage PARP, and decreased the ratio of Bcl-2/Bax. Hence, these data indicates that noscapine selectively suppresses HCC cell growth through apoptosis induction, providing evidence for application of noscapine as a novel agent against human hepatocellular carcinoma.

  16. Wu-Ling-San formula inhibits the crystallization of calcium oxalate in vitro.

    PubMed

    Chen, Yu-Cheng; Ho, Chien-Yi; Chen, Lieh-Der; Hsu, Sheng-Feng; Chen, Wen-Chi

    2007-01-01

    Urinary stone disease is a common disease and has a high rate of recurrence. There is no ideal long-term medical treatment to prevent the recurrence of urinary stones. Wu-Ling-San (WLS) formula has been used for centuries in China for long-term treatment of urological diseases. However, no pharmacological studies have been conducted to evaluate its effect on urinary stone disease. Therefore, using a photospectrometer, we studied the effects of WLS on nucleation, growth and aggregation of calcium oxalate in vitro. The results showed that WLS extract significantly slowed the speed of calcium oxalate (CaOx) crystal nucleation. WLS extracts at concentrations of 6.25, 12.5, 25, and 50 mg/ml inhibited nucleation of calcium oxalate crystallization by 344, 387, 543, and 943%, respectively. WLS extracts did not inhibit the growth of CaOx crystallization; however, WLS extracts at concentrations of 12.5 and 25 mg/ml significantly inhibited the aggregation of CaOx crystallization by 74.24% and 75.05%, respectively. WLS extract at a concentration of 50 mg/ml inhibited CaOx aggregation by 92.49%. In conclusion, our results indicate that WLS extract inhibited calcium oxalate nucleation and aggregation, and may have the potential to prevent stone recurrence.

  17. Antibiotic drug tigecycline reduces neuroblastoma cells proliferation by inhibiting Akt activation in vitro and in vivo.

    PubMed

    Zhong, Xiaoxia; Zhao, Erhu; Tang, Chunling; Zhang, Weibo; Tan, Juan; Dong, Zhen; Ding, Han-Fei; Cui, Hongjuan

    2016-06-01

    As the first member of glycylcycline bacteriostatic agents, tigecycline is approved as a novel expanded-spectrum antibiotic, which is clinically available. However, accumulating evidence indicated that tigecycline was provided with the potential application in cancer therapy. In this paper, tigecycline was shown to exert an anti-proliferative effect on neuroblastoma cell lines. Furthermore, it was found that tigecycline induced G1-phase cell cycle arrest instead of apoptosis by means of Akt pathway inhibition. In neuroblastoma cell lines, the Akt activator insulin-like growth factor-1 (hereafter referred to as IGF-1) reversed tigecycline-induced cell cycle arrest. Besides, tigecycline inhibited colony formation and suppressed neuroblastoma cells xenograft formation and growth. After tigecycline treatment in vivo, the Akt pathway inhibition was confirmed as well. Collectively, our data provided strong evidences that tigecycline inhibited neuroblastoma cells growth and proliferation through the Akt pathway inhibition in vitro and in vivo. In addition, these results were supported by previous studies concerning the application of tigecycline in human tumors treatment, suggesting that tigecycline might act as a potential candidate agent for neuroblastoma treatment.

  18. Flavonoid Ampelopsin Inhibits the Growth and Metastasis of Prostate Cancer In Vitro and in Mice

    PubMed Central

    Ni, Feng; Gong, Yi; Li, Linglin; Abdolmaleky, Hamid M.; Zhou, Jin-Rong

    2012-01-01

    The objective of this study was to evaluate the chemopreventive effect of a novel flavonoid, ampelopsin (AMP) on the growth and metastasis of prostate cancer cells. AMP showed the more potent activity in inhibiting the proliferation of androgen-sensitive LNCaP and, to less extent, androgen-independent PC-3 human prostate cancer cell lines in vitro, primarily by induction of apoptosis associated with down-regulation of bcl-2. On the other hand, AMP showed much less activity in inhibiting the proliferation of normal prostate epithelial cells than that of prostate cancer cell lines. AMP also inhibited the migration and invasion of PC-3 cells in vitro associated with down-regulation of CXCR4 expression. In the animal study using an orthotopic prostate tumor model, AMP (150 and 300 mg/kg body weight) inhibited the growth of PC-3 tumors and lymph node and lung metastases in a dose-dependent manner. Compared to the control mice, mice treated with AMP at 300 mg/kg BW had reduced final tumor weight by 49.2% (P<0.05), lymph node metastases by 54.5% (P = 0.3) and lung metastases by 93% (P<0.05), but had no apparent alteration on food intake or body weight. The in vivo anti-growth and anti-metastasis activities of AMP were associated with induction of apoptosis and inhibition of proliferation of prostate cancer cells, reduction of prostate tumor angiogenesis, and reduction of CXCR4 expression. Our results provide supporting evidence to warrant further investigation to develop AMP as a novel efficacious and safe candidate agent against progression and metastasis of prostate cancer. PMID:22693649

  19. QSAR modeling of in vitro inhibition of cytochrome P450 3A4.

    PubMed

    Mao, Boryeu; Gozalbes, Rafael; Barbosa, Frédérique; Migeon, Jacques; Merrick, Sandra; Kamm, Kelly; Wong, Eric; Costales, Chester; Shi, Wei; Wu, Cheryl; Froloff, Nicolas

    2006-01-01

    We report the QSAR modeling of cytochrome P450 3A4 (CYP3A4) enzyme inhibition using four large data sets of in vitro data. These data sets consist of marketed drugs and drug-like compounds all tested in four assays measuring the inhibition of the metabolism of four different substrates by the CYP3A4 enzyme. The four probe substrates are benzyloxycoumarin, testosterone, benzyloxyresorufin, and midazolam. We first show that using state-of-the-art QSAR modeling approaches applied to only one of these four data sets does not lead to predictive models that would be useful for in silico filtering of chemical libraries. We then present the development and the testing of a multiple pharmacophore hypothesis (MPH) that is formulated as a conceptual extension of the traditional QSAR approach to modeling the promiscuous binding of a large variety of drugs to CYP3A4. In the simplest form, the MPH approach takes advantage of the multiple substrate data sets and identifies the binding of test compounds as either proximal or distal relative to that of a given substrate. Application of the approach to the in silico filtering of test compounds for potential inhibitors of CYP3A4 is also presented. In addition to an improvement in the QSAR modeling for the inhibition of CYP3A4, the results from this modeling approach provide structural insights into the drug-enzyme interactions. The existence of multiple inhibition data sets in the BioPrint database motivates the original development of the concept of a multiple pharmacophore hypothesis and provides a unique opportunity for formulating alternative strategies of QSAR modeling of the inhibition of the in vitro metabolism of CYP3A4.

  20. Zinc binds to and directly inhibits protein phosphatase 2A in vitro.

    PubMed

    Xiong, Yan; Luo, Dan-Ju; Wang, Xiu-Lian; Qiu, Mei; Yang, Yang; Yan, Xiong; Wang, Jian-Zhi; Ye, Qi-Fa; Liu, Rong

    2015-06-01

    Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn(2+) has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn(2+) on PP2A and their direct interaction in vitro. The results showed that Zn(2+) mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 μmol/L) of Zn(2+) inhibited PP2A directly. Further Zn(2+)-IDA-agarose affinity binding assays showed that Zn(2+) bound to and inhibited PP2Ac(51-270) but not PP2Ac(1-50) or PP2Ac(271-309). Taken together, Zn(2+) inhibits PP2A directly through binding to PP2Ac(51-270) in vitro.

  1. Cadmium restores in vitro splicing activity inhibited by zinc-depletion.

    PubMed

    Lee, Myeong Jin; Ayaki, Hitoshi; Goji, Junko; Kitamura, Keiko; Nishio, Hisahide

    2006-10-01

    Zinc (Zn)-depletion inhibits the second step of RNA splicing, namely exon-ligation. To investigate the effects of cadmium (Cd) and other metal ions on RNA splicing inhibited by Zn-depletion, we measured in vitro splicing activities in the presence of these metals. Zn-depletion in the splicing reaction mixture was achieved by addition of a Zn-chelator, 1,10-phenanthroline. Cd(II) at 1, 5 and 10 microM restored the splicing activity to 2, 24 and 72% of that in the control reaction mixture, while higher concentrations of Cd(II) decreased the splicing activity, and more than 50 microM Cd(II) showed a complete absence of spliced products. Hg(II) also restored the splicing activity, albeit to a lesser extent, since 5 and 10 microM Hg(II) restored the splicing activity to 3 and 4% of the control value. The other metal ions examined in this study, Co(II), Cu(II), Mg(II) and Mn(II), did not show any restoration of the splicing activity. We concluded that Cd(II) could restore the in vitro splicing activity inhibited by Zn-depletion, although higher concentrations of Cd(II) prevented progress of the RNA splicing reaction. These results suggest that Cd(II) has a bifunctional property regarding RNA splicing, and is stimulatory at low concentrations and inhibitory at high concentrations.

  2. The Aplidin analogs PM01215 and PM02781 inhibit angiogenesis in vitro and in vivo.

    PubMed

    Borjan, Bojana; Steiner, Normann; Karbon, Silvia; Kern, Johann; Francesch, Andrés; Hermann, Martin; Willenbacher, Wolfgang; Gunsilius, Eberhard; Untergasser, Gerold

    2015-10-19

    Novel synthesized analogs of Aplidin, PM01215 and PM02781, were tested for antiangiogenic effects on primary human endothelial cells in vitro and for inhibition of angiogenesis and tumor growth in vivo. Antiangiogenic activity of both derivatives was evaluated by real-time cell proliferation, capillary tube formation and vascular endothelial growth factor (VEGF)-induced spheroid sprouting assays. Distribution of endothelial cells in the different phases of the cell cycle was analyzed by flow cytometry. Aplidin analogs were tested in vivo in chicken chorioallantoic membrane (CAM) assays. Both derivatives inhibited angiogenic capacities of human endothelial cells (HUVECs) in vitro at low nanomolar concentrations. Antiangiogenic effects of both analogs were observed in the CAM. In addition, growth of human multiple myeloma xenografts in vivo in CAM was significantly reduced after application of both analogs. On the molecular level, both derivatives induced cell cycle arrest in G1 phase. This growth arrest of endothelial cells correlated with induction of the cell cycle inhibitor p16(INK4A) and increased senescence-associated beta galactosidase activity. In addition, Aplidin analogs induced oxidative stress and decreased production of the vascular maturation factors Vasohibin-1 and Dickkopf-3. From these findings we conclude that both analogs are promising agents for the development of antiangiogenic drugs acting independent on classical inhibition of VEGF signaling.

  3. Mullerian Inhibiting Substance Suppresses Proliferation and Induces Apoptosis and Autophagy in Endometriosis Cells In Vitro

    PubMed Central

    Borahay, Mostafa A.; Lu, Fangxian; Ozpolat, Bulent; Tekedereli, Ibrahim; Gurates, Bilgin; Karipcin, Sinem; Kilic, Gokhan S.

    2013-01-01

    Objective. To determine the effects of Mullerian inhibiting substance (MIS) treatment on endometriosis cells through study of apoptosis and autophagy. Design. Experimental in vitro study. Setting. University research laboratory. Cell Line. CRL-7566 endometriosis cell line. This line was established from a benign ovarian cyst taken from a patient with endometriosis. Interventions. In vitro treatment with MIS. Main Outcome Measures. The main outcome measures were cellular viability, proliferation, cell-cycle arrest, and induction of apoptosis and autophagy in endometriotic cells. Results. MIS treatment inhibited proliferation of endometriosis cells and induced apoptosis, as indicated by Annexin V staining, and induced caspase-9 cleavage and cell-cycle arrest, as evidenced by increased expression of p27 CDK-inhibitor. MIS treatment also induced autophagy in endometriosis cells as demonstrated by a significant increase in LC3-II induction, a hallmark of autophagy. Conclusions. MIS inhibits cell growth and induces autophagy, as well as apoptosis, in ectopic endometrial cell lines. Our results suggest that MIS may have a potential as a novel approach for medical treatment of endometriosis. Further studies may be needed to test the efficacy of MIS treatment in animal models and to develop MIS treatment specifically targeted to the endometriosis. PMID:23853725

  4. In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo.

    PubMed

    Scott, H V; Gero, A M; O'Sullivan, W J

    1986-01-01

    The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID50 values (concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 +/- 8.7, 6.4 +/- 5.3 and 6.3 +/- 0.5 microM, respectively. Comparative ID50 values for chloroquine were 13.5 +/- 4.2, 22.8 +/- 7.6 and 343 +/- 114 microM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 microM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5'-phosphate decarboxylase (E.C. 4.1.1.23).

  5. Fucoidan inhibits LPS-induced inflammation in vitro and during the acute response in vivo.

    PubMed

    Park, Jisang; Cha, Jeong-Dan; Choi, Kyung-Min; Lee, Kyung-Yeol; Han, Kang Min; Jang, Yong-Suk

    2017-02-01

    Studies have been focused on natural products with antibacterial and anti-inflammatory activities, such as fucoidan. Many in vivo studies have evaluated the effect of fucoidan on tumor growth, diabetes, obesity, ischemia reperfusion, and oxidative stress. However, the effects of fucoidan on bacteria-induced gingival inflammation and periodontitis have not been reported. We previously characterized the anti-inflammatory effect of fucoidan in vitro. Here, we confirmed the anti-inflammatory activity of fucoidan in a macrophage cell line in terms of its inhibition of the expression of inflammatory mediators and pro-inflammatory cytokines. Additionally, we confirmed the ability of fucoidan to inhibit gingival inflammation, expression of pro-inflammatory cytokines, and neutrophil recruitment in the gingival tissue of mice injected with LPS prepared from P. gingivalis. Interestingly, however, fucoidan did not inhibit the expression of pro-inflammatory cytokines in a P. gingivalis-infected mouse model of periodontitis. Additionally, fucoidan treatment did not lead to clearance of P. gingivalis or improvement of P. gingivalis infection-mediated bone loss in the periodontitis model. We conclude that fucoidan exerts anti-inflammatory effects in vitro and in vivo, together with a limited antibacterial effect in vivo.

  6. A natural small molecule voacangine inhibits angiogenesis both in vitro and in vivo

    SciTech Connect

    Kim, Yonghyo; Jung, Hye Jin; Kwon, Ho Jeong

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Voacangine exhibits potent anti-angiogenic activity both in vitro and in vivo. Black-Right-Pointing-Pointer Voacangine inhibits tumor-induced angiogenesis by suppressing HIF-1{alpha}. Black-Right-Pointing-Pointer Voacangine could be the basis for the development of novel anti-angiogenic agents. -- Abstract: Angiogenesis, the formation of new blood vessels from pre-existing ones, plays a critical role in normal and pathological phenotypes, including solid tumor growth and metastasis. Accordingly, the development of new anti-angiogenic agents is considered an efficient strategy for the treatment of cancer and other human diseases linked with angiogenesis. We have identified voacangine, isolated from Voacanga africana, as a novel anti-angiogenic agent. Voacangine inhibits the proliferation of HUVECs at an IC{sub 50} of 18 {mu}M with no cytotoxic effects. Voacangine significantly suppressed in vitro angiogenesis, such as VEGF-induced tube formation and chemoinvasion. Moreover, the compound inhibits in vivo angiogenesis in the chorioallantoic membrane at non-toxic doses. In addition, voacangine decreased the expression levels of hypoxia inducible factor-1{alpha} and its target gene, VEGF, in a dose-dependent manner. Taken together, these results suggest that the naturally occurring compound, voacangine, is a novel anti-angiogenic compound.

  7. TQ inhibits hepatocellular carcinoma growth in vitro and in vivo via repression of Notch signaling

    PubMed Central

    Ke, Xiquan; Zhao, Yan; Lu, Xinlan; Wang, Zhe; Liu, Yuanyuan; Ren, Mudan; Lu, Guifang; Zhang, Dan; Sun, Zhenguo; Xu, Zhipeng; Song, Jee Hoon; Cheng, Yulan; Meltzer, Stephen J.; He, Shuixiang

    2015-01-01

    Thymoquinone (TQ) has been reported to possess anti-tumor activity in various types of cancer. However, its effects and molecular mechanism of action in hepatocellular carcinoma (HCC) are still not completely understood. We observed that TQ inhibited tumor cell growth in vitro, where treatment with TQ arrested the cell cycle in G1 by upregulating p21 and downregulating cyclinD1 and CDK2 expression; moreover, TQ induced apoptosis by decreasing expression of Bcl-2 and increasing expression of Bax. Simultaneously, TQ demonstrated a suppressive impact on the Notch pathway, where overexpression of NICD1 reversed the inhibitory effect of TQ on cell proliferation, thereby attenuating the repressive effects of TQ on the Notch pathway, cyclinD1, CDK2 and Bcl-2, and also diminishing upregulation of p21 and Bax. In a xenograft model, TQ inhibited HCC growth in nude mice; this inhibitory effect in vivo, as well as of HCC cell growth in vitro, was associated with a discernible decline in NICD1 and Bcl-2 levels and a dramatic rise in p21 expression. In conclusion, TQ inhibits HCC cell growth by inducing cell cycle arrest and apoptosis, achieving these effects by repression of the Notch signaling pathway, suggesting that TQ represents a potential preventive or therapeutic agent in HCC patients. PMID:26416455

  8. The HIV protease inhibitor nelfinavir inhibits Kaposi's sarcoma-associated herpesvirus replication in vitro.

    PubMed

    Gantt, Soren; Carlsson, Jacquelyn; Ikoma, Minako; Gachelet, Eliora; Gray, Matthew; Geballe, Adam P; Corey, Lawrence; Casper, Corey; Lagunoff, Michael; Vieira, Jeffrey

    2011-06-01

    Kaposi's sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.

  9. In vitro actinomycete biofilm development and inhibition by the polyene antibiotic, nystatin, on IUD copper surfaces.

    PubMed

    Shanmughapriya, Santhanam; Francis, Arumugam Lency; Kavitha, Senthil; Natarajaseenivasan, Kalimuthusamy

    2012-01-01

    The presence of intrauterine contraceptive devices (IUDs) gives a solid surface for attachment and an ideal niche for biofilm to form and flourish. Pelvic actinomycosis is often associated with the use of IUDs. Treatment of IUD-associated pelvic actinomycosis requires the immediate removal of the IUD. Therefore, this article presents in vitro evidence to support the use of novel antibiotics in the treatment of actinomycete biofilms. Twenty one clinical actinomycetes isolates from endocervical swabs of IUD wearers were assessed for their biofilm forming ability. An in vitro biofilm model with three isolates, Streptomyces strain A4, Nocardia strain C15 and Nocardia strain C17 was subjected to treatment with nystatin. Inhibition of biofilm formation by nystatin was found to be concentration dependent, with MBIC50 values in the range 0.08-0.16 mg ml(-1). Furthermore, at a concentration of 0.16 mg ml(-1), nystatin inhibited the twitching motility of the isolates, providing evidence for a possible mechanism of biofilm inhibition.

  10. Inhibition of enteroaggregative Escherichia coli cell adhesion in-vitro by designed peptides.

    PubMed

    Gupta, Deepika; Sarkar, Subendu; Sharma, Monica; Thapa, B R; Chakraborti, Anuradha

    2016-09-01

    Enteroaggregative Escherichia coli (EAEC) bears remarkable capacity to adhere the host intestinal mucosal surface and results in acute or persistent childhood diarrhea worldwide. In this study, an attempt has been made to inhibit EAEC cell adherence in-vitro using synthetic peptides. E. coli isolates (n = 54) were isolated from the stool samples of clinically diagnosed pediatric diarrheal patients. 92.8% isolates showed different types of aggregative adherence patterns with HEp-2 cells. AAF-II (Aggregative Adherence Fimbriae-II) EAEC exhibited the maximum ability to form biofilm and intracellular survival. Peptides were designed against the high antigenic epitopic regions of AAF-II adhesin of EAEC O42 using prediction algorithms like BcePred and ProPred software to block the EAEC cell adhesion in-vitro. Peptides P2 (DITITPATNRDVNV) and P3 (MRIKAWGEANHGQL) demonstrated higher inhibition of EAEC cell adhesion than P1 (GMQGSITPAIPLRPG). Interestingly, increasing the pre-incubation time of the peptides with HEp-2 cells from 1 h to 2 h showed the maximum inhibition. The data suggested the potential role of P2 and P3 peptides in successfully blocking the binding of AAF-II EAEC with HEp-2 cell receptors. Hence, the peptides may be efficacious in designing new chemotherapeutic for the management of EAEC mediated diarrhea.

  11. Inhibition of the adenine nucleotide translocator by N-acetyl perfluorooctane sulfonamides in vitro

    SciTech Connect

    O'Brien, Timothy M. Oliveira, Paulo J.; Wallace, Kendall B.

    2008-03-01

    N-alkyl perfluorooctane sulfonamides have been widely used as surfactants on fabrics and papers, fire retardants, and anti-corrosion agents, among many other commercial applications. The global distribution and environmental persistence of these compounds has generated considerable interest regarding potential toxic effects. We have previously reported that perfluorooctanesulfonamidoacetate (FOSAA) and N-ethylperfluorooctanesulfonamidoacetate (N-EtFOSAA) induce the mitochondrial permeability transition (MPT) in vitro. In this study we tested the hypothesis that FOSAA and N-EtFOSAA interact with the adenine nucleotide translocator (ANT) resulting in a functional inhibition of the translocator and induction of the MPT. Respiration and membrane potential of freshly isolated liver mitochondria from Sprague-Dawley rats were measured using an oxygen electrode and a tetraphenylphosphonium-selective (TPP{sup +}) electrode, respectively. Mitochondrial swelling was measured spectrophotometrically. The ANT ligands bongkregkic acid (BKA) and carboxyatractyloside (cATR) inhibited uncoupling of mitochondrial respiration caused by 10 {mu}M N-EtFOSAA, 40 {mu}M FOSAA, and the positive control 8 {mu}M oleic acid. ADP-stimulated respiration and depolarization of mitochondrial membrane potential were inhibited by cATR, FOSAA, N-EtFOSAA, and oleic acid, but not by FCCP. BKA inhibited calcium-dependent mitochondrial swelling induced by FOSAA, N-EtFOSAA, and oleic acid. Seventy-five micromolar ADP also inhibited swelling induced by the test compounds, but cATR induced swelling was not inhibited by ADP. Results of this investigation indicate that N-acetyl perfluorooctane sulfonamides interact directly with the ANT to inhibit ADP translocation and induce the MPT, one or both of which may account for the metabolic dysfunction observed in vivo.

  12. Production of mouse monoclonal antibodies which inhibit in vitro adherence of Entamoeba histolytica trophozoites.

    PubMed Central

    Ravdin, J I; Petri, W A; Murphy, C F; Smith, R D

    1986-01-01

    Adherence by axenic Entamoeba histolytica trophozoites to mammalian cells is mediated by an N-acetylgalactosamine (GalNAc)-inhibitable adhesin on the surface of the parasite. We isolated 35 hybridoma cell lines producing antibodies to E. histolytica as indicated by ELISA with sonicated amebic protein or by immunofluorescence assay with fixed whole trophozoites. Tissue culture supernatants were further screened for subcloning by the ability to bind to Chinese hamster ovary (CHO) cells which were first exposed to a partially purified soluble preparation of the amebic GalNAc-inhibitable lectin. Eight tissue culture supernatants were positive in this assay. Antibodies from four subcloned cell lines (D3-14, H8-5, I12-2, and I1-21) inhibited amebic adherence to CHO cells (P less than 0.01). Of the original 35 tissue culture supernatants, 3 also inhibited amebic adherence (P less than 0.01; F1, F14, and J10); monoclonal antibodies in these supernatants did not bind to lectin-exposed CHO cells. Three purified monoclonal antibodies (H8-5, I12-2, and I1-21) inhibited amebic adherence at greater than or equal to 2 micrograms/10(4) amebae (P less than 0.05). None of these inhibitory monoclonal antibodies immunoprecipitated with a soluble amebic protein preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Monoclonal antibodies which inhibit in vitro adherence by E. histolytica will be useful in purification of the GalNAc-inhibitable lectin. PMID:2873102

  13. Nucleosomes inhibit both transcriptional initiation and elongation by RNA polymerase III in vitro.

    PubMed Central

    Morse, R H

    1989-01-01

    To examine the effect of nucleosomes on in vitro transcription, purified chicken erythrocyte core histones and plasmid DNA bearing the Xenopus 5S RNA gene were assembled into nucleosomes and used as templates for transcription in a Xenopus oocyte nuclear extract. Plasmids having a nucleosome incorporating a specific region of the gene were selected by treating the reconstituted molecules with restriction endonucleases. In this way, it was shown that a nucleosome on or close to the internal control region of the 5S RNA gene inhibits transcription. Furthermore, experiments with 5S maxigenes showed that RNA polymerase III, in contrast to SP6 RNA polymerase, will not transcribe through a nucleosome in vitro. Images PMID:2792088

  14. Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro

    PubMed Central

    Reisinger, Nicole; Schaumberger, Simone; Nagl, Veronika; Hessenberger, Sabine; Schatzmayr, Gerd

    2014-01-01

    The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application. PMID:25290524

  15. Milk thistle extract and silymarin inhibit lipopolysaccharide induced lamellar separation of hoof explants in vitro.

    PubMed

    Reisinger, Nicole; Schaumberger, Simone; Nagl, Veronika; Hessenberger, Sabine; Schatzmayr, Gerd

    2014-10-06

    The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application.

  16. An Engineered Arginase FC Protein Inhibits Tumor Growth In Vitro and In Vivo

    PubMed Central

    Li, Lihua; Wang, Yan; Chen, Jun; Cheng, Bi; Hu, Jiehua; Zhou, Yuehua; Gao, Xin; Gao, Liucun; Mei, Xifan; Sun, Meiyan; Zhang, Zhuomei; Song, Haifeng

    2013-01-01

    Arginine is a semiessential amino acid required for the growth of melanoma and hepatocellular carcinoma, and the enzymatic removal of arginine by pegylated arginine deiminase (ADI) or arginase is being tested clinically. Here, we report a genetically engineered arginase FC fusion protein exhibiting a prolonged half-life and enhanced efficacy. The use of this enzyme to treat different tumor lines both inhibited cell proliferation and impaired cellular migration in vitro and in vivo. Our data reinforce the hypothesis that nutritional depletion is a key strategy for cancer treatment. PMID:23737831

  17. In vitro inhibition of sperm motility by some local mineral water drinks.

    PubMed

    Oyelola, O O; Ayangade, S O; Amole, F

    1987-10-01

    The effect of eight common local mineral water drinks in Ile-Ife, Nigeria (seven cola and one lemon-based) on sperm motility in vitro was carried out. There was no significant difference in the pH values of all the drinks, the pH ranged between 2.30-2.50. After one minute, Pepsi-Cola had the least inhibitory effect on sperm motility, Bitter-lemon had the strongest effect, while the other brands had varying effects between the two extremes. The percentage motility inhibition by the mineral water drinks is also a function of the initial total sperm count in the semen.

  18. New indole-isoxazolone derivatives: Synthesis, characterisation and in vitro SIRT1 inhibition studies.

    PubMed

    Panathur, Naveen; Gokhale, Nikhila; Dalimba, Udayakumar; Koushik, Pulla Venkat; Yogeeswari, Perumal; Sriram, Dharmarajan

    2015-07-15

    A new series of indole-isoxazolone hybrids bearing substituted amide, substituted [(1,2,3-triazol-4-yl)methoxy]methyl group or substituted benzylic ether at position-2 of the indole nucleus was synthesised using a facile synthetic route and the molecules were characterised using spectroscopic techniques. The molecules were screened against three human cancer cell lines to evaluate their in vitro cytotoxic property. Most of the trifluoromethyl substituted derivatives exhibited better growth inhibition activity than their methyl substituted analogues. The SIRT1 inhibition activity of two potent molecules (I17 and I18) was investigated and the SIRT1 IC50 values are 35.25 and 37.36 μM, respectively for I17 and I18. The molecular docking studies with SIRT1 enzyme revealed favourable interactions of the molecule I17 with the amino acids constituting the receptor enzyme.

  19. Phentolamine inhibits angiogenesis in vitro: Suppression of proliferation migration and differentiation of human endothelial cells.

    PubMed

    Pan, Liangli; Liu, Chenyang; Kong, Yanan; Piao, Zhengguo; Cheng, Biao

    2016-06-16

    It is widely known that the β-adrenergic receptor (AR) blocker (propranolol) inhibits human endothelial cell (EC) angiogenesis in vitro, but how the α-AR antagonist (phentolamine) affects human EC angiogenesis has not yet been studied. Here, we show for the first time that both human dermal microvascular ECs (HDMECs) and human brain microvascular ECs (HBMECs) express α-ARs. Moreover, our results indicate that phentolamine inhibits the proliferation, migration, and tubulogenesis of HDMECs and HBMECs. Finally, VEGFR-2 and Ang1/2 expression of HDMECs was suppressed by phentolamine. Together, these results indicate that phentolamine impairs several critical events of neovascularization, and α-ARs, as well as the VEGF/VEGFR-2 and Ang/Tie-2 signaling pathways, may be involved in these processes. Our results suggest a novel therapeutic strategy for the use of α-blockers in the treatment of human angiogenesis-dependent diseases.

  20. Transintestinal secretion of ciprofloxacin, grepafloxacin and sparfloxacin: in vitro and in situ inhibition studies.

    PubMed

    Rodríguez-Ibáñez, M; Nalda-Molina, R; Montalar-Montero, M; Bermejo, M V; Merino, V; Garrigues, T M

    2003-03-01

    The influence of the secretion process on the absorption of ciprofloxacin, grepafloxacin and sparfloxacin has been evaluated by means of inhibition studies. Two well known P-glycoprotein inhibitors (cyclosporine, verapamil), a mixed inhibitor of P-glycoprotein and the organic cation transporter OCT1 (quinidine) and a well established MRP substrate (p-aminohipuric acid) have been selected in order to distinguish the possible carriers implicated. An in situ rat gut perfusion model and CACO-2 permeability studies are used. Both methods suggest the involvement of several types of efflux transporters for every fluoroquinolone. The relevance of the secretory pathway depends on the intrinsic permeability of the quinolone. The in vitro model seems to be more suitable for discriminating mechanisms underlying the absorption process, while in situ studies are less sensitive to inhibition studies.

  1. Baicalein Inhibits Staphylococcus aureus Biofilm Formation and the Quorum Sensing System In Vitro.

    PubMed

    Chen, Yan; Liu, Tangjuan; Wang, Ke; Hou, Changchun; Cai, Shuangqi; Huang, Yingying; Du, Zhongye; Huang, Hong; Kong, Jinliang; Chen, Yiqiang

    2016-01-01

    Biofilm formed by Staphylococcus aureus significantly enhances antibiotic resistance by inhibiting the penetration of antibiotics, resulting in an increasingly serious situation. This study aimed to assess whether baicalein can prevent Staphylococcus aureus biofilm formation and whether it may have synergistic bactericidal effects with antibiotics in vitro. To do this, we used a clinically isolated strain of Staphylococcus aureus 17546 (t037) for biofilm formation. Virulence factors were detected following treatment with baicalein, and the molecular mechanism of its antibiofilm activity was studied. Plate counting, crystal violet staining, and fluorescence microscopy revealed that 32 μg/mL and 64 μg/mL baicalein clearly inhibited 3- and 7-day biofilm formation in vitro. Moreover, colony forming unit count, confocal laser scanning microscopy, and scanning electron microscopy showed that vancomycin (VCM) and baicalein generally enhanced destruction of biofilms, while VCM alone did not. Western blotting and real-time quantitative polymerase chain reaction analyses (RTQ-PCR) confirmed that baicalein treatment reduced staphylococcal enterotoxin A (SEA) and α-hemolysin (hla) levels. Most strikingly, real-time qualitative polymerase chain reaction data demonstrated that 32 μg/mL and 64 μg/mL baicalein downregulated the quorum-sensing system regulators agrA, RNAIII, and sarA, and gene expression of ica, but 16 μg/mL baicalein had no effect. In summary, baicalein inhibited Staphylococcus aureus biofilm formation, destroyed biofilms, increased the permeability of vancomycin, reduced the production of staphylococcal enterotoxin A and α-hemolysin, and inhibited the quorum sensing system. These results support baicalein as a novel drug candidate and an effective treatment strategy for Staphylococcus aureus biofilm-associated infections.

  2. β-catenin knockdown inhibits the proliferation of human glioma cells in vitro and in vivo

    PubMed Central

    WANG, ZHONG; CHEN, QIANXUE

    2016-01-01

    β-catenin is a crucial oncogene that is capable of regulating cancer progression. The aim of the present study was to clarify whether β-catenin was associated with the proliferation and progress of glioma. In order to knockdown the expression of β-catenin in human U251 glioma cells, three pairs of small interfering (si)RNA were designed and synthesized and the most effective siRNA was selected and used for silencing the endogenous β-catenin, which was detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Proliferation was subsequently detected using a methylthiazolyl-tetrazolium bromide assay and the results demonstrated that knockdown of β-catenin significantly inhibited the proliferation of U251 cells in a time- and dose-dependent manner (P<0.01). Cell apoptosis rate was analyzed using flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide staining demonstrated that β-catenin siRNA significantly increased the apoptosis of U251 cells (P<0.01). Furthermore, the results of an in vitro scratch assay demonstrated that β-catenin silencing suppressed the proliferation of U251 cells, as compared with the control group (P<0.01). In vivo, β-catenin expression levels in U251 cells were significantly inhibited (P<0.01) following β-catenin short hairpin (sh)RNA lentiviral-vector transfection, as detected by western blot analysis and RT-qPCR. Tumorigenicity experiments demonstrated that β-catenin inhibition significantly increased the survival rate of nude mice. The results of the present study demonstrated that knockdown of β-catenin expression significantly inhibited the progression of human glioma cancer cells, in vitro and in vivo; thus suggesting that β-catenin silencing may be a novel therapy for the treatment of human glioma. PMID:26998037

  3. Amphiphilic suramin dissolves Matrigel, causing an 'inhibition' artefact within in vitro angiogenesis assays.

    PubMed

    Prigozhina, Natalie L; Heisel, Andrew J; Seldeen, Jordan R; Cosford, Nicholas D P; Price, Jeffrey H

    2013-12-01

    The field of study concerning promotion and/or inhibition of angiogenesis has gathered much attention in the scientific community. A great deal of work has been invested towards defining reproducible assays to gauge for promotion or inhibition of angiogenesis in response to drug treatments or growth conditions. Two common components of these assays were noted by our group to have an unexpected and previously unreported interaction. Suramin is a commercially available compound, commonly used as a positive control for in vitro angiogenic inhibition assays. Matrigel is a popular extracellular substrate that supports angiogenic network formation when endothelial cells are cultured on its surface. However, our group demonstrated that suramin alone (without the presence of cells) will actively dissolve Matrigel, causing the extracellular matrix to transition from the gel-like physical state to a more liquid state. This causes cells on the Matrigel to congregate and sink to the bottom of the well. Therefore, previous observations of inhibition of endothelial cell angiogenesis through the incubation with suramin (including previous observations made by our group) are, largely, an artefact caused by suramin and matrix interaction rather than suramin and cells interaction, as previously reported. Our results suggest that the presence of sulphate groups and amphiphilic properties of suramin are likely responsible for the disruption of the matrix layer. We believe that this information is of prime importance to anyone using similar in vitro models, or employing suramin in any therapy or drug development assays. © 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.

  4. Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays.

    PubMed

    Wilson, Danny W; Crabb, Brendan S; Beeson, James G

    2010-06-03

    Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development. The D10 P. falciparum line was transfected to express green fluorescent protein (GFP). In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry. Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts. Flow cytometry based assays using GFP-fluorescent parasites proved sensitive and highly reproducible for

  5. Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays

    PubMed Central

    2010-01-01

    Background Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development. Methods The D10 P. falciparum line was transfected to express green fluorescent protein (GFP). In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry. Results Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts. Conclusions Flow cytometry based assays using GFP-fluorescent parasites proved

  6. Effects of salvianolic acid B on in vitro growth inhibition and apoptosis induction of retinoblastoma cells

    PubMed Central

    Liu, Xing-An

    2012-01-01

    AIM To observe the effects of salvianolic acid B (SalB) on in vitro growth inhibition and apoptosis induction of retinoblastoma HXO-RB44 cells. METHODS The effects of SalB on the HXO-RB44 cells proliferation in vitro were observed by MTT colorimetric method. The morphological changes of apoptosis before and after the treatment of SalB were observed by Hoechst 33258 fluorescent staining method. Apoptosis rate and cell cycle changes of HXO-RB44 cells were detected by flow cytometer at 48 hours after treated by SalB. The expression changes of Caspase-3 protein in HXO-RB44 cells were detected by Western Blot. RESULTS SalB significantly inhibited the growth of HXO-RB44 cells, while the inhibition was in a concentration-and time-dependent manner. The results of fluorescent staining method indicated that HXO-RB44 cells showed significant phenomenon of apoptosis including karyorrhexis, fragmentation and the formation of apoptotic bodies, etc. after 24, 48 and 72 hours co-culturing of SalB and HXO-RB44 cells. The results of flow cytometer showed that the apoptosis rate and the proportion of cells in S phase were gradually increased at 48 hours and 72 hours after treated by different concentrations of SalB. Western Blot strip showed that the expression of Caspase-3 protein in HXO-RB44 cells was gradually increased with the increase of the concentration of SalB. CONCLUSION SalB can significantly affect on HXO-RB44 cells growth inhibition and apoptosis induction which may be achieved through the up-regulation of Caspase-3 expression and the induction of cell cycle arrest. PMID:22773971

  7. In vitro inhibition of Eimeria tenella sporozoite invasion into host cells by probiotics.

    PubMed

    Hessenberger, S; Schatzmayr, G; Teichmann, K

    2016-10-15

    The aim was to study the effects of probiotics isolated from the intestinal tract of livestock animals on Eimeria tenella invasion into Madin-Darby bovine kidney (MDBK) cells in vitro. E. tenella sporozoites were purified and labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester before seeding on cell cultures, and invasion was evaluated by fluorescence microscopy. Two protocols (A and B) were used. In protocol A, Enterococcus faecium # 589 or Lactobacillus salivarius subsp. salivarius # 505 were added together with sporozoites to MDBK cell cultures and invasion was evaluated after incubation for approximately 20h. Viable, dead, or spent culture supernatants of probiotics were tested. In protocol B, viable probiotics were incubated with MDBK cells for one hour before sporozoites were added and invasion was evaluated after two more hours of incubation. Parasite invasion of viable, dead, or spent culture supernatant of E. faecium # 589 was assessed. Using protocol A, it was shown that parasite invasion was inhibited by viable (80%) or dead (75%) E. faecium # 589. While inhibition by viable L. salivarius subsp. salivarius # 505 was not valid at the highest concentration and not significant at the other test concentrations, dead cells inhibited parasite invasion up to 45%. Spent culture supernatants of both probiotics had no influence on parasite invasion. Using protocol B, it was shown that viable Bifidobacterium animalis subsp. animalis # 503, E. faecium # 497, E. faecium # 589, L. reuteri # 514, L. salivarius subsp. salivarius # 505, and Bacillus subtilis # 588 inhibited parasite invasion into MDBK cells up to 80%. Anticoccidial activity was strain-specific for E. faecium strains, and the strongest effect was shown by E. faecium # 589. Anticoccidial effects of some of the tested probiotics have already been shown in vivo, which makes them candidates to prevent coccidiosis. These findings have now been confirmed in vitro. The used parasite invasion

  8. Walnut leaf extract inhibits PTP1B and enhances glucose-uptake in vitro.

    PubMed

    Pitschmann, Anna; Zehl, Martin; Atanasov, Atanas G; Dirsch, Verena M; Heiss, Elke; Glasl, Sabine

    2014-03-28

    Walnut, Juglans regia L. (Juglandaceae), is one of the medicinal plants used to treat diabetic symptoms in Austrian folk medicine. The air-dried green leaves are either used as aqueous decoctions or liquor preparations and are consumed on a daily basis. We investigated the hypoglycemic effect of a methanolic Juglans regia leaf extract on glucose uptake, protein tyrosine phosphatase 1B (PTP1B) inhibition and peroxisome proliferator-activated receptor gamma (PPARγ) activation. Hypoglycemic activity was assessed by glucose-uptake in C2C12 myocytes, inhibition of PTP1B and activation of PPARγ. Phytochemical characterization of the extract was carried out by LC-MS and GC-MS. Methanolic Juglans regia leaf extract enhanced the glucose uptake rate in C2C12 myocytes at concentrations of 25µg/mL compared to untreated cells. This activity may partly be explained by the inhibition of PTP1B but not PPARγ agonism. LC-MS analyses revealed chlorogenic acid (1), 3-p-coumaroylquinic acid (2), a trihydroxynaphthalene-hexoside (3), as well as eight flavonoids (4-11) as main phenolic constituents in the active extract. The finding that Juglans regia leaf extract enhances glucose uptake and inhibits PTP1B provides an in vitro-based rationale for the traditional use of walnut leaf preparations against elevated blood-glucose levels. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. The rhizobacterium Arthrobacter agilis produces dimethylhexadecylamine, a compound that inhibits growth of phytopathogenic fungi in vitro.

    PubMed

    Velázquez-Becerra, Crisanto; Macías-Rodríguez, Lourdes I; López-Bucio, José; Flores-Cortez, Idolina; Santoyo, Gustavo; Hernández-Soberano, Christian; Valencia-Cantero, Eduardo

    2013-12-01

    Plant diseases caused by fungal pathogens such as Botrytis cinerea and the oomycete Phytophthora cinnamomi affect agricultural production worldwide. Control of these pests can be done by the use of fungicides such as captan, which may have deleterious effects on human health. This study demonstrates that the rhizobacterium Arthrobacter agilis UMCV2 produces volatile organic compounds that inhibit the growth of B. cinerea in vitro. A single compound from the volatile blends, namely dimethylhexadecylamine (DMHDA), could inhibit the growth of both B. cinerea and P. cinnamomi when supplied to the growth medium in low concentrations. DMHDA also inhibited the growth of beneficial fungi Trichoderma virens and Trichoderma atroviride but at much higher concentrations. DMHDA-related aminolipids containing 4, 8, 10, 12, and 14 carbons in the alkyl chain were tested for their inhibitory effect on the growth of the pathogens. The results show that the most active compound from those tested was dimethyldodecylamine. This effect correlates with a decrease in the number of membrane lipids present in the mycelium of the pathogen including eicosanoic acid, (Z)-9-hexadecenoic acid, methyl ester, and (Z)-9-octadecenoic acid, methyl ester. Strawberry leaflets treated with DMHDA were not injured by the compound. These data indicate that DMHDA and related compounds, which can be produced by microorganisms may effectively inhibit the proliferation of certain plant pathogens.

  10. Crude Aloe vera Gel Shows Antioxidant Propensities and Inhibits Pancreatic Lipase and Glucose Movement In Vitro

    PubMed Central

    Taukoorah, Urmeela; Mahomoodally, M. Fawzi

    2016-01-01

    Aloe vera gel (AVG) is traditionally used in the management of diabetes, obesity, and infectious diseases. The present study aimed to investigate the inhibitory potential of AVG against α-amylase, α-glucosidase, and pancreatic lipase activity in vitro. Enzyme kinetic studies using Michaelis-Menten (K m) and Lineweaver-Burk equations were used to establish the type of inhibition. The antioxidant capacity of AVG was evaluated for its ferric reducing power, 2-diphenyl-2-picrylhydrazyl hydrate scavenging ability, nitric oxide scavenging power, and xanthine oxidase inhibitory activity. The glucose entrapment ability, antimicrobial activity, and total phenolic, flavonoid, tannin, and anthocyanin content were also determined. AVG showed a significantly higher percentage inhibition (85.56 ± 0.91) of pancreatic lipase compared to Orlistat. AVG was found to increase the Michaelis-Menten constant and decreased the maximal velocity (V max) of lipase, indicating mixed inhibition. AVG considerably inhibits glucose movement across dialysis tubes and was comparable to Arabic gum. AVG was ineffective against the tested microorganisms. Total phenolic and flavonoid contents were 66.06 ± 1.14 (GAE)/mg and 60.95 ± 0.97 (RE)/mg, respectively. AVG also showed interesting antioxidant properties. The biological activity observed in this study tends to validate some of the traditional claims of AVG as a functional food. PMID:26880905

  11. In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles

    SciTech Connect

    Park, Margriet V.D.Z. Annema, Wijtske; Salvati, Anna; Lesniak, Anna; Elsaesser, Andreas; Barnes, Clifford; McKerr, George; Howard, C. Vyvyan; Lynch, Iseult; Dawson, Kenneth A.; Piersma, Aldert H.; Jong, Wim H. de

    2009-10-01

    While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 {mu}g/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

  12. Induction of sister chromatid exchanges and inhibition of cellular proliferation in vitro. I. Caffeine

    SciTech Connect

    Guglielmi, G.E.; Vogt, T.F.; Tice, R.R.

    1982-01-01

    While many agents have been examined for their ability to induce SCE's, complete dose-response information has often been lacking. We have reexamined the ability of one such compound - caffeine - to induce SCEs and also to inhibit cellular proliferation in human peripheral lymphocytes in vitro. An acute exposure to caffeine prior to the DNA synthetic period did not affect either SCE frequency or the rate of cellular proliferation. Chronic exposure to caffeine throughout the culture period lead to both a dose-dependent increase in SCEs (SCE/sub d/ or doubling dose = 2.4 mM; SCE/sub 10/ or the dose capable of inducing 10 SCE = 1.4 mM) and a dose-dependent inhibition of cellular proliferation (IC/sub 50/ or the 50% inhibition concentration = 2.6 mM). The relative proportion of first generation metaphase cells, an assessment of proliferative inhibiton, increased linearly with increasing caffeine concentrations. However, SCE frequency increased nonlinearly over the same range of caffeine concentrations. Examination of the ratio of nonsymmetrical to symmetrical SCEs in third generation metaphase cells indicated that caffeine induced SCEs in equal frequency in each of three successive generations. The dependency of SCE induction and cellular proliferative inhibition on caffeine's presence during the DNA synthetic period suggests that caffeine may act as an antimetabolite in normal human cells.

  13. [IL-37 inhibits the proliferation, invasion and migration of SMMC-7721 cells in vitro].

    PubMed

    Zhang, Guoqing; Tang, Chengyoug; Tan, Jingyu; Liu, Rui; Zhou, Mi; Wu, Zhongjun

    2015-10-01

    To analyze the impact of interleukin-37 (IL-37) on proliferation, migration and invasion of SMMC-7721 hepatocellular carcinoma cells and the related molecular mechanism. SMMC-7721 cells cultured in vitro were divided into the (50, 100, 200, 500) ng/mL recombinant human IL-37 (rhIL-37) treated groups and control group. Cell proliferation ability was detected by CCK-8 assay. Wound healing assay was used to observe cell migration ability. Transwell(TM) assay was performed to test the cell invasion ability. The levels of signal transducers and activators of transcription 3 (STAT3), phospho-STAT3 (p-STAT3) and matrix metalloproteinase-2 (MMP-2) were determined using Western blotting. CCK-8 assay showed that rhIL-37 inhibited the proliferation of SMMC-7721 cells. Wound healing assay revealed that rhIL-37 inhibited the migration of SMMC-7721 cells. Transwell(TM) indicated that rhIL-37 inhibited the invasion of SMMC-7721 cells. Western blotting demonstrated that rhIL-37 down-regulated the expressions of p-STAT3 and MMP-2. IL-37 can inhibit the proliferation, migration and invasion of SMMC-7721 cells. It may be related with the down-regulation of p-STAT3 and MMP-2.

  14. Inhibition of human topoisomerase II in vitro by bioactive benzene metabolites.

    PubMed Central

    Frantz, C E; Chen, H; Eastmond, D A

    1996-01-01

    Benzene is a clastogenic and carcinogenic agent that induces acute myelogenous leukemia in humans and multiple of tumors in animals. Previous research has indicated that benzene must first be metabolized to one or more bioactive species to exert its myelotoxic and genotoxic effects. To better understand the possible role of individual benzene metabolites in the leukemogenic process, as well as to further investigate inhibition of topoisomerase II by benzene metabolites, a series of known and putative benzene metabolites, phenol, 4,4'-biphenol, 2,2'-biphenol, hydroquinone, catechol, 1,2,4-benzenetriol, 1,4-benzoquinone, and trans-trans-muconaldehyde were tested for inhibitory effects in vitro on the human topoisomerase II enzyme. With minor modifications of the standard assay conditions, 1,4-benzoquinone and trans-trans-muconaldehyde were shown to be directly inhibitory, whereas all of the phenolic metabolites were shown to inhibit enzymatic activity following bioactivation using a peroxidase activation system. The majority of compounds tested inhibited topoisomerase II at concentrations at or below 10 microM. These results confirm and expand upon previous findings from our laboratory and indicate that many of the metabolites of benzene could potentially interfere with topoisomerase II. Since other inhibitors of topoisomerase II have been shown to induce leukemia in humans, inhibition of this enzyme by benzene metabolites may also play a role in the carcinogenic effects of benzene. PMID:9118913

  15. In Vitro α-Amylase Inhibition and Antioxidant Activities of Methanolic Extract of Amaranthus Caudatus Linn

    PubMed Central

    Kumar, Ashok; Khan, Saleemulla

    2011-01-01

    Objectives The present study was aimed to investigate the α-amylase inhibition and antioxidant activities of methanolic extract of Amaranthus caudatus Linn (MeAc). Methods Methanolic extract of Amaranthus caudatus was screened for α-amylase inhibition activity by CNPG3 method (2-chloro-p-nitrophenyl-α-D-maltotrioside) and antioxidant activity was evaluated by 1,1-diphenyl-2-picryl-hydrazile (DPPH) free radical scavenging, superoxide dismutase (SOD) scavenging, hydroxyl free radical scavenging, nitric oxide (NO) radical scavenging, and 2.2’-azinobis-3-ethylbenzothiazole-6-sulfonic acid (ABTS) radical scavenging assays. MeAc was also screened for non enzymatic hemoglycosylation. Results The methanolic extract of Amaranthus caudatus showed potent α-amylase inhibition activity (IC50 19.233 µg/ml). MeAc showed significant antioxidant activity in all the in vitro antioxidant models. Furthermore, the MeAc was found to be extremely effective in scavenging ABTS radical activity (IC50 48.75±1.1 µg/ml) when compared to DPPH (IC50 77.5±0.4 µg/ml), SOD (IC50 62.5±2.1 µg/ml), hydroxyl (IC50 88.50±1.8 µg/ml) and NO (IC50 67.5±2.2 µg/ml) scavenging activity. Conclusions The methanolic extract of A. caudatus showed potent α-amylase inhibition and antioxidant activities. PMID:22043408

  16. Crude Aloe vera Gel Shows Antioxidant Propensities and Inhibits Pancreatic Lipase and Glucose Movement In Vitro.

    PubMed

    Taukoorah, Urmeela; Mahomoodally, M Fawzi

    2016-01-01

    Aloe vera gel (AVG) is traditionally used in the management of diabetes, obesity, and infectious diseases. The present study aimed to investigate the inhibitory potential of AVG against α-amylase, α-glucosidase, and pancreatic lipase activity in vitro. Enzyme kinetic studies using Michaelis-Menten (K m ) and Lineweaver-Burk equations were used to establish the type of inhibition. The antioxidant capacity of AVG was evaluated for its ferric reducing power, 2-diphenyl-2-picrylhydrazyl hydrate scavenging ability, nitric oxide scavenging power, and xanthine oxidase inhibitory activity. The glucose entrapment ability, antimicrobial activity, and total phenolic, flavonoid, tannin, and anthocyanin content were also determined. AVG showed a significantly higher percentage inhibition (85.56 ± 0.91) of pancreatic lipase compared to Orlistat. AVG was found to increase the Michaelis-Menten constant and decreased the maximal velocity (V max) of lipase, indicating mixed inhibition. AVG considerably inhibits glucose movement across dialysis tubes and was comparable to Arabic gum. AVG was ineffective against the tested microorganisms. Total phenolic and flavonoid contents were 66.06 ± 1.14 (GAE)/mg and 60.95 ± 0.97 (RE)/mg, respectively. AVG also showed interesting antioxidant properties. The biological activity observed in this study tends to validate some of the traditional claims of AVG as a functional food.

  17. Salicylanilide pyrazinoates inhibit in vitro multidrug-resistant Mycobacterium tuberculosis strains, atypical mycobacteria and isocitrate lyase.

    PubMed

    Krátký, Martin; Vinšová, Jarmila; Novotná, Eva; Stolaříková, Jiřina

    2014-03-12

    The development of antimicrobial agents represents an up-to-date topic. This study investigated in vitro antimycobacterial activity, mycobacterial isocitrate lyase inhibition and cytotoxicity of salicylanilide pyrazinoates. They may be considered being mutual prodrugs of both antimycobacterial active salicylanilides and pyrazinoic acid (POA), an active metabolite of pyrazinamide, in which these esters are likely hydrolysed without presence of pyrazinamidase/nicotinamidase. Minimum inhibitory concentrations (MICs) of the esters were within the range 0.5-8 μmol/l for Mycobacterium tuberculosis and 1-32 μmol/l for nontuberculous mycobacteria (Mycobacterium avium, Mycobacterium kansasii). All esters showed a weak inhibition (8-17%) of isocitrate lyase at the concentration of 10 μmol/l. The most active pyrazinoates showed MICs for multidrug-resistant tuberculosis strains in the range of 0.125-2 μmol/l and no cross-resistance with clinically used drugs, thus being the most in vitro efficacious salicylanilide esters with 4-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate superiority (MICs⩽0.25 μmol/l). This promising activity is likely due to an additive or synergistic effect of released POA and salicylanilides. Selectivity indexes for the most active salicylanilide pyrazinoates ranged up to 64, making some derivatives being attractive candidates for the next research; 4-bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl pyrazine-2-carboxylate showed the most convenient toxicity profile.

  18. Dehydroepiandrosterone (DHEA) treatment in vitro inhibits adipogenesis in human omental but not subcutaneous adipose tissue.

    PubMed

    Rice, S P L; Zhang, L; Grennan-Jones, F; Agarwal, N; Lewis, M D; Rees, D A; Ludgate, M

    2010-05-14

    Dehydroepiandrosterone (DHEA), a precursor sex steroid, circulates in sulphated form (DHEAS). Serum DHEAS concentrations are inversely correlated with metabolic syndrome components and in vivo/in vitro studies suggest a role in modulating adipose mass. To investigate further, we assessed the in vitro biological effect of DHEA in white (3T3-L1) and brown (PAZ6) preadipocyte cell lines and human primary preadipocytes. DHEA (from 10(-8)M) caused concentration-dependent proliferation inhibition of 3T3-L1 and PAZ6 preadipocytes. Cell cycle analysis demonstrated unaltered apoptosis but indicated blockade at G1/S or G2/M in 3T3-L1 and PAZ6, respectively. Preadipocyte cell-line adipogenesis was not affected. In human primary subcutaneous and omental preadipocytes, DHEA significantly inhibited proliferation from 10(-8)M. DHEA 10(-7)M had opposing effects on adipogenesis in the two fat depots. Subcutaneous preadipocyte differentiation was unaffected or increased whereas omental preadipocytes showed significantly reduced adipogenesis. We conclude that DHEA exerts fat depot-specific differences which modulate body composition by limiting omental fat production.

  19. Aged black garlic extract induces inhibition of gastric cancer cell growth in vitro and in vivo.

    PubMed

    Wang, Xin; Jiao, Fei; Wang, Qin-Wen; Wang, Juan; Yang, Ke; Hu, Rong-Rong; Liu, Han-Chen; Wang, Hong-Yang; Wang, Yi-Shan

    2012-01-01

    There is mounting evidence that garlic extracts possess significant anticancer actions. However, no studies have been reported on the effects of aged black garlic extracts (ABGE) on gastric cancer in vitro or in vivo. To examine the potential action of ABGE against gastric cancer, the present study evaluated its effect on the inhibition of cell proliferation and induction of apoptosis in SGC-7901 human gastric cancer cells. Additionally, we performed an in vivo study by inoculating the murine foregastric carcinoma cell line in Kunming mice and treating them with various doses of ABGE (0, 200, 400 and 800 mg/kg, intraperitoneally) for 2 weeks. Dose-dependent apoptosis was detected in ABGE-treated cells in in vitro studies. In tumor-bearing mice, significant antitumor effects of ABGE were observed, such as growth inhibition of inoculated tumors. Further investigation of serum superoxide dismutases, glutathione peroxidase, interleukin-2 and the increased indices of spleen and thymus indicated that the anticancer action of ABGE may be partly due to its antioxidant and immunomodulative effects.

  20. Terminalia bellirica Extract Inhibits Low-Density Lipoprotein Oxidation and Macrophage Inflammatory Response in Vitro

    PubMed Central

    Tanaka, Miori; Kishimoto, Yoshimi; Saita, Emi; Suzuki-Sugihara, Norie; Kamiya, Tomoyasu; Taguchi, Chie; Iida, Kaoruko; Kondo, Kazuo

    2016-01-01

    The deciduous tree Terminalia bellirica found in Southeast Asia is extensively used in traditional Indian Ayurvedic medicine for the treatment of hypertension, rheumatism, and diabetes. The anti-atherogenic effect of Terminalia bellirica fruit has not been fully elucidated. Here, we investigated the effect of Terminalia bellirica extract (TBE) on low-density lipoprotein (LDL) oxidation and inflammation in macrophages. TBE showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (EC50: 7.2 ± 1.2 μg/mL) and 15-lipoxygenase inhibitory activity. TBE also significantly inhibited free radical-induced LDL oxidation compared to the solvent control in vitro. In THP-1 macrophages, TBE treatment resulted in significant decreases of the mRNA expression of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and lectin-like oxidized LDL receptor-1 (LOX-1). TBE also reduced matrix metalloproteinase (MMP)-9 secretion and intracellular reactive oxygen species (ROS) production in THP-1 macrophages. These results show that TBE has the inhibitory effects on LDL oxidation and macrophage inflammatory response in vitro, suggesting that its in vivo use might inhibit atherosclerosis plaque progression. PMID:27314393

  1. In vitro transcription and translation inhibition via DNA functionalized gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Conde, J.; de la Fuente, J. M.; Baptista, P. V.

    2010-12-01

    The use of gold nanoparticles (AuNPs) has been gaining momentum as vectors for gene silencing strategies, combining the AuNPs' ease of functionalization with DNA and/or siRNA, high loading capacity and fast uptake by target cells. Here, we used AuNP functionalized with thiolated oligonucleotides to specifically inhibit transcription in vitro, demonstrating the synergetic effect between AuNPs and a specific antisense sequence that blocks the T7 promoter region. Also, AuNPs efficiently protect the antisense oligonucleotide against nuclease degradation, which can thus retain its inhibitory potential. In addition, we demonstrate that AuNPs functionalized with a thiolated oligonucleotide complementary to the ribosome binding site and the start codon, effectively shut down in vitro translation. Together, these two approaches can provide for a simple yet robust experimental set up to test for efficient gene silencing of AuNP-DNA conjugates. What is more, these results show that appropriate functionalization of AuNPs can be used as a dual targeting approach to an enhanced control of gene expression—inhibition of both transcription and translation.

  2. Human adipose tissue-derived mesenchymal stem cells inhibit melanoma growth in vitro and in vivo.

    PubMed

    Ahn, Jin-Ok; Coh, Ye-Rin; Lee, Hee-Woo; Shin, Il-Seob; Kang, Sung-Keun; Youn, Hwa-Young

    2015-01-01

    The effects of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the growth of human malignancies, including melanoma, are controversial and the underlying mechanisms are not yet-well understood. The aim of the present study was to investigate the in vitro and in vivo anti-tumor effects of human AT-MSCs on human melanoma. The inhibitory effect of AT-MSC-conditioned medium (AT-MSC-CM) on the growth of A375SM and A375P (human melanoma) cells was evaluated using a cell viability assay. Cell-cycle arrest and apoptosis in melanoma cells were investigated by flow cytometry and western blot analysis. To evaluate the in vivo anti-tumor effect of AT-MSCs, CM-DiI-labeled AT-MSCs were circumtumorally injected in tumor-bearing athymic mice and tumor size was measured. AT-MSC-CM inhibited melanoma growth by altering cell-cycle distribution and inducing apoptosis in vitro. AT-MSCs suppressed tumor growth in tumor-bearing athymic mice and fluorescence analysis showed that AT-MSCs migrated efficiently to tumor tissues. AT-MSCs inhibit the growth of melanoma suggesting promise as a novel therapeutic agent for melanoma. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  3. Inhibition of adenovirus multiplication by inosine pranobex and interferon α in vitro

    PubMed Central

    Lasek, Witold; Janyst, Michał; Młynarczyk, Grażyna

    2016-01-01

    There are no specific antivirals designed for adenoviral infections. Due to many cases of adenovirus infections worldwide, epidemic nature of some types of adenoviruses, and growing number of patients with severe adenoviral infections resulting from dysfunction the immune system, the need for searching an effective and safe therapy is increasing. Inosine pranobex exerts antiviral effects which are both direct and secondary to immunomodulatory activity. In the present study we evaluated in vitro effect of inosine pranobex and interferon α (IFN-α) on replication of HAdV-2 and HAdV-5. The effectiveness of inosine pranobex under these conditions has not been previously reported. In conducted study we reported that inosine pranobex reduced the titer of infectious HAdV-2 and HAdV-5 in vitro. Higher concentrations of IP strongly inhibited multiplication of viruses. Combination of inosine pranobex and IFN-α display higher efficacy than either treatment alone and suggest that both agents may increase therapeutic effectiveness without augmenting toxic effects. Combination index calculations showed that inosine pranobex and INF-α synergistically inhibit HAdV-2 and HAdV-5 titers in A549 cells. PMID:26862302

  4. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity.

    PubMed

    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li

    2016-04-22

    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  5. Sinusoidal electromagnetic fields promote bone formation and inhibit bone resorption in rat femoral tissues in vitro.

    PubMed

    Zhou, Jian; Ma, Xiao-Ni; Gao, Yu-Hai; Yan, Juan-Li; Shi, Wen-Gui; Xian, Cory J; Chen, Ke-Ming

    2016-01-01

    Effects of sinusoidal electromagnetic fields (SEMFs) on bone metabolism have not yet been well defined. The present study investigated SEMF effects on bone formation and resorption in rat femur bone tissues in vitro. Cultured femur diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues were treated with 50 Hz 1.8 mT SEMFs 1.5 h per day for up to 12 days and treatment effects on bone formation and resorption markers and associated gene expression were examined. Treatment with SEMFs caused a significant increase in alkaline phosphatase (ALP) activity and inhibited the tartrate-resistant acid phosphatase (TRACP) activity in the femoral diaphyseal or metaphyseal tissues. SEMFs also significantly increased levels of mRNA expression of osterix (OSX), insulin-like growth factor (IGF-1) and ALP in the bone tissues. SEMF treatment decreased glucose content and increased lactic acid contents in the culture conditioned medium. In addition, treatment with SEMFs decreased mRNA expression levels of bone resorption-related genes TRACP, macrophage colony stimulating factor (M-CSF) and cathepsin K (CTSK) in the cultured bone tissues. In conclusion, the current study demonstrated that treatment with 1.8 mT SEMFs at 1.5 h per day promoted bone formation, increased metabolism and inhibited resorption in both metaphyseal and diaphyseal bone tissues in vitro.

  6. Mifepristone Inhibits Ovarian Cancer Cancer Cell Growth In Vitro and In Vivo

    PubMed Central

    Goyeneche, Alicia A.; Carón, Rubén W.; Telleria, Carlos M.

    2008-01-01

    Purpose These studies were designed to determine whether the synthetic steroid mifepristone inhibits ovarian cancer growth in vitro and in vivo and the molecular mechanisms involved. Experimental Design The effect of mifepristone on ovarian cancer cell growth in vitro was studied in ovarian cancer cell lines of different genetic backgrounds (SK-OV-3, Caov-3, OV2008, and IGROV-1). In addition, the growth inhibition capacity of mifepristone on ovarian carcinoma xenografts was tested in nude mice. Results Mifepristone inhibited ovarian cancer cell proliferation in a dose- and time-dependent manner. The cytostatic effect of mifepristone was confirmed in a clonogenic survival assay and was not linked to loss of viability. Mifepristone blocked DNA synthesis, arrested the cell cycle at the G1-to-S transition, up-regulated cyclin-dependent kinase (cdk) inhibitors p21cip1 and p27kip1, down-regulated transcription factor E2F1, decreased expression of the E2F1 regulated genes, cdk1 (cdc2) and cyclin A, and modestly decreased cdk2 and cyclin E levels. The abrupt arrest in cell growth induced by mifepristone correlated with reduced cdk2 activity, increased association of cdk2 with p21cip1 and p27kip1, increased nuclear localization of the cdk inhibitors, and reduced nuclear abundance of cdk2 and cyclin E. In vivo, mifepristone significantly delayed the growth of ovarian carcinoma xenografts in a dose-dependent manner and without apparent toxic effects for the animals. Conclusions These preclinical studies demonstrate that mifepristone is effective as a single agent in vitro and in vivo, inhibiting the growth of human epithelial ovarian cancer cells. Mifepristone markedly reduces cdk2 activity likely due to increased association of cdk2 with the cdk inhibitors p21cip1 and p27kip1 and reduced nuclear cdk2/cyclin E complex availability. Acting as a cytostatic agent mifepristone promises to be of translational significance in ovarian cancer therapeutics. PMID:17545545

  7. Kaempferol inhibits the growth and metastasis of cholangiocarcinoma in vitro and in vivo.

    PubMed

    Qin, Youyou; Cui, Wu; Yang, Xuewei; Tong, Baifeng

    2016-03-01

    Kaempferol is a flavonoid that has been reported to exhibit antitumor activity in various malignant tumors. However, the role of kaempferol on cholangiocarcinoma (CCA) is largely unknown. In this article, we found that kaempferol inhibited proliferation, reduced colony formation ability, and induced apoptosis in HCCC9810 and QBC939 cells in vitro. Results from transwell assay and wound-healing assay demonstrated that kaempferol significantly suppressed the migration and invasion abilities of HCCC9810 and QBC939 cells in vitro. Kaempferol was found to decrease the expression of Bcl-2 and increase the expressions of Bax, Fas, cleaved-caspase 3, cleaved-caspase 8, cleaved-caspase 9, and cleaved-PARP. In addition, kaempferol also downregulated the levels of phosphorylated AKT, TIMP2, and MMP2. In vivo, it was found that the volume of subcutaneous xenograft (0.15 cm(3)) in the kaempferol-treated group was smaller than that (0.6 cm(3)) in the control group. Kaempferol also suppressed the number and volume of metastasis foci in the lung metastasis model, with no marked effects on body weight of mice. Immunohistochemistry assay showed that the number of Ki-67-positive cells was lower in the kaempferol-treated group than that in the control group. We further confirmed that the changes of apoptosis- and invasion-related proteins after kaempferol treatment in vivo were similar to the results in vitro. These data suggest that kaempferol may be a promising candidate agent for the treatment of CCA.

  8. The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays

    PubMed Central

    Tanner, Rachel; O’Shea, Matthew K.; White, Andrew D.; Müller, Julius; Harrington-Kandt, Rachel; Matsumiya, Magali; Dennis, Mike J.; Parizotto, Eneida A.; Harris, Stephanie; Stylianou, Elena; Naranbhai, Vivek; Bettencourt, Paulo; Drakesmith, Hal; Sharpe, Sally; Fletcher, Helen A.; McShane, Helen

    2017-01-01

    The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays. PMID:28256545

  9. A cortical astrocyte subpopulation inhibits axon growth in vitro and in vivo.

    PubMed

    Liu, Rui; Wang, Zhe; Gou, Lin; Xu, Hanpeng

    2015-08-01

    Astrocytes are the most heterogeneous and predominant glial cell type in the central nervous system. However, the functional significance of this heterogeneity remains to be elucidated. Following injury, damaged astrocytes inhibit axonal regeneration in vivo and in vitro. Cultured primary astrocytes are commonly considered good supportive substrates for neuron attachment and axon regeneration. However, it is not known whether different populations of cells in the heterogeneous astrocyte culture affect neuron behavior in the same way. In the present study, the effect of astrocyte heterogeneity on neuronal attachment and neurite outgrowth was examined using an in vitro and in vivo coculture system. In vitro, neonatal cortical astrocytes were co-cultured with purified dorsal root ganglia (DRG) neurons and astrocyte growth morphology, neuron attachment and neurite growth were evaluated. The results demonstrated that the heterogeneous astrocyte cells showed two different types of growth pattern, typical and atypical. Typical astrocytes were supportive to neuron attachment and neurite growth, which was consistent with previous studies, whereas atypical astrocytes inhibited neuron attachment and neurite growth. These inhibitory astrocytes exhibited a special growth pattern with various shapes and sizes, a high cell density, few oligodendrocytes on the top layer and occupied a smaller growth area compared with typical astrocytes. Neurites extended freely on typical supportive astrocyte populations, however, moved away when they reached atypical astrocyte growth pattern. Neurons growing on the atypical astrocyte pattern demonstrated minimal neurite outgrowth and these neurites had a dystrophic appearance, however, neuronal survival was unaffected. Immunocytochemistry studies demonstrated that these atypical inhibitory astrocytes were glial fibrillary acidic protein (GFAP) positive cells. The existence of inhibitory astrocyte subpopulations in normal astrocytes reflects the

  10. Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

    PubMed

    Xu, Cheng; Guo, Tz-Chun; Mutoloki, Stephen; Haugland, Øyvind; Marjara, Inderjit S; Evensen, Øystein

    2010-09-01

    Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-alpha and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-alpha induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-alpha is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-alpha results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-gamma has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-alpha, while postinfection antiviral responses or postinfection treatment with rIFN-alpha is not able to limit viral replication.

  11. alpha-Chaconine inhibits angiogenesis in vitro by reducing matrix metalloproteinase-2.

    PubMed

    Lu, Ming-Kun; Chen, Pei-Hsieng; Shih, Yuan-Wei; Chang, Ya-Ting; Huang, En-Tze; Liu, Cheng-Ruei; Chen, Pin-Shern

    2010-01-01

    alpha-Chaconine, a naturally occurring steroidal glycoalkaloid in potato sprouts, was found to possess anti-carcinogenic properties, such as inhibiting proliferation, migration, invasion, and inducing apoptosis of tumor cells. However, the effect of alpha-chaconine on tumor angiogenesis remains unclear. In the present study, we examined the effect of alpha-chaconine on angiogenesis in vitro. Data demonstrated that alpha-chaconine inhibited proliferation of bovine aortic endothelial cells (BAECs) in a dose-dependent manner. When treated with non-toxic doses of alpha-chaconine, cell migration, invasion and tube formation were markedly suppressed. Furthermore, alpha-chaconine reduced the expression and activity of matrix metalloproteinase-2 (MMP-2), which is involved in angiogenesis. Our biochemical assays indicated that alpha-chaconine potently suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), phosphatidylinositide-3 kinase (PI3K) and Akt, while it did not affect phosphorylation of extracellular signal regulating kinase (ERK) and p38. In addition, alpha-chaconine significantly increased the cytoplasmic level of inhibitors of kappaBalpha (IkappaBalpha) and decreased the nuclear level of nuclear factor kappa B (NF-kappaB), suggesting that alpha-chaconine could inhibit NF-kappaB activity. Furthermore, the treatment of inhibitors specific for JNK (SP600125), PI3K (LY294002) or NF-kappaB (pyrrolidine dithiocarbamate) to BAECs reduced tube formation. Taken together, the results suggested that alpha-chaconine inhibited migration, invasion and tube formation of BAECs by reducing MMP-2 activities, as well as JNK and PI3K/Akt signaling pathways and inhibition of NF-kappaB activity. These findings reveal a new therapeutic potential for alpha-chaconine on anti-angiogenic therapy.

  12. Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

    SciTech Connect

    Xue, Gang; Zou, Xi; Zhou, Jin-Yong; Sun, Wei; Wu, Jian; Xu, Jia-Li; Wang, Rui-Ping

    2013-09-20

    Highlights: •Raddeanin A is a triterpenoid saponin in herb medicine Anemone raddeana Regel. •Raddeanin A can inhibit 3 kinds of gastric cancer cells’ proliferation and invasion. •Caspase-cascades’ activation indicates apoptosis induced by Raddeanin A. •MMPs, RECK, Rhoc and E-cad are involved in Raddeanin A-induced invasion inhibition. -- Abstract: Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A’s dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug.

  13. Progesterone inhibits the growth of human neuroblastoma: in vitro and in vivo evidence.

    PubMed

    Atif, Fahim; Sayeed, Iqbal; Yousuf, Seema; Ishrat, Tauheed; Hua, Fang; Wang, Jun; Brat, Daniel J; Stein, Donald G

    2011-01-01

    We investigated the antitumorogenic effects of progesterone (P4) in a human neuroblastoma (SK-N-AS) cell line in vitro and in a mouse xenograft model of neuroblastoma. The safety of P4 was tested in rat primary cortical neurons and human foreskin fibroblasts (HFF-1). At high doses, P4 significantly (P < 0.05) decreased SK-N-AS cell viability in vitro, and this effect was not blocked either by 5α-reductase inhibitor, finasteride or the P4 receptor antagonist RU486. Even at very high doses, P4 did not induce any cell death in healthy primary cortical neurons or HFF-1. The bioavailability of P4 24 h after the last injection in the serum of treated animals was significantly (P < 0.05) higher (10-33 μg/mL) than in untreated animals. In nude mice, P4 (50 and 100 mg/kg) inhibited neuroblastoma growth by ~50% over 8 d of treatment. No drug toxicity was observed in the mice, as measured by body weight and activity. P4 suppressed the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-9, MMP-2), which are involved in tumor vascular development. High-dose P4 inhibited tumor growth by suppressing cell proliferation and inducing apoptosis, as evidenced by the expression of proliferating cell nuclear antigen and cleaved caspase-3. P4 significantly increased the expression of P4 receptor isoform-A and suppressed phospho-Akt (Ser437) expression. In conclusion, at high doses, P4 effectively inhibits the growth of solid neuroblastoma tumor and has high bioavailability, selective toxicity and a high margin of safety, making it a possible candidate for further study as a potential clinical treatment of neuroblastoma.

  14. Alstonia scholaris R. Br. Significantly Inhibits Retinoid-Induced Skin Irritation In Vitro and In Vivo

    PubMed Central

    Lee, Soo-Jin; Cho, Sun-A; An, Su-Sun; Na, Yong-Joo; Park, Nok-Hyun; Kim, Han-Sung; Lee, Chan-Woo; Kim, Han-Kon; Kim, Eun-Kyung; Jang, Young-Pyo; Kim, Jin-Woong

    2012-01-01

    Topical retinoids inhibit matrix metalloproteinases and accelerate collagen synthesis, thereby triggering antiaging effects in the skin. However, topical retinoids can cause severe skin reactions, including scaling, erythema, papules, and inflammation. The present study demonstrates that the ethanolic bark extract of Alstonia scholaris R. Br. can significantly inhibit all-trans retinoic acid-induced inflammation in human HaCat keratinocyte cells. Furthermore, two representative retinoid-induced proinflammatory cytokines, monocyte chemoattractant protein-1 and interleukin-8, were significantly suppressed by A. scholaris extract (by 82.1% and 26.3% at 100 ppm, and dose-dependently across the tested concentrations) in vitro. In a cumulative irritation patch test, A. scholaris extract decreased retinol-induced skin irritation, while strengthening the ability of retinoids to inhibit matrix metalloproteinase-1 expression, which is strongly associated with aging effects. These results suggest that A. scholaris is a promising compound that may increase the antiaging function of retinoids while reducing their ability to cause skin irritation. PMID:21912567

  15. Galangin, a flavonol derived from Rhizoma Alpiniae Officinarum, inhibits acetylcholinesterase activity in vitro.

    PubMed

    Guo, Ava J Y; Xie, Heidi Q; Choi, Roy C Y; Zheng, Ken Y Z; Bi, Cathy W C; Xu, Sherry L; Dong, Tina T X; Tsim, Karl W K

    2010-09-06

    Acetylcholinesterase (AChE) inhibitors are widely used for the treatment of Alzheimer's disease (AD). Several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from plants, suggesting that herbs could potentially serve as sources for novel AChE inhibitors. Here, we searched potential AChE inhibitors from flavonoids, a group of naturally occurring compounds in plants or traditional Chinese medicines (TCM). Twenty-one flavonoids, covered different subclasses, were tested for their potential function in inhibiting AChE activity from the brain in vitro. Among all the tested flavonoids, galangin, a flavonol isolated from Rhizoma Alpiniae Officinarum, the rhizomes of Alpiniae officinarum (Hance.) showed an inhibitory effect on AChE activity with the highest inhibition by over 55% and an IC(50) of 120 microM and an enzyme-flavonoid inhibition constant (K(i)) of 74 microM. The results suggest that flavonoids could be potential candidates for further development of new drugs against AD.

  16. Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

    SciTech Connect

    Horwitz, M.S.; Friefeld, B.R.; Keiser, H.D.

    1982-12-01

    Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60/sup 0/C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases ..cap alpha.., BETA, ..gamma.. and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.

  17. Inhibition of ZEB1 expression induces redifferentiation of adult human β cells expanded in vitro.

    PubMed

    Sintov, Elad; Nathan, Gili; Knoller, Sarah; Pasmanik-Chor, Metsada; Russ, Holger A; Efrat, Shimon

    2015-08-12

    In-vitro expansion of functional adult human β-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of β-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded β-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), β-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human β-like cells for cell therapy of diabetes.

  18. An in vitro screening with emerging contaminants reveals inhibition of carboxylesterase activity in aquatic organisms.

    PubMed

    Solé, Montserrat; Sanchez-Hernandez, Juan C

    2015-12-01

    Pharmaceuticals and personal care products (PPCPs) form part of the new generation of pollutants present in many freshwater and marine ecosystems. Although environmental concentrations of these bioactive substances are low, they cause sublethal effects (e.g., enzyme inhibition) in non-target organisms. However, little is known on metabolism of PPCPs by non-mammal species. Herein, an in vitro enzyme trial was performed to explore sensitivity of carboxylesterase (CE) activity of aquatic organisms to fourteen PPCPs. The esterase activity was determined in the liver of Mediterranean freshwater fish (Barbus meridionalis and Squalius laietanus), coastal marine fish (Dicentrarchus labrax and Solea solea), middle-slope fish (Trachyrhynchus scabrus), deep-sea fish (Alepocephalus rostratus and Cataetix laticeps), and in the digestive gland of a decapod crustacean (Aristeus antennatus). Results showed that 100μM of the lipid regulators simvastatin and fenofibrate significantly inhibited (30-80% of controls) the CE activity of all target species. Among the personal care products, nonylphenol and triclosan were strong esterase inhibitors in most species (36-68% of controls). Comparison with literature data suggests that fish CE activity is as sensitive to inhibition by some PPCPs as that of mammals, although their basal activity levels are lower than in mammals. Pending further studies on the interaction between PPCPs and CE activity, we postulate that this enzyme may act as a molecular sink for certain PPCPs in a comparable way than that described for the organophosphorus pesticides.

  19. Suppression of local invasion of ameloblastoma by inhibition of matrix metalloproteinase-2 in vitro

    PubMed Central

    Wang, Anxun; Zhang, Bin; Huang, Hongzhang; Zhang, Leitao; Zeng, Donglin; Tao, Qian; Wang, Jianguang; Pan, Chaobin

    2008-01-01

    Background Ameloblastomas are odontogenic neoplasms characterized by local invasiveness. This study was conducted to address the role of matrix metalloproteinase-2 (MMP-2) in the invasiveness of ameloblastomas. Methods Plasmids containing either MMP-2 siRNA or tissue inhibitor of metalloproteinase-2 (TIMP-2) cDNA were created and subsequently transfected into primary ameloblastoma cells. Zymography, RT-PCR, and Western blots were used to assess MMP-2 activity and expression of MMP-2 and TIMP-2, as well as protein levels. Results Primary cultures of ameloblastoma cells expressed cytokeratin (CK) 14 and 16, and MMP-2, but only weakly expressed CK18 and vimentin. MMP-2 mRNA and protein levels were significantly inhibited by RNA interference (P < 0.05). Both MMP-2 siRNA and TIMP-2 overexpression inhibited MMP-2 activity and the in vitro invasiveness of ameloblastoma. Conclusion These results indicate that inhibition of MMP-2 activity suppresses the local invasiveness of ameloblastoma cells. This mechanism may serve as a novel therapeutic target in ameloblastomas pursuant to additional research. PMID:18588710

  20. In vitro and in vivo evaluation of the inhibition potential of risperidone toward clozapine biotransformation

    PubMed Central

    Chetty, Manoranjenni; d'Esposito, Fabrizio; Zhang, Wei V; Glen, John; Dore, Glenys; Stankovic, Zvijezdana; Edwards, Robert J; Ramzan, Iqbal; Murray, Michael

    2009-01-01

    AIMS To study the impact of risperidone (RISP) on clozapine (CLZ) biotransformation in vitro in microsomal fractions containing varying expression of CYP oxidases and in vivo in patients. METHODS Human liver microsomes (n= 11) were assessed for expression of CYPs 1A2, 2D6 and 3A4, because these enzymes mediate RISP and CLZ oxidation. Inhibition of CLZ oxidation by RISP was assessed. Plasma CLZ elimination was estimated in patients with schizophrenia who received either CLZ alone or the CLZ–RISP combination (n= 10 per group). RESULTS (i) The CYP3A4 and CYP1A2 inhibitors ketoconazole and fluvoxamine inhibited CLZ oxidation to varying extents in individual microsomal fractions. (ii) RISP did not inhibit CLZ oxidation, regardless of variations in CYP expression. (iii) RISP co-administration did not impair CLZ clearance. CONCLUSIONS No evidence was found for CYP-mediated inhibitory or pharmacokinetic interactions between RISP and CLZ. Occasional literature reports of such interactions may involve other pathways that participate in CLZ disposition. PMID:19843060

  1. Benzyl-isothiocyanate Induces Apoptosis and Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells in vitro

    PubMed Central

    Zhu, Mingyue; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Lin, Bo; Guo, Junli; Li, Mengsen

    2017-01-01

    Despite consideration of benzyl isothiocyanate(BITC) is applied to prevention and therapeutic of cancer, the role of BITC in inducing apoptosis, and inhibiting migration and invasion of hepatocellular carcinoma(HCC) cells is still unclear. In this study, we aim to explore the effects of BITC on the growth, migration and invasion of HCC cells in vitro. When human HCC cell lines, Bel 7402 and HLE, were treated with an optimal concentration of BITC for 48 hours, the results indicated that BITC inhibits growth and promotes apoptosis of HCC cells; BITC has a significant inhibitory effect on the migration and invasion of HCC cells. BITC stimulated expression of caspase-3/8 and PARP-1, and suppressed expression of survivin, MMP2/9 and CXCR4. BITC also inhibited the enzymatic activities of MMP2 and MMP9. Altogether, BITC was able to induce apoptosis and suppress the invasive and migratory abilities of Bel 7402 and HLE cells. The role mechanism of BITC might involve an up-regulating the expression of apoptosis-related proteins and down-regulating the expression of metastasis-related proteins. BITC may be applied as a novel chemotherapy for HCC patients. PMID:28243328

  2. Cryptoporic acid E from Cryptoporus volvatus inhibits influenza virus replication in vitro.

    PubMed

    Gao, Li; Han, Jiayuan; Si, Jianyong; Wang, Junchi; Wang, Hexiang; Sun, Yipeng; Bi, Yuhai; Liu, Jinhua; Cao, Li

    2017-07-01

    Influenza virus infection is a global public health issue. The efficacy of antiviral agents for influenza virus has been limited by the emergence of drug-resistant virus strains. Thus, there is an urgent need to identify novel antiviral therapies. Our previous studies have found that Cryptoporus volvatus extract can potently inhibit influenza virus replication in vitro and in vivo. However, the effective component of Cryptoporus volvatus, which mediates the antiviral activity, hasn't been identified. Here, we identified a novel anti-influenza virus molecule, Cryptoporic acid E (CAE), from Cryptoporus volvatus. Our results showed that CAE had broad-spectrum anti-influenza activity against 2009 pandemic strain A/Beijing/07/2009 (H1N1/09pdm), seasonal strain A/Beijing/CAS0001/2007(H3N2), mouse adapted strains A/WSN/33 (H1N1), and A/PR8/34 (H1N1). We further investigated the mode of CAE action. Time-course-analysis indicated that CAE exerted its inhibition mainly at the middle stages of the replication cycle of influenza virus. Subsequently, we confirmed that CAE inhibited influenza virus RNA polymerase activity and blocked virus RNA replication and transcription in MDCK cells. In addition, we found that CAE also impaired influenza virus infectivity by directly targeting virus particles. Our data suggest that CAE is a major effective component of Cryptoporus volvatus. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Alstonia scholaris R. Br. Significantly Inhibits Retinoid-Induced Skin Irritation In Vitro and In Vivo.

    PubMed

    Lee, Soo-Jin; Cho, Sun-A; An, Su-Sun; Na, Yong-Joo; Park, Nok-Hyun; Kim, Han-Sung; Lee, Chan-Woo; Kim, Han-Kon; Kim, Eun-Kyung; Jang, Young-Pyo; Kim, Jin-Woong

    2012-01-01

    Topical retinoids inhibit matrix metalloproteinases and accelerate collagen synthesis, thereby triggering antiaging effects in the skin. However, topical retinoids can cause severe skin reactions, including scaling, erythema, papules, and inflammation. The present study demonstrates that the ethanolic bark extract of Alstonia scholaris R. Br. can significantly inhibit all-trans retinoic acid-induced inflammation in human HaCat keratinocyte cells. Furthermore, two representative retinoid-induced proinflammatory cytokines, monocyte chemoattractant protein-1 and interleukin-8, were significantly suppressed by A. scholaris extract (by 82.1% and 26.3% at 100 ppm, and dose-dependently across the tested concentrations) in vitro. In a cumulative irritation patch test, A. scholaris extract decreased retinol-induced skin irritation, while strengthening the ability of retinoids to inhibit matrix metalloproteinase-1 expression, which is strongly associated with aging effects. These results suggest that A. scholaris is a promising compound that may increase the antiaging function of retinoids while reducing their ability to cause skin irritation.

  4. Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro.

    PubMed

    Xue, Gang; Zou, Xi; Zhou, Jin-Yong; Sun, Wei; Wu, Jian; Xu, Jia-Li; Wang, Rui-Ping

    2013-09-20

    Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A's dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Mycobacterial DNA gyrB inhibitors: Ligand based pharmacophore modelling and in vitro enzyme inhibition studies.

    PubMed

    Saxena, Shalini; Renuka, Janupally; Jeankumar, Variam Ullas; Yogeeswari, Perumal; Sriram, Dharmarajan

    2014-01-01

    Among the topoisomerases, DNA gyrase belongs to the type II classes that catalysing DNA supercoiling or relaxation, catenation or decatenation, knotting or unknotting. It is one of the validated targets for anti-tubercular drug discovery and inhibitors from this group are also active against non-replicating, persistent mycobacteria, which might be important for shortening the duration of TB therapy. From past few years, extensive research was carried out towards potent DNA gyrase inhibitor design. The current review focuses on the most of potent series of DNA gyrase inhibitors and its structure activity relationships (SAR). The current manuscript also reports the current research on identification of potent DNA gyrase inhibitors using ligand based virtual screening approaches. The pharmacophore model was developed and validated against 65 known Mycobacterium smegmatics (MS) DNA Gyrase inhibitors. Validated pharmacophore model consists of HBA, HY, and RA features were essential for DNA Gyrase inhibition and this model was used to screen virtual screening to retrieve potential inhibitors from our in house database. Finally, 15 hits were ranked as potential leads based on pharmacophoric fit value and estimated activity. Furthermore, in-vitro enzymatic inhibition studies were performed for these 15 most promising candidates and these compounds were found to exhibit inhibition at 30 µM.

  6. In vitro acetylcholinesterase inhibition by psoralen using molecular docking and enzymatic studies

    PubMed Central

    Somani, Gauresh; Kulkarni, Chinmay; Shinde, Prashant; Shelke, Rupesh; Laddha, Kirti; Sathaye, Sadhana

    2015-01-01

    Introduction: Alzheimer's disease (AD) has increased at an alarming rate and is now a worldwide health problem. Inhibitors of acetylcholinesterase (AChE) leading to inhibition of acetylcholine breakdown constitute the main therapeutic strategy for AD. Psoralen was investigated as inhibitor of AChE enzyme in an attempt to explore its potential for the management of AD. Materials and Methods: Psoralen was isolated from powdered Psoralea corylifolia fruits. AChE enzyme inhibitory activity of different concentrations of psoralen was investigated by use of in vitro enzymatic and molecular docking studies. Further, the enzyme kinetics were studied using Lineweaver-Burk plot. Results: Psoralen was found to inhibit AChE enzyme activity in a concentration-dependent manner. Kinetic studies showed psoralen inhibits AChE in a competitive manner. Molecular docking study revealed that psoralen binds well within the binding site of the enzyme showing interactions such as π-π stacking and hydrogen bonding with residues present therein. Conclusion: The result of AChE enzyme inhibitory activity of the psoralen in this study is promising. It could be further explored as a potential candidate for further development of new drugs against AD. PMID:25709334

  7. Inhibition of calcium oxalate crystallisation in vitro by an extract of Bergenia ciliata

    PubMed Central

    Saha, Sarmistha; Verma, Ramtej J.

    2013-01-01

    Objective To evaluate the effectiveness of an extract obtained from the rhizomes of Bergenia ciliata (Saxifragaceae) on the inhibition of calcium oxalate (CaOx) crystallisation in vitro. Materials and methods A hydro-alcoholic extract (30:70, v/v) of rhizomes of B. ciliata was prepared at different concentrations (1–10 mg/mL). The crystallisation of CaOx monohydrate (COM) was induced in a synthetic urine system. The nucleation and aggregation of COM crystals were measured using spectrophotometric methods. The rates of nucleation and aggregation were evaluated by comparing the slope of the turbidity of a control system with that of one exposed to the extract. The results were compared with a parallel study conducted with a marketed poly-herbal combination, Cystone, under identical concentrations. Crystals generated in the urine were also analysed by light microscopy. Statistical differences and percentage inhibitions were calculated and assessed. Results The extract of B. ciliata was significantly more effective in inhibiting the nucleation and aggregation of COM crystals in a dose-dependent manner than was Cystone. Moreover, the extract induced more CaOx dihydrate crystals, with a significant reduction in the number and size of COM crystals. Conclusion An extract of the traditional herb B. ciliata has an excellent inhibitory activity on crystalluria and therefore might be beneficial in dissolving urinary stones. However, further study in animal models of urolithiasis is needed to evaluate its potential anti-urolithiatic activity. PMID:26558080

  8. The flavonoid component isorhamnetin in vitro inhibits proliferation and induces apoptosis in Eca-109 cells.

    PubMed

    Ma, Gang; Yang, Chunlei; Qu, Yi; Wei, Huaying; Zhang, Tongtong; Zhang, Najuan

    2007-04-25

    Isorhamnetin is one member of flavonoid components which has been used in the treatment of heart disease. Recently the in vitro anti-cancer effect of isorhamnetin on human esophageal squamous carcinoma cell line Eca-109 was investigated in our lab. When Eca-109 cells were in vitro exposed to the graded doses of isorhamnetin (0-80 microg/ml) for 48 h, respectively, isorhamnetin exhibited cytostatic effect on the treated cells, with an IC(50) of 40+/-0.08 microg/ml as estimated by MTT assay. Inhibition on proliferation by isorhamnetin was detected by trypan blue exclusion assay, clone formation test, immunocytochemical assay of PCNA and (3)H-thymidine uptake analysis. Cell cycle distribution was measured by FCM. It was found that the viability of Eca-109 cells was significantly hampered by isorhamnetin. Compared with the negative control group, the treated group which was exposed to isorhamnetin had increased population in G(0)/G(1) phase from 74.6 to 84 while had a significant reduction in G(2)/M phase from 11.9 to 5.8. In addition to its cytostatic effect, isorhamnetin also showed stimulatory effect on apoptosis. Typical apoptotic morphology such as condensation and fragmentation of nuclei and blebbing membrane of the apoptotic cells could be observed through transmission electron microscope. Moreover, the sharp increase in apoptosis rate between the control and treated group were detected by FCM from 6.3 to 16.3. To explore the possible molecular mechanisms that underlie the growth inhibition and apoptosis stimulatory effects of isorhamnetin, the expressions of six proliferation- and death-related genes were detected by FCM. Expressions of bcl-2, c-myc and H-ras were downregulated whereas Bax, c-fos and p53 were upregulated. However, the in vivo experiments were required to further confirm the anti-cancer effects of isorhamnetin. In conclusion, isorhamnetin appears to be a potent drug against esophageal cancer due to its in vitro potential to not only inhibit

  9. Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

    PubMed Central

    Xue, Hongxia; Gan, Fang; Qian, Gang; Hu, Junfa; Hao, Shu; Xu, Jing; Chen, Xingxiang; Huang, Kehe

    2017-01-01

    This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress. PMID:28071725

  10. Shikonin Exerts Antitumor Activity via Proteasome Inhibition and Cell Death Induction in vitro and in vivo

    PubMed Central

    Yang, Huanjie; Zhou, Ping; Huang, Hongbiao; Chen, Di; Ma, Ningfang; Cui, Cindy Qiuzhi; Shen, Shouxing; Dong, Weihua; Zhang, Xiaoyan; Lian, Wen; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2009-01-01

    Dysregulation of the ubiquitin-proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell-killing activity, and results from a clinical study using a shikonin-containing mixture demonstrated its safety and efficacy for the treatment of late-stage lung cancer. In the present study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C1 and C4 of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin-like activity of purified 20S proteasome (IC50 12.5 μmol/L) and tumor cellular 26S proteasome (IC50 between 2-16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC-3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB-α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC-3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin, and inhibition of the proteasome activity by shikonin contributes to its anti-tumor property. PMID:19165859

  11. In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues.

    PubMed

    Maiale, Santiago J; Marina, María; Sánchez, Diego H; Pieckenstain, Fernando L; Ruiz, Oscar A

    2008-10-01

    Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affinity for spermidine and spermine (K(m) approximately 5.5 and 1.2 microM, respectively), and also oxidized norspermidine (K(m) approximately 64.0 microM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-aminooxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a K(i) value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetramine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15-triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with K(i) values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K(i) value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited ( approximately 85%) S. sclerotiorum mycelial growth on Czapek-Dox medium. Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.

  12. The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

    NASA Astrophysics Data System (ADS)

    Shimakami, Tetsuro; Honda, Masao; Shirasaki, Takayoshi; Takabatake, Riuta; Liu, Fanwei; Murai, Kazuhisa; Shiomoto, Takayuki; Funaki, Masaya; Yamane, Daisuke; Murakami, Seishi; Lemon, Stanley M.; Kaneko, Shuichi

    2014-04-01

    Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

  13. Rosmarinus officinalis L. essential oil inhibits in vivo and in vitro leukocyte migration.

    PubMed

    Nogueira de Melo, Gessilda Alcantara; Grespan, Renata; Fonseca, Jefferson Pitelli; Farinha, Thiago Oliveira; Silva, Expedito Leite; Romero, Adriano Lopes; Bersani-Amado, Ciomar A; Cuman, Roberto Kenji Nakamura

    2011-09-01

    Rosmarinus officinalis L. (Lamiaceae), popularly known as rosemary, is used for food flavoring and in folk medicine as an antispasmodic, analgesic, antirheumatic, diuretic, and antiepileptic agent. Few studies have shown the anti-inflammatory effects of rosemary essential oil (REO). This study evaluated the effects of REO on leukocyte migration through in vivo leukocyte migration and in vitro chemotaxis assay. REO was analyzed by using gas chromatography-mass spectometry, and the main components identified were camphor (27.59%), 1,8-cineole (15.74%), α-pinene (16.58%), and β-myrcene (10.02%). In rats, administration of REO reduced the number of leukocytes that rolled, adhered, and migrated to the scrotal chamber after carrageenan injection. All doses of REO tested significantly inhibited leukocyte chemotaxis induced by casein. The effects of REO on leukocyte migration highlight an important mechanism of the anti-inflammatory action of rosemary.

  14. Dehydroepiandrosterone inhibits the spontaneous release of superoxide radical by alveolar macrophages in vitro in asbestosis

    SciTech Connect

    Rom, W.N.; Harkin, T. )

    1991-08-01

    Asbestosis is characterized by an alveolar macrophage alveolitis with injury and fibrosis of the lower respiratory tract. Alveolar macrophages recovered by bronchoalveolar lavage spontaneously release exaggerated amounts of oxidants including superoxide anion and hydrogen peroxide that may mediate alveolar epithelial cell injury. Dehydroepiandrosterone (DHEA) is a normally occurring adrenal androgen that inhibits glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate shunt necessary for NADPH generation and superoxide anion formation. In this regard, the authors hypothesized that DHEA may reduce asbestos-induced oxidant release. DHEA added in vitro to alveolar macrophages lavaged from 11 nonsmoking asbestos workers significantly reduced superoxide anion release. DHEA is an antioxidant and potential anticarcinogenic agent that may have a therapeutic role in reducing the increased oxidant burden in asbestos-induced alveolitis of the lower respiratory tract.

  15. In vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants.

    PubMed

    de Andrade-Neto, Valter F; Pohlit, Adrian M; Pinto, Ana Cristina S; Silva, Ellen Cristina C; Nogueira, Karla L; Melo, Márcia R S; Henrique, Marycleuma C; Amorim, Rodrigo C N; Silva, Luis Francisco R; Costa, Mônica R F; Nunomura, Rita C S; Nunomura, Sergio M; Alecrim, Wilson D; Alecrim, M das Graças C; Chaves, F Célio M; Vieira, Pedro Paulo R

    2007-06-01

    In the present study, a quassinoid, neosergeolide, isolated from the roots and stems of Picrolemma sprucei (Simaroubaceae), the indole alkaloids ellipticine and aspidocarpine, isolated from the bark of Aspidosperma vargasii and A. desmanthum (Apocynaceae), respectively, and 4-nerolidylcatechol, isolated from the roots of Pothomorphe peltata (Piperaceae), all presented significant in vitro inhibition (more active than quinine and chloroquine) of the multi-drug resistant K1 strain of Plasmodium falciparum. Neosergeolide presented activity in the nanomolar range. This is the first report on the antimalarial activity of these known, natural compounds. This is also the first report on the isolation of aspidocarpine from A. desmanthum. These compounds are good candidates for pre-clinical tests as novel lead structures with the aim of finding new antimalarial prototypes and lend support to the traditional use of the plants from which these compounds are derived.

  16. Aloe-emodin inhibits proliferation of adult human keratinocytes in vitro.

    PubMed

    Popadic, Dusan; Savic, Emina; Ramic, Zorica; Djordjevic, Vladimir; Trajkovic, Vladimir; Medenica, Ljiljana; Popadic, Svetlana

    2012-01-01

    Aloe-emodin (AE) is a plant-derived hydroxyanthraquinone with several biological activities. It is present in a variety of skin-conditioning agents containing aloe extracts, but its influence on keratinocyte growth was not examined so far. We investigated the influence of AE on human keratinocyte proliferation and apoptosis in vitro. AE significantly inhibited proliferation of cultivated human keratinocytes at 5 μM concentration, as revealed by incorporation of radioactive thymidine. The antiproliferative effect of AE was accompanied with induction of apoptosis, but not necrosis, as demonstrated by flow cytometric analysis and lactate dehydrogenase release assay. Based on the half maximal inhibitory concentration values, we demonstrated that AE may impair proliferation of keratinocytes at concentrations far below the industry standards for commercial products containing aloe extracts. Therefore, further research of AE effects on the human skin and proper labeling of products are necessary for maximizing benefits from aloe extracts and to avoid undesired responses.

  17. Inhibition of the in-vitro growth of Mycobacterium tuberculosis by a phytosiderophore.

    PubMed

    Rajiv, J; Dam, T; Kumar, S; Bose, M; Aggarwal, K K; Babu, C R

    2001-10-01

    Non-compliance by patients and poor clinical management due to the use of incorrect regimens are the main reasons for the development of drug resistance by mycobacterial strains. New strategies for the control of multi-drug-resistant mycobacterial strains have become a necessity for proper management of tuberculosis, which, according to the WHO report (1997), is estimated to remain among the top 10 mortality-causing diseases of the twenty-first century. One of the strategies is the use of iron-sequestering agents like siderophores as active therapeutic agents in the treatment of tuberculosis. This report describes for the first time the inhibition of the growth of Mycobacterium tuberculosis H37Ra in vitro by a phytosiderophore isolated from the root washings of Tephrosia purpurea. This finding may help in the establishment of a new drug regimen which will be more effective in the treatment of tuberculosis.

  18. Inhibition of iron induced lipid peroxidation and antioxidant activity of Indian spices and Acacia in vitro.

    PubMed

    Yadav, Amit Singh; Bhatnagar, Deepak

    2010-03-01

    The spices used in the Indian foods such as Star anise (Illicium verum), Bay leaves (Cinnamomum zeylanicum) and Cobra's saffron (Mesua ferrea), and Acacia (Acacia catechu), which have medicinal value, were used as test samples, to find their effect on in vitro lipid peroxidation (LPO). Rat liver post mitochondrial supernatant (PMS) in Tris HCl buffer, pH 7.4 was incubated for 0 and 1 h, with various test extracts in three different oxidant systems. The results show that addition of test samples to FeCl(3) medium at 0 h significantly stop the initiation of the LPO. However, the propagation phase of LPO was inhibited by Cobra's saffron and Acacia and not by Star anise and Bay leaves. The test samples also showed strong reducing power and superoxide radical scavenging activity. Cobra's saffron and Acacia showed the highest antioxidant activity, probably due to the higher polyphenol content as compared to other test samples.

  19. In vitro inhibition of caprine herpesvirus 1 by acyclovir and mizoribine.

    PubMed

    Elia, G; Camero, M; Decaro, N; Lovero, A; Martella, V; Tempesta, M; Buonavoglia, C; Crescenzo, G

    2015-04-01

    Caprine herpesvirus 1 (CpHV-1) infection in goats induces genital vesicular-ulcerative lesions that strictly resemble the lesions induced by herpesvirus 2 in the human host. The immunosuppressive drug Mizoribine (MIZ) was found to increase the antiviral activity of Acyclovir (ACV) against herpesvirus infections, raising interesting perspectives on new combined therapeutic strategies. In this study the anti-CpHV-1 activity in vitro of ACV alone or in combination with MIZ was characterized. When applied alone at non-toxic concentrations, ACV had a slight effect on CpHV-1 replication while in combination with MIZ a dose-dependent inhibition of the virus yield was observed with an IC50 of ACV of 28.5 µM. These findings suggest that combined therapy of ACV and MIZ is potentially exploitable in the treatment of genital infection by herpesviruses.

  20. Evaluation of toxicity of river sediments by in vitro enzyme inhibition

    SciTech Connect

    Tabata, Masako; Ozawa, Keiko; Ohtakara, Akiko; Nakabayashi, Hiromi; Suzuki, Shizuo )

    1990-06-01

    To confirm the presence of toxic substances in aquatic environments, various toxicological assays have been carried out. Latent toxicity cannot be evaluated only by chemical analysis since natural samples contain diverse kinds of compounds. On the other hand a biological assay should be appropriate for evaluating directly latent toxicity. Many biological toxic examinations have been conducted on fish, shellfish, algae, zooplankton and others using the whole body. Another method for evaluating of toxicity of environmental samples has been to use enzyme inhibition in vitro. The authors have investigated the distribution of cholinesterase inhibitor in the river sediment and evaluated the latent toxicity of aquatic environment samples. The present paper reports the inhibitory effects of solvent extracts from Tama and Ayase River sediments on three enzymes: alkaline phosphatase and glucose-6-phosphate dehydrogenase that diagnose for a functional disorder of a liver and {beta}-glucuronidase for that of a kidney used in clinical inspection.

  1. Ficus religiosa L. bark extracts inhibit human rhinovirus and respiratory syncytial virus infection in vitro.

    PubMed

    Cagno, Valeria; Civra, Andrea; Kumar, Ravi; Pradhan, Subhankar; Donalisio, Manuela; Sinha, Barij Nayan; Ghosh, Manik; Lembo, David

    2015-12-24

    Ficus religiosa L. is one of the most relevant members of the family of Moraceae. It is the most sacred tree of South Asia, and it is used in traditional Ayurvedic and Unani medicine to cure respiratory disorders like cough, wheezing and asthma. Some studies were performed to investigate the anti-asthmatic potential of F. religiosa bark, leaves and fruit extracts but none of them tested their antiviral activity against viruses responsible for the exacerbation of wheezing and asthma. The present study was undertaken to investigate the antiviral activity of F. religiosa L. extracts against respiratory viruses such as human respiratory syncytial virus (RSV) and human rhinovirus (HRV). The antiviral activity of F. religiosa L. was tested in vitro by plaque reduction and virus yield assays and the major mechanism of action was investigated by virus inactivation and time-of-addition assays. F. religiosa L. methanol bark extract was the most active against HRV with an EC50 of 5.52 µg/mL. This extract likely inhibited late steps of replicative cycle. Water bark extract was the most active against RSV with an EC50 between 2.23 and 4.37 µg/mL. Partial virus inactivation and interference with virus attachment were both found to contribute to the anti-RSV activity. Replication of both viruses was inhibited in viral yield reduction assays. The results of the present study demonstrate that F. religiosa L. is endowed with antiviral activity against RSV and HRV in vitro. Further work remains to be done to identify the active components and to assess the therapeutic potential in vivo. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Anthocyanins inhibit trastuzumab-resistant breast cancer in vitro and in vivo.

    PubMed

    Li, Xin; Xu, Jinmei; Tang, Xi; Liu, Yilun; Yu, Xiaoping; Wang, Zhi; Liu, Weihua

    2016-05-01

    Trastuzumab (Herceptin®) is a recombinant humanized monoclonal antibody that is targeted against the human epidermal growth factor receptor 2 (HER2) tyrosine kinase receptor. Trastuzumab has been successfully used to treat patients with HER2-positive breast cancer, which accounts for ~25% of invasive breast cancer. However, the majority of patients who initially respond to trastuzumab demonstrate disease progression within 1 year of treatment. Therefore, identifying alternative drugs that overcome trastuzumab resistance and target HER2 may increase the magnitude and duration of response. Through a high‑throughput screening approach, we previously identified numerous anthocyanins that exert activity in HER2‑positive human breast cancer cell lines. The present study aimed to evaluate the anti‑tumor properties of anthocyanins against parental HER2‑positive cells and derivative trastuzumab‑resistant cells in vitro and in vivo. Cell proliferation, western blotting, Annexin V staining, migration and invasion assays were used to determine the effects of anthocyanins in vitro. Cyanidin-3-glucoside and peonidin-3-glucoside were able to inhibit phosphorylation of HER2, induce apoptosis, suppress migration and invasion, and inhibit tumor cell growth. Coupled with the fact that anthocyanins have been used for decades as supplements for the treatment of various types of cancer in Asia, the present study may have established a framework for the development and testing of anthocyanins as a novel treatment paradigm used to overcome classical trastuzumab-resistance and to improve the outcome of this disease.

  3. Diphenyl diselenide, a simple glutathione peroxidase mimetic, inhibits human LDL oxidation in vitro.

    PubMed

    de Bem, Andreza Fabro; Farina, Marcelo; Portella, Rafael de Lima; Nogueira, Cristina Wayne; Dinis, Teresa C P; Laranjinha, João A N; Almeida, Leonor M; Rocha, João Batista Teixeira

    2008-11-01

    Oxidative modification of low-density lipoprotein (LDL) represents an important factor in atherogenesis. In the present study, we have investigated the antioxidant capability of diphenyl diselenide (PhSe)(2), a simple organoseleno compound, against copper (Cu2+) and peroxyl radical-induced human LDL oxidation in vitro. In initial studies using human serum, (PhSe)(2) caused a dose-dependent inhibition of Cu(2+)-induced lipid peroxidation, which was correlated to thiol consumption. (PhSe)(2) increased lipid peroxidation lag phase and decreased lipid peroxidation rate in isolated human LDL, evaluated by measuring both conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) levels. Consistent with these observations, (PhSe)(2) showed a marked inhibitory effect on 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH)-induced oxidation of LDL or parinaric acid (PnA) incorporated into LDL. (PhSe)(2) also displayed a dose-dependent protective effect against Cu(2+)-induced lipid peroxidation in rat aortic slices. Interestingly, besides the antioxidant effects of (PhSe)(2) toward the lipid moieties of LDL, which was related to its thiol-peroxidase activity, protein moieties from human isolated LDL were also protected against Cu(2+)-induced oxidation. The results presented herein are the first to show that (i) (PhSe)(2) inhibits lipid peroxidation in human isolated LDL in vitro, (ii) this phenomenon is related to its thiol-peroxidase activity, and (iii) this chalcogen also prevents the oxidation of protein moieties of human LDL. Taken together, such data render (PhSe)(2) a promising molecule for pharmacological studies with respect to the atherogenic process.

  4. Interleukin-34 inhibits hepatitis B virus replication in vitro and in vivo.

    PubMed

    Cheng, Sheng-Tao; Tang, Hua; Ren, Ji-Hua; Chen, Xiang; Huang, Ai-Long; Chen, Juan

    2017-01-01

    The hepatitis B virus (HBV) infection could activate the immune system and induce extensive inflammatory response. As the most important inflammatory factor, interleukins are critical for anti-viral immunity. Here we investigated whether interleukin-34 (IL-34) play a role in HBV infection. In this study, we first found that both serum IL-34 and IL-34 mRNA in PBMCs in chronic HBV patients was significantly decreased compared to the healthy controls. Furthermore, both IL-34 protein and mRNA levels were declined hepatoma cells expressing HBV. In addition, the clinical parameters analysis found that serum IL-34 was significantly associated with HBV DNA (P = 0.0066), ALT (P = 0.0327), AST (P = 0.0435), TB (P = 0.0406), DB (P = 0.0368) and AFP (P = 0.0225). Correlation analysis also found that serum IL-34 negatively correlated with HBV DNA copies, ALT and AST. In vitro studies found that IL-34 treatment in HepAD38 and HepG2.2.15 cells markedly inhibited HBV DNA, total RNA, 3.5kb mRNA and HBc protein. In vivo studies further demonstrated IL-34 treatment in HBV transgenic mice exhibited greater inhibition on HBV DNA, total RNA, 3.5kb mRNA and HBc protein, suggesting the effect to IL-34 on HBV is likely due to host innate or adaptive immune response. Our study identified a novel interleukin, IL-34, which has anti-viral activity in HBV replication in hepatocytes in vitro and in vivo. These data suggest a rationale for the use of IL-34 in the HBV treatment.

  5. In vitro crystallization, characterization and growth-inhibition study of urinary type struvite crystals

    NASA Astrophysics Data System (ADS)

    Chauhan, Chetan K.; Joshi, Mihir J.

    2013-01-01

    The formation of urinary stones, known as nephrolithiasis or urolithiasis, is a serious, debilitating problem throughout the world. Struvite—NH4MgPO4·6H2O, ammonium magnesium phosphate hexahydrate, is one of the components of urinary stones (calculi). Struvite crystals with different morphologies were grown by in vitro single diffusion gel growth technique with different growth parameters. The crystals were characterized by powder XRD, FT-IR, thermal analysis and dielectric study. The powder XRD results of struvite confirmed the orthorhombic crystal structure. The FT-IR spectrum proved the presence of water of hydration, metal-oxygen bond, N-H bond and P-O bond. For thermal analysis TGA, DTA and DSC were carried out simultaneously. The kinetic and thermodynamic parameters of dehydration/decomposition process were calculated. Vickers micro-hardness and related mechanical parameters were also calculated. The in vitro growth inhibition studies of struvite by the juice of Citrus medica Linn as well as the herbal extracts of Commiphora wightii, Boerhaavia diffusa Linn and Rotula aquatica Lour were carried out and found potent inhibitors of struvite.

  6. In vitro toxicities of experimental jet fuel system ice-inhibiting agents.

    PubMed

    Geiss, K T; Frazier, J M

    2001-07-02

    One research emphasis within the Department of Defense has been to seek the replacement of operational compounds with alternatives that pose less potential risk to human and ecological systems. Alternatives to glycol ethers, such as diethylene glycol monomethyl ether (M-DE), were investigated for use as jet fuel system ice-inhibiting agents (FSIIs). This group of chemicals includes three derivatives of 1,3-dioxolane-4-methanol (M-1, M-2, and M-3) and a 1,3-dioxane (M-27). In addition, M-DE was evaluated as a reference compound. Our approach was to implement an in vitro test battery based on primary rat hepatocyte cultures to perform initial toxicity evaluations. Hepatocytes were exposed to experimental chemicals (0, 0.001, 0.01, 0.1, 1, 10 mM dosages) for periods up to 24 h. Samples were assayed for lactate dehydrogenase (LDH) release, MTT dye reduction activity, glutathione level, and rate of protein synthesis as indicators of toxicity. Of the compounds tested, M-1, especially at the 10-mM dose, appeared to be more potent than the other chemicals, as measured by these toxicity assays. M-DE, the current FSII, elicited little response in the toxicity assays. Although some variations in toxicity were observed at the 10-mM dose, the in vitro toxicities of the chemicals tested (except for M-1) were not considerably greater than that of M-DE.

  7. In vitro inhibition of bovine enamel demineralization by enamel matrix derivative.

    PubMed

    Ran, Jin Mei; Ieong, Cheng Cheng; Xiang, Chen Yang; Lv, Xue Ping; Xue, Jing; Zhou, Xue Dong; Li, Wei; Zhang, Ling Lin

    2014-01-01

    This study aimed to determine whether enamel matrix derivative (Emdogain) affects the demineralization of bovine enamel in vitro and to assess the agent's anti-caries potential. Bovine enamel blocks were prepared and randomly divided into three groups (n = 15 per group), which were treated with distilled water (negative control), NaF (positive control), or Emdogain. All three groups were pH-cycled 12 times over 6 days. The percentage of surface enamel microhardness reduction (%SMHR), calcium demineralization rate (CDR), surface roughness, lesion depth and mineral loss after demineralization were examined. Surface morphology of specimens was studied by scanning electron microscopy. The Emdogain and positive control groups showed similar surface roughness, lesion depths and mineral loss, which were significantly lower than those in the negative control group. In addition, the enamel surfaces of both the Emdogain and NaF groups showed much narrower intercrystalline spaces than the surfaces of the negative control group, which exhibited extensive microfractures along the crystal edges. %SMHR differed significantly among all three groups, with the smallest value in the Emdogain group and the greatest in the negative control group. These results indicate that enamel matrix derivative (Emdogain) can significantly inhibit demineralization of bovine enamel in vitro, suggesting that it has potential as an anti-caries agent.

  8. Resveratrol inhibits development of experimental endometriosis in vivo and reduces endometrial stromal cell invasiveness in vitro.

    PubMed

    Bruner-Tran, Kaylon L; Osteen, Kevin G; Taylor, Hugh S; Sokalska, Anna; Haines, Kaitlin; Duleba, Antoni J

    2011-01-01

    Endometriosis is a common gynecologic disorder characterized by ectopic attachment and growth of endometrial tissues. Resveratrol is a natural polyphenol with antiproliferative and anti-inflammatory properties. Our objective was to study the effects of resveratrol on human endometriotic implants in a nude mouse model and to examine its impact on human endometrial stromal (HES) cell invasiveness in vitro. Human endometrial tissues were obtained from healthy donors. Endometriosis was established in oophorectomized nude mice by intraperitoneal injection of endometrial tissues. Mice were treated with 17β-estradiol (8 mg, silastic capsule implants) alone (n = 16) or with resveratrol (6 mg/mouse; n = 20) for 10-12 and 18-20 days beginning 1 day after tissue injection. Mice were killed and endometrial implants were evaluated. A Matrigel invasion assay was used to examine the effects of resveratrol on HES cells. We assessed number and size of endometriotic implants in vivo and Matrigel invasion in vitro. Resveratrol decreased the number of endometrial implants per mouse by 60% (P < 0.001) and the total volume of lesions per mouse by 80% (P < 0.001). Resveratrol (10-30 μM) also induced a concentration-dependent reduction of invasiveness of HES by up to 78% (P < 0.0001). Resveratrol inhibits development of endometriosis in the nude mouse and reduces invasiveness of HES cells. These observations may aid in the development of novel treatments of endometriosis.

  9. Melissa officinalis extract inhibits attachment of herpes simplex virus in vitro.

    PubMed

    Astani, Akram; Reichling, Jürgen; Schnitzler, Paul

    2012-01-01

    Extracts and essential oils of medicinal plants are increasingly of interest as novel drugs for antiherpetic agents, since the herpes simplex virus (HSV) might develop resistance to commonly used antiviral drugs. An aqueous extract of Melissa officinalis as well as phenolic extract compounds, i.e. caffeic acid, p-coumaric acid and rosmarinic acid were examined for their antiviral activity against herpes simplex virus type 1 (HSV-1) in vitro. When drugs were added to HSV-1-infected cells, no antiviral effect was observed as determined by plaque reduction assay and analysis of expression of viral protein ICP0. However, the Melissa extract demonstrated a high virucidal activity against HSV-1, even at very low concentrations of 1.5 μg/ml, whereas similar results for phenolic compounds were only achieved at 100 times higher concentrations. Besides the virucidal activity, the Melissa extract and rosmarinic acid inhibited HSV-1 attachment to host cells in a dose-dependent manner. These results indicate that rosmarinic acid was the main contributor to the antiviral activity of Melissa extract. However, the selectivity index of Melissa extract of 875 against HSV is superior to the selectivity indices of single constituents. Melissa extract exhibits low toxicity, is virucidal and affects HSV-1 attachment to host cells in vitro. Copyright © 2012 S. Karger AG, Basel.

  10. Marine compounds inhibit growth of multiple myeloma in vitro and in vivo

    PubMed Central

    Steiner, Normann; Ribatti, Domenico; Willenbacher, Wolfgang; Jöhrer, Karin; Kern, Johann; Marinaccio, Christian; Aracil, Miguel; García-Fernández, Luis F.; Gastl, Guenther

    2015-01-01

    Purpose The prognosis of patients with multiple myeloma (MM) is still dismal despite recent improvements achieved by introducing new therapeutic agents. However, there remains an urgent need for progress in myeloma drug development. We here show that novel marine-derived compounds can exert potent anti-myeloma activity. Experimental Design Nine marine-derived compounds were applied at low nM concentrations (0.1-100 nM) to MM cell lines (OPM-2, NCI-H929, U266, RPMI-8226), to primary human myeloma cells and to peripheral blood mononuclear cells. Apoptosis was determined by flow cytometry. In addition, eGFP-transgenic MM cell lines growing with mesenchymal cells from bone marrow were used to visualize tumors by fluorescence stereomicroscopy. Anti-myelomaactivities were studied in vitro in 3D spheroids and in vivo in myeloma xenografts on chicken embryos. Tumor size was analyzed by measuring GFP content with a GFP ELISA. Anti-angiogenic activities of compounds were tested in an in vivo gelatin sponge assay with conditioned media from primary bone marrow-derived endothelial cells. Results We identified a subset of marine compounds with strong anti-myeloma activity in vitro and in vivo. Moreover, some of the compounds inhibited myeloma-related angiogenesis in the in vivo gelatin sponge assay. They merit further drug development to improve treatment options for MM. PMID:25860931

  11. Viscolin Inhibits In Vitro Smooth Muscle Cell Proliferation and Migration and Neointimal Hyperplasia In Vivo

    PubMed Central

    Chen, Chin-Chuan; Liang, Chan-Jung; Leu, Yann-Lii; Chen, Yuh-Lien; Wang, Shu-Huei

    2016-01-01

    Viscolin, an extract of Viscum coloratum, has anti-inflammatory and anti-proliferative properties against harmful stimuli. The aim of the study was to examine the anti-proliferative effects of viscolin on platelet derived growth factor-BB (PDGF)-treated human aortic smooth muscle cells (HASMCs) and identify the underlying mechanism responsible for these effects. Viscolin reduced the PDGF-BB-induced HASMC proliferation and migration in vitro; it also arrested HASMCs in the G0/G1 phase by decreasing the protein expression of Cyclin D1, CDK2, Cyclin E, CDK4, and p21Cip1 as detected by Western blot analysis. These effects may be mediated by reduced PDGF-induced phosphorylation of ERK1/2, JNK, and P38, but not AKT as well as inhibition of PDGF-mediated nuclear factor (NF)-κB p65 and activator protein 1 (AP-1)/c-fos activation. Furthermore, viscolin pre-treatment significantly reduced neointimal hyperplasia of an endothelial-denuded femoral artery in vivo. Taken together, viscolin attenuated PDGF–BB-induced HASMC proliferation in vitro and reduced neointimal hyperplasia in vivo. Thus, viscolin may represent a therapeutic candidate for the prevention and treatment of vascular proliferative diseases. PMID:27977759

  12. Inhibition of fungal and bacterial plant pathogens in vitro and in planta with ultrashort cationic lipopeptides.

    PubMed

    Makovitzki, Arik; Viterbo, Ada; Brotman, Yariv; Chet, Ilan; Shai, Yechiel

    2007-10-01

    Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.

  13. Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro

    PubMed Central

    Paul, Amber M.; Shi, Yongliang; Acharya, Dhiraj; Douglas, Jessica R.; Cooley, Amanda; Anderson, John F.; Huang, Faqing

    2014-01-01

    Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP–siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP–siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy. PMID:24828333

  14. Inhibition of Fungal and Bacterial Plant Pathogens In Vitro and In Planta with Ultrashort Cationic Lipopeptides▿

    PubMed Central

    Makovitzki, Arik; Viterbo, Ada; Brotman, Yariv; Chet, Ilan; Shai, Yechiel

    2007-01-01

    Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection. PMID:17720828

  15. Skeletal unloading inhibits the in vitro proliferation and differentiation of rat osteoprogenitor cells

    NASA Technical Reports Server (NTRS)

    Kostenuik, P. J.; Halloran, B. P.; Morey-Holton, E. R.; Bikle, D. D.

    1997-01-01

    Loss of weight bearing in the growing rat decreases bone formation, osteoblast numbers, and bone maturation in unloaded bones. These responses suggest an impairment of osteoblast proliferation and differentiation. To test this assumption, we assessed the effects of skeletal unloading using an in vitro model of osteoprogenitor cell differentiation. Rats were hindlimb elevated for 0 (control), 2, or 5 days, after which their tibial bone marrow stromal cells (BMSCs) were harvested and cultured. Five days of hindlimb elevation led to significant decreases in proliferation, alkaline phosphatase (AP) enzyme activity, and mineralization of BMSC cultures. Differentiation of BMSCs was analyzed by quantitative competitive polymerase chain reaction of cDNA after 10, 15, 20, and 28 days of culture. cDNA pools were analyzed for the expression of c-fos (an index of proliferation), AP (an index of early osteoblast differentiation), and osteocalcin (a marker of late differentiation). BMSCs from 5-day unloaded rats expressed 50% less c-fos, 61% more AP, and 35% less osteocalcin mRNA compared with controls. These data demonstrate that cultured osteoprogenitor cells retain a memory of their in vivo loading history and indicate that skeletal unloading inhibits proliferation and differentiation of osteoprogenitor cells in vitro.

  16. GBT440 Inhibits Sickling of Sickle Cell Trait Blood Under In Vitro Conditions Mimicking Strenuous Exercise

    PubMed Central

    Dufu, Kobina; Lehrer-Graiwer, Josh; Ramos, Eleanor; Oksenberg, Donna

    2016-01-01

    In sickle cell trait (SCT), hemoglobin A (HbA) and S (HbS) are co-expressed in each red blood cell (RBC). While homozygous expression of HbS (HbSS) leads to polymerization and sickling of RBCs resulting in sickle cell disease (SCD) characterized by hemolytic anemia, painful vaso-occlusive episodes and shortened life-span, SCT is considered a benign condition usually with minor or no complications related to sickling. However, physical activities that cause increased tissue oxygen demand, dehydration and/or metabolic acidosis leads to increased HbS polymerization and life-threatening complications including death. We report that GBT440, an agent being developed for the treatment of SCD, increases the affinity of oxygen for Hb and inhibits in vitro polymerization of a mixture of HbS and HbA that simulates SCT blood. Moreover, GBT440 prevents sickling of SCT blood under in vitro conditions mimicking strenuous exercise with hypoxia, dehydration and acidosis. Together, our results indicate that GBT440 may have the potential to protect SCT individuals from sickling-related complications during conditions that favor HbS polymerization. PMID:27757216

  17. Inhibition of Drp1 provides neuroprotection in vitro and in vivo.

    PubMed

    Grohm, J; Kim, S-W; Mamrak, U; Tobaben, S; Cassidy-Stone, A; Nunnari, J; Plesnila, N; Culmsee, C

    2012-09-01

    Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.

  18. Boswellia carterii Extract Inhibits TH1 Cytokines and Promotes TH2 Cytokines In Vitro

    PubMed Central

    Chevrier, Marc R.; Ryan, Abigail E.; Lee, David Y.-W.; Zhongze, Ma; Wu-Yan, Zhang; Via, Charles S.

    2005-01-01

    Traditional herbal formulas used to treat inflammatory arthritis in China and India include Boswellia carterii or Boswellia serrata. They both contain boswellic acids (BAs) which have been shown to exhibit anti-inflammatory and antiarthritic properties. This study tests the hypothesis that mixtures of BAs derived from B. carterii have immunomodulatory properties. B. carterii plant resin obtained from China was prepared as an ethanol extract, and the presence of seven BAs was confirmed by column chromatography, high-performance liquid chromatography, and UV laser desorption/ionization tandem mass spectroscopy. The extract was then tested for its ability to alter in vitro production of TH1 cytokines (interleukin-2 [IL-2] and gamma interferon) and TH2 cytokines (IL-4 and IL-10) by murine splenocytes. Delivery of the resin extract using ethanol as a solvent resulted in significant cellular toxicity not seen with the addition of ethanol alone. By contrast, delivery of the resin extract using a sesame oil solvent resulted in a dose-dependent inhibition of TH1 cytokines coupled with a dose-dependent potentiation of TH2 cytokines. These results indicate that a purified mixture of BAs from B. carterii plant resin exhibits carrier-dependent immunomodulatory properties in vitro. PMID:15879017

  19. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    SciTech Connect

    Scaife, R.M. ); Wilson, L. ); Purich, D.L. )

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  20. Skeletal unloading inhibits the in vitro proliferation and differentiation of rat osteoprogenitor cells

    NASA Technical Reports Server (NTRS)

    Kostenuik, P. J.; Halloran, B. P.; Morey-Holton, E. R.; Bikle, D. D.

    1997-01-01

    Loss of weight bearing in the growing rat decreases bone formation, osteoblast numbers, and bone maturation in unloaded bones. These responses suggest an impairment of osteoblast proliferation and differentiation. To test this assumption, we assessed the effects of skeletal unloading using an in vitro model of osteoprogenitor cell differentiation. Rats were hindlimb elevated for 0 (control), 2, or 5 days, after which their tibial bone marrow stromal cells (BMSCs) were harvested and cultured. Five days of hindlimb elevation led to significant decreases in proliferation, alkaline phosphatase (AP) enzyme activity, and mineralization of BMSC cultures. Differentiation of BMSCs was analyzed by quantitative competitive polymerase chain reaction of cDNA after 10, 15, 20, and 28 days of culture. cDNA pools were analyzed for the expression of c-fos (an index of proliferation), AP (an index of early osteoblast differentiation), and osteocalcin (a marker of late differentiation). BMSCs from 5-day unloaded rats expressed 50% less c-fos, 61% more AP, and 35% less osteocalcin mRNA compared with controls. These data demonstrate that cultured osteoprogenitor cells retain a memory of their in vivo loading history and indicate that skeletal unloading inhibits proliferation and differentiation of osteoprogenitor cells in vitro.

  1. Evaluation of human D-amino acid oxidase inhibition by anti-psychotic drugs in vitro.

    PubMed

    Shishikura, Miho; Hakariya, Hitomi; Iwasa, Sumiko; Yoshio, Takashi; Ichiba, Hideaki; Yorita, Kazuko; Fukui, Kiyoshi; Fukushima, Takeshi

    2014-06-01

    It is of importance to determine whether antipsychotic drugs currently prescribed for schizophrenia exert D-amino acid oxidase (DAO)-inhibitory effects. We first investigated whether human (h)DAO can metabolize D-kynurenine (D-KYN) to produce the fluorescent compound kynurenic acid (KYNA) by using high-performance liquid chromatography with mass spectrometry, and fluorescence spectrometry. After confirmation of KYNA production from D-KYN by hDAO, 8 first- and second-generation antipsychotic drugs, and 6 drugs often prescribed concomitantly, were assayed for hDAO-inhibitory effects by using in vitro fluorometric methods with D-KYN as the substrate. DAO inhibitors 3-methylpyrazole-5-carboxylic acid and 4H-thieno[3,2-b]pyrrole-5-carboxylic acid inhibited KYNA production in a dose-dependent manner. Similarly, the second-generation antipsychotics blonanserin and risperidone were found to possess relatively strong hDAO-inhibitory effects in vitro (5.29 ± 0.47 μM and 4.70 ± 0.17 μM, respectively). With regard to blonanserin and risperidone, DAO-inhibitory effects should be taken into consideration in the context of their in vivo pharmacotherapeutic efficacy.

  2. Glycerol Monolaurate Inhibits Candida and Gardnerella vaginalis In Vitro and In Vivo but Not Lactobacillus▿

    PubMed Central

    Strandberg, Kristi L.; Peterson, Marnie L.; Lin, Ying-Chi; Pack, Melinda C.; Chase, David J.; Schlievert, Patrick M.

    2010-01-01

    We investigated the effects of glycerol monolaurate (GML) on Lactobacillus, Candida, and Gardnerella vaginalis human vaginal microflora. Our previous work demonstrated that 6 months of GML treatment vaginally does not alter lactobacillus counts in monkeys. Candida and G. vaginalis are commonly associated with vaginal infections in women, many becoming chronic or recurrent. In vitro growth inhibition studies determined the effects of GML (0 to 500 μg/ml) against multiple Candida species and G. vaginalis. A randomized, double-blind study investigated the effects of GML on vaginal microflora Lactobacillus, Candida, and G. vaginalis in colonized or infected women (n = 36). Women self-administered intravaginal gels containing 0% (n = 14), 0.5% (n = 13), or 5% (n = 9) GML every 12 h for 2 days. Vaginal swabs were collected before and immediately after the first gel administration and 12 h after the final gel administration. Swabs were tested for Lactobacillus, Candida, G. vaginalis, and GML. In vitro GML concentrations of 500 μg/ml were candicidal for all species tested, while a concentration of 10 μg/ml was bactericidal for G. vaginalis. Control and GML gels applied vaginally in women did not alter vaginal pH or Lactobacillus counts. Control gels reduced G. vaginalis counts but not Candida counts, whereas GML gels reduced both Candida and G. vaginalis. No adverse events were reported by participating women. GML is antimicrobial for Candida and G. vaginalis in vitro. Vaginal GML gels in women do not affect Lactobacillus negatively but significantly reduce Candida and G. vaginalis. PMID:20008774

  3. Glycerol monolaurate inhibits Candida and Gardnerella vaginalis in vitro and in vivo but not Lactobacillus.

    PubMed

    Strandberg, Kristi L; Peterson, Marnie L; Lin, Ying-Chi; Pack, Melinda C; Chase, David J; Schlievert, Patrick M

    2010-02-01

    We investigated the effects of glycerol monolaurate (GML) on Lactobacillus, Candida, and Gardnerella vaginalis human vaginal microflora. Our previous work demonstrated that 6 months of GML treatment vaginally does not alter lactobacillus counts in monkeys. Candida and G. vaginalis are commonly associated with vaginal infections in women, many becoming chronic or recurrent. In vitro growth inhibition studies determined the effects of GML (0 to 500 microg/ml) against multiple Candida species and G. vaginalis. A randomized, double-blind study investigated the effects of GML on vaginal microflora Lactobacillus, Candida, and G. vaginalis in colonized or infected women (n=36). Women self-administered intravaginal gels containing 0% (n=14), 0.5% (n=13), or 5% (n=9) GML every 12 h for 2 days. Vaginal swabs were collected before and immediately after the first gel administration and 12 h after the final gel administration. Swabs were tested for Lactobacillus, Candida, G. vaginalis, and GML. In vitro GML concentrations of 500 microg/ml were candicidal for all species tested, while a concentration of 10 microg/ml was bactericidal for G. vaginalis. Control and GML gels applied vaginally in women did not alter vaginal pH or Lactobacillus counts. Control gels reduced G. vaginalis counts but not Candida counts, whereas GML gels reduced both Candida and G. vaginalis. No adverse events were reported by participating women. GML is antimicrobial for Candida and G. vaginalis in vitro. Vaginal GML gels in women do not affect Lactobacillus negatively but significantly reduce Candida and G. vaginalis.

  4. Thymoquinone inhibits proliferation in gastric cancer via the STAT3 pathway in vivo and in vitro

    PubMed Central

    Zhu, Wen-Qian; Wang, Jun; Guo, Xu-Feng; Liu, Zhou; Dong, Wei-Guo

    2016-01-01

    AIM: To elucidate the mechanism of thymoquinone (TQ)-induced apoptosis in human gastric cancer cells in vitro and in vivo. METHODS: HGC27, BGC823, and SGC7901 cells were cultured in vitro and treated with TQ (0, 10, 25, 50, 75, 100, 125 μmol/L) for 12 h, 24 h, and 36 h, and then the proliferation inhibitory rates were detected by methylthiazole tetrazolium assay. Apoptosis was observed after Hoechst staining. The protein expressions of signal transducer and activator of transcription (STAT)3, p-STAT3, STAT5, p-STAT5, phospho-janus-activated kinase 2 (JAK2), JAK2, p-Src, Src, glyceraldehyde-3-phosphate dehydrogenase, lamin-A, survivin, Cyclin D, Bcl-2, Bax, peroxisome proliferator activated receptor, and caspase-3,7,9 were detected by western blot. Cell cycle and apoptosis were determined with flow cytometry. TQ induced dose-dependent apoptotic cell death in HGC27 cells was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) analysis and Hoechst 33258. RESULTS: TQ inhibited the phosphorylation of STAT3 but not STAT5. TQ-induced downregulation of STAT3 activation was associated with a reduction in JAK2 and c-Src activity. TQ also downregulated the expression of STAT3-regulated genes, such as Bcl-2, cyclin D, survivin, and vascular endothelial growth factor, and activated caspase-3,7,9. Consistent with the in vitro results, TQ was significantly effective as an antitumor agent in a xenograft tumor mouse model. CONCLUSION: This study provides strong evidence that downregulation of the STAT3 signaling pathway mediates TQ-induced apoptosis in gastric cancer. PMID:27122665

  5. Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells▿ †

    PubMed Central

    Bellecave, Pantxika; Cazenave, Christian; Rumi, Julie; Staedel, Cathy; Cosnefroy, Ophélie; Andreola, Marie-Line; Ventura, Michel; Tarrago-Litvak, Laura; Astier-Gin, Thérèse

    2008-01-01

    We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection. PMID:18347106

  6. Sulfhydryl dependence in primary explant hematopoietic cells. Inhibition of growth in vitro with vitamin B12 compounds.

    PubMed Central

    Toohey, J I

    1975-01-01

    Primary explants of P388, EL-4, and L1210 murine leukemia cells and of normal mouse bone marrow are shown to require sulfhydryl compounds for proliferation in vitro. Nine extablished cell lines show no stimulation by these compounds. Leukemia cells can lose the sulfhydryl dependence after various periods of adaptation to in vitro culture. Various sulfhydryl compounds have widely differing potencies in promoting in vitro proliferation of dependent cells. The effect appears to be specific for sulfhydryl groups in the reduced form. Vitamin B12 compounds inhibit the growth of sulfhydryl-requiring cells, apparently by catalyzing the oxidation of the sulfhydryl groups. PMID:1054516

  7. Lack of reproducible growth inhibition by Schlafen1 and Schlafen2 in vitro.

    PubMed

    Zhao, Liang; Neumann, Brent; Murphy, Kathleen; Silke, John; Gonda, Thomas J

    2008-01-01

    The Schlafen gene family has been implicated in lymphoid and myeloid maturation and differentiation as well as inflammation. However, little is known about the functions of this gene family except that anti-proliferative activities, particularly for Schlafen1, the prototype member of the family, have been reported. This was shown mainly by ectopic expression of Schlafen1 in murine fibroblasts resulting in growth inhibition and a G1 cell cycle arrest apparently via repression of Cyclin D1 expression. However, we have been unable to reproduce these findings. Schlafen1 and Schlafen2 failed to inhibit cell proliferation, cause G1 cell cycle arrest, or affect Cyclin D1 level in murine fibroblasts. This was regardless of whether overexpression was constitutive, induced or from transient transfections. Moreover, in our hands, Schlafen1 and -2 do not appear to regulate the activity of Cyclin D1 promoter. Importantly, we also showed that Schlafen1 and -2 do not play anti-proliferative roles in more physiologically-relevant myeloid cell lines. We therefore suggest that Schlafen1 and Schlafen2 might not have obligatory anti-proliferative activities, at least in vitro, and that efforts to explore their functions should be directed to other aspects, such as haemopoietic development and immune response.

  8. TFF1 inhibits proliferation and induces apoptosis of gastric cancer cells in vitro.

    PubMed

    Ge, Yanli; Zhang, Junjie; Cao, Jianchun; Wu, Qiong; Sun, Longe; Guo, Likun; Wang, Zhirong

    2012-05-01

    Trefoil Factor Family (TFF) plays an essential role in the intestinal epithelial restitution, but the relationship between TFF1 and gastric cancer (GC) is still unclear. The present study aimed to determine the role of TFF1 in repairing gastric mucosa and in the pathogenesis of GC. The TFF1 expression in different gastric mucosas was measured with immunohistochemistry. Then, siRNA targeting TFF1 or plasmids expressing TFF1 gene were transfected into BGC823 cells, SGC7901 cells and GES-1 cells. The cell proliferation was detected with MTT assay and apoptosis and cell cycle measured by flow cytometry. From normal gastric mucosa to mucosa with dysplasia and to gastric cancer, the TFF1 expression had a decreasing trend. Down-regulation of TFF1 expression significantly reduced the apoptosis of three cell lines and markedly facilitated their proliferation but had no significant effect on cell cycle. Over-expression of TFF1 could promote apoptosis of three cell lines and inhibit proliferation but had no pronounced effect on cell cycle. TFF1 can inhibit proliferation and induce apoptosis of GC cells in vitro.

  9. Graphene oxide inhibits malaria parasite invasion and delays parasitic growth in vitro.

    PubMed

    Kenry; Lim, Ying Bena; Nai, Mui Hoon; Cao, Jianshu; Loh, Kian Ping; Lim, Chwee Teck

    2017-09-28

    The interactions between graphene oxide (GO) and various biological entities have been actively investigated in recent years, resulting in numerous potential bioapplications of these nanomaterials. Despite this, the biological interactions between GO and disease-causing protozoan parasites have not been well elucidated and remain relatively unexplored. Here, we investigate the in vitro interactions between GO nanosheets and a particular species of malaria parasites, Plasmodium falciparum (P. falciparum). We hypothesize that GO nanosheets may exhibit antimalarial characteristic via action mechanisms of physical obstruction of P. falciparum parasites as well as nutrient depletion. To ascertain this, we characterize the physical interactions between GO nanosheets, red blood cells (RBCs), and malarial parasites as well as the adsorption of several biomolecules necessary for parasitic survival and growth on GO nanosheets. Subsequent to establishing the origin of this antimalarial behavior of GO nanosheets, their efficiency in inhibiting parasite invasion is evaluated. We observe that GO nanosheets at various tested concentrations significantly inhibit the invasion of malaria parasites into RBCs. Furthermore, GO nanosheets delay parasite progression from the ring to the trophozoite stage. Overall, this study may further shed light on the graphene-parasite interactions and potentially facilitate the development of nanomaterial-based strategies for combating malaria.

  10. Chronic exposure of low dose salinomycin inhibits MSC migration capability in vitro

    PubMed Central

    SCHERZAD, AGMAL; HACKENBERG, STEPHAN; FROELICH, KATRIN; RAK, KRISTEN; HAGEN, RUDOLF; TAEGER, JOHANNES; BREGENZER, MAXIMILLIAN; KLEINSASSER, NORBERT

    2016-01-01

    Salinomycin is a polyether antiprotozoal antibiotic that is used as a food additive, particularly in poultry farming. By consuming animal products, there may be a chronic human exposure to salinomycin. Salinomycin inhibits the differentiation of preadipocytes into adipocytes. As human mesenchymal stem cells (MSC) may differentiate into different mesenchymal cells, it thus appeared worthwhile to investigate whether chronic salinomycin exposure impairs the functional properties of MSC and induces genotoxic effects. Bone marrow MSC were treated with low-dose salinomycin (100 nM) (MSC-Sal) for 4 weeks, while the medium containing salinomycin was changed every other day. Functional changes were evaluated and compared to MSC without salinomycin treatment (MSC-control). MSC-Sal and MSC-control were positive for cluster of differentiation 90 (CD90), CD73 and CD44, and negative for CD34. There were no differences observed in cell morphology or cytoskeletal structures following salinomycin exposure. The differentiation into adipocytes and osteocytes was not counteracted by salinomycin, and proliferation capability was not inhibited following salinomycin exposure. The migration of MSC-Sal was attenuated significantly as compared to the MSC-control. There were no genotoxic effects after 4 weeks of salinomycin exposure. The present study shows an altered migration capacity as a sign of functional impairment of MSC induced by chronic salinomycin exposure. Further in vitro toxicological investigations, particularly with primary human cells, are required to understand the impact of chronic salinomycin consumption on human cell systems. PMID:26998269

  11. Chronic exposure of low dose salinomycin inhibits MSC migration capability in vitro.

    PubMed

    Scherzad, Agmal; Hackenberg, Stephan; Froelich, Katrin; Rak, Kristen; Hagen, Rudolf; Taeger, Johannes; Bregenzer, Maximillian; Kleinsasser, Norbert

    2016-03-01

    Salinomycin is a polyether antiprotozoal antibiotic that is used as a food additive, particularly in poultry farming. By consuming animal products, there may be a chronic human exposure to salinomycin. Salinomycin inhibits the differentiation of preadipocytes into adipocytes. As human mesenchymal stem cells (MSC) may differentiate into different mesenchymal cells, it thus appeared worthwhile to investigate whether chronic salinomycin exposure impairs the functional properties of MSC and induces genotoxic effects. Bone marrow MSC were treated with low-dose salinomycin (100 nM) (MSC-Sal) for 4 weeks, while the medium containing salinomycin was changed every other day. Functional changes were evaluated and compared to MSC without salinomycin treatment (MSC-control). MSC-Sal and MSC-control were positive for cluster of differentiation 90 (CD90), CD73 and CD44, and negative for CD34. There were no differences observed in cell morphology or cytoskeletal structures following salinomycin exposure. The differentiation into adipocytes and osteocytes was not counteracted by salinomycin, and proliferation capability was not inhibited following salinomycin exposure. The migration of MSC-Sal was attenuated significantly as compared to the MSC-control. There were no genotoxic effects after 4 weeks of salinomycin exposure. The present study shows an altered migration capacity as a sign of functional impairment of MSC induced by chronic salinomycin exposure. Further in vitro toxicological investigations, particularly with primary human cells, are required to understand the impact of chronic salinomycin consumption on human cell systems.

  12. Inhibition of phosphatidylcholine-specific phospholipase C prevents bone marrow stromal cell senescence in vitro.

    PubMed

    Sun, Chunhui; Wang, Nan; Huang, Jie; Xin, Jie; Peng, Fen; Ren, Yinshi; Zhang, Shangli; Miao, Junying

    2009-10-01

    Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long-term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and levels of integrin beta4, caveolin-1 and ROS with BMSC senescence. The activity of PC-PLC and levels of integrin beta4, caveolin-1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC-PLC activity with D609 significantly decreased the number of senescence-associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin beta4, caveolin-1 and ROS. The data suggest that PC-PLC is involved in senescence of BMSCs, and its function is associated with integrin beta4, caveolin-1 and ROS.

  13. Thiazolidinediones inhibit the growth of PC12 cells both in vitro and in vivo

    SciTech Connect

    Kim, Sang Wan; Choi, Ok Kyung; Chang, Mee Soo; Shin, Chan Soo; Park, Kyong Soo; Kim, Seong Yeon

    2008-06-27

    Thiazolidinediones (TZDs) have recently been proposed as a therapy for PPAR{gamma}-expressing tumors. Pheochromocytoma (PHEO) is associated with high morbidity and mortality due to excess catecholamine production, and few effective drug therapies currently exist. We investigated the effects of TZDs on PHEO both in vitro and in vivo. PPAR{gamma} protein was expressed in human adrenal PHEO tissues as well as in rat PHEO cells, PC12. TZDs, including rosiglitazone (RGZ) and pioglitazone (PGZ), inhibited proliferation of PC12 cells in a dose-dependent manner and increased casapse-3 expression of PC12 cells. TZDs also reduced expression of cyclin E and cyclin-dependent kinase2. RGZ inhibited nerve growth factor-induced neurite outgrowth and reduced expression of catecholamine-synthesizing enzymes. Finally, rat PHEO growth generated by subcutaneous injection of PC12 cells was slowed in an RGZ-treated mouse. These data suggest that TZDs may be a promising therapeutic approach for medical treatment for PHEO.

  14. Warifteine, an Alkaloid Purified from Cissampelos sympodialis, Inhibits Neutrophil Migration In Vitro and In Vivo

    PubMed Central

    Lima, Thaline F. A.; Rocha, Juliana D. B.; Guimarães-Costa, Anderson B.; Barbosa-Filho, José M.; Decoté-Ricardo, Débora; Saraiva, Elvira M.; Arruda, Luciana B.; Piuvezam, Marcia R.; Peçanha, Ligia M. T.

    2014-01-01

    Cissampelos sympodialis Eichl is a plant from the Northeast and Southeast of Brazil. Its root infusion is popularly used for treatment of inflammatory and allergic diseases. We investigated whether warifteine, its main alkaloid, would have anti-inflammatory effect due to a blockage of neutrophil function. In vivo warifteine treatment inhibited casein-induced neutrophil migration to the peritoneal cavity but did not inhibit neutrophil mobilization from the bone marrow. Analysis of the direct effect of warifteine upon neutrophil adherence and migration in vitro demonstrated that the alkaloid decreased cell adhesion to P and E-selectin-transfected cells. In addition, fLMP-induced neutrophil migration in a transwell system was blocked by warifteine; this effect was mimicked by cAMP mimetic/inducing substances, and warifteine increased intracellular cAMP levels in neutrophils. The production of DNA extracellular traps (NETs) was also blocked by warifteine but there was no alteration on PMA-induced oxidative burst or LPS-stimulated TNFα secretion. Taken together, our data indicate that the alkaloid warifteine is a potent anti-inflammatory substance and that it has an effect on neutrophil migration through a decrease in both cell adhesion and migration. PMID:24995347

  15. Regulation of Persistent Activity by Background Inhibition in an In Vitro Model of a Cortical Microcircuit

    PubMed Central

    Fellous, Jean-Marc; Sejnowski, Terrence J.

    2010-01-01

    We combined in vitro intracellular recording from prefrontal cortical neurons with simulated synaptic activity of a layer 5 prefrontal microcircuit using a dynamic clamp. During simulated in vivo background conditions, the cell responded to a brief depolarization with a sequence of spikes that outlasted the depolarization, mimicking the activity of a cell recorded during the delay period of a working memory task in the behaving monkey. The onset of sustained activity depended on the number of action potentials elicited by the cue-like depolarization. Too few spikes failed to provide enough NMDA drive to elicit sustained reverberations; too many spikes activated a slow intrinsic hyperpolarization current that prevented spiking; an intermediate number of spikes produced sustained activity. When high dopamine levels were simulated by depolarizing the cell and by increasing the amount of NMDA current, the cell exhibited spontaneous ‘up-states’ that terminated by the activation of a slow intrinsic hyperpolarizing current. The firing rate during the delay period could be effectively modulated by the standard deviation of the inhibitory background synaptic noise without significant changes in the background firing rate before cue onset. These results suggest that the balance between fast feedback inhibition and slower AMPA and NMDA feedback excitation is critical in initiating persistent activity and that the maintenance of persistent activity may be regulated by the amount of correlated background inhibition. PMID:14576214

  16. Hepcidin inhibits in vitro erythroid colony formation at reduced erythropoietin concentrations

    PubMed Central

    Dallalio, Gail; Law, Erin; Means, Robert T.

    2006-01-01

    The anemia of chronic disease (ACD) results from 3 major processes: slightly shortened red cell survival, impaired reticuloendothelial system iron mobilization, and impaired erythropoiesis. Hepcidin is an acute-phase protein with specific iron regulatory properties, which, along with the anemia seen with increased hepcidin expression, have led many to consider it the major mediator of ACD. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. Erythroid colony formation in vitro was inhibited by hepcidin at erythropoietin (Epo) concentrations less than or equal to 0.5 U/mL but not at Epo 1.0 U/mL. At Epo concentrations of 0.3 U/mL, HCD57 erythroleukemia cells exposed to hepcidin exhibit decreased expression of the antiapoptotic protein pBad compared with controls. These studies suggest that hepcidin may contribute to anemia in ACD not only through effects on iron metabolism, but also through inhibition of erythroid progenitor proliferation and survival. PMID:16332970

  17. Inhibition of Histone Deacetylases Induces Bovine Leukemia Virus Expression In Vitro and In Vivo

    PubMed Central

    Merezak, C.; Reichert, M.; Van Lint, C.; Kerkhofs, P.; Portetelle, D.; Willems, L.; Kettmann, R.

    2002-01-01

    Packaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only ∼2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation. PMID:11967319

  18. Curcumin and Boswellia serrata gum resin extract inhibit chikungunya and vesicular stomatitis virus infections in vitro.

    PubMed

    von Rhein, Christine; Weidner, Tatjana; Henß, Lisa; Martin, Judith; Weber, Christopher; Sliva, Katja; Schnierle, Barbara S

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever, and severe arthritis that can persist for years. CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions including Europe and the United States of America. CHIKV has recently caused large outbreaks in Latin America. No treatment or licensed CHIKV vaccine exists. Traditional medicines are known to have anti-viral effects; therefore, we examined whether curcumin or Boswellia serrata gum resin extract have antiviral activity against CHIKV. Both compounds blocked entry of CHIKV Env-pseudotyped lentiviral vectors and inhibited CHIKV infection in vitro. In addition, vesicular stomatitis virus vector particles and viral infections were also inhibited to the same extent, indicating a broad antiviral activity. Although the bioavailability of these compounds is rather poor, they might be used as a lead structure to develop more effective antiviral drugs or might be used topically to prevent CHIKV spread in the skin after mosquito bites.

  19. Ultrahigh molecular weight polyethylene wear debris inhibits osteoprogenitor proliferation and differentiation in vitro.

    PubMed

    Chiu, Richard; Ma, Ting; Smith, R Lane; Goodman, Stuart B

    2009-04-01

    Polyethylene wear debris induces progressive osteolysis by increasing bone degradation and suppressing bone formation. Polyethylene particles inhibit the function of mature osteoblasts, but whether polyethylene particles also interfere with the proliferation and differentiation of osteoprogenitor cells is unknown. In this study, we investigated the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on the osteogenic activity of primary murine bone marrow osteoprogenitors and MC3T3-E1 preosteoblastic cells in vitro. Submicron-sized UHMWPE particles generated from wear simulator tests were isolated from serum-containing solution by density gradient centrifugation. The particles were coated onto the surface of culture wells at concentrations of 0.038, 0.075, 0.150, 0.300, and 0.600% v/v in a layer of type I collagen matrix. Primary murine bone marrow cells and MC3T3-E1 preosteoblasts were seeded onto the particle-collagen matrix and induced to differentiate in osteogenic medium for 20 days. Exposure of both cell populations to UHMWPE particles resulted in a dose-dependent decrease in mineralization, proliferation, alkaline phosphatase activity, and osteocalcin production when compared with control cells cultured on collagen matrix without particles. Complete suppression of osteogenesis was observed at particle concentrations > or =0.150% v/v. This study demonstrated that UHMWPE particles inhibit the osteogenic activity of osteoprogenitor cells, which may result in reduced periprosthetic bone regeneration and repair. Copyright 2008 Wiley Periodicals, Inc.

  20. CHLORHEXIDINE INHIBITS L1 CELL ADHESION MOLECULE MEDIATED NEURITE OUTGROWTH IN VITRO

    PubMed Central

    Milstone, Aaron M.; Bamford, Penny; Aucott, Susan W.; Tang, Ningfeng; White, Kimberly R.; Bearer, Cynthia F.

    2013-01-01

    Background Chlorhexidine is a skin disinfectant that reduces skin and mucous membrane bacterial colonization and inhibits organism growth. Despite numerous studies assessing chlorhexidine safety in term infants, residual concerns have limited its use in hospitalized neonates, especially low birth weight preterm infants. The aim of this study was to assess the potential neurotoxicity of chlorhexidine on the developing central nervous system using a well-established in vitro model of neurite outgrowth that includes laminin and L1 cell adhesion molecule (L1) as neurite outgrowth promoting substrates. Methods Cerebellar granule neurons are plated on either poly L-lysine, L1 or laminin. Chlorhexidine, hexachlorophene or their excipients are added to the media. Neurons are grown for 24 h, then fixed and neurite length measured. Results Chlorhexidine significantly reduced the length of neurites grown on L1 but not laminin. Chlorhexidine concentrations as low as 125 ng/ml statistically significantly reduced neurite length on L1. Hexachlorophene did not affect neurite length. Conclusion Chlorhexidine at concentrations detected in the blood following topical applications in preterm infants specifically inhibited L1 mediated neurite outgrowth of cerebellar granule neurons. It is now vital to determine whether the blood brain barrier is permeable to chlorhexidine in preterm infants. PMID:24126818

  1. Lactobacillus crispatus inhibits the infectivity of Chlamydia trachomatis elementary bodies, in vitro study.

    PubMed

    Nardini, Paola; Ñahui Palomino, Rogers Alberto; Parolin, Carola; Laghi, Luca; Foschi, Claudio; Cevenini, Roberto; Vitali, Beatrice; Marangoni, Antonella

    2016-06-29

    Lactobacillus species dominate the vaginal microbiota of healthy reproductive-age women and protect the genitourinary tract from the attack of several infectious agents. Chlamydia trachomatis, a leading cause of sexually transmitted disease worldwide, can induce severe sequelae, i.e. pelvic inflammatory disease, infertility and ectopic pregnancy. In the present study we investigated the interference of Lactobacillus crispatus, L. gasseri and L. vaginalis, known to be dominant species in the vaginal microbiome, with the infection process of C. trachomatis. Lactobacilli exerted a strong inhibitory effect on Chlamydia infectivity mainly through the action of secreted metabolites in a concentration/pH dependent mode. Short contact times were the most effective in the inhibition, suggesting a protective role of lactobacilli in the early steps of Chlamydia infection. The best anti-Chlamydia profile was shown by L. crispatus species. In order to delineate metabolic profiles related to anti-Chlamydia activity, Lactobacillus supernatants were analysed by (1)H-NMR. Production of lactate and acidification of the vaginal environment seemed to be crucial for the activity, in addition to the consumption of the carbonate source represented by glucose. The main conclusion of this study is that high concentrations of L. crispatus inhibit infectivity of C. trachomatis in vitro.

  2. Lactobacillus crispatus inhibits the infectivity of Chlamydia trachomatis elementary bodies, in vitro study

    PubMed Central

    Nardini, Paola; Ñahui Palomino, Rogers Alberto; Parolin, Carola; Laghi, Luca; Foschi, Claudio; Cevenini, Roberto; Vitali, Beatrice; Marangoni, Antonella

    2016-01-01

    Lactobacillus species dominate the vaginal microbiota of healthy reproductive-age women and protect the genitourinary tract from the attack of several infectious agents. Chlamydia trachomatis, a leading cause of sexually transmitted disease worldwide, can induce severe sequelae, i.e. pelvic inflammatory disease, infertility and ectopic pregnancy. In the present study we investigated the interference of Lactobacillus crispatus, L. gasseri and L. vaginalis, known to be dominant species in the vaginal microbiome, with the infection process of C. trachomatis. Lactobacilli exerted a strong inhibitory effect on Chlamydia infectivity mainly through the action of secreted metabolites in a concentration/pH dependent mode. Short contact times were the most effective in the inhibition, suggesting a protective role of lactobacilli in the early steps of Chlamydia infection. The best anti-Chlamydia profile was shown by L. crispatus species. In order to delineate metabolic profiles related to anti-Chlamydia activity, Lactobacillus supernatants were analysed by 1H-NMR. Production of lactate and acidification of the vaginal environment seemed to be crucial for the activity, in addition to the consumption of the carbonate source represented by glucose. The main conclusion of this study is that high concentrations of L. crispatus inhibit infectivity of C. trachomatis in vitro. PMID:27354249

  3. JAK2 and AMP-kinase inhibition in vitro by food extracts, fractions and purified phytochemicals.

    PubMed

    Martin, Harry; Burgess, Elaine J; Smith, Wendy A; McGhie, Tony K; Cooney, Janine M; Lunken, Rona C M; de Guzman, Erika; Trower, Tania; Perry, Nigel B

    2015-01-01

    We have identified a range of food phytochemicals that inhibit Janus Kinase 2 (JAK2) and Adenosine Monophosphate Kinase (AMPK). A mutated and dysregulated form of JAK2, a tyrosine kinase, is associated with several diseases including Crohn's disease. Using an in vitro, time-resolved fluorescence (TR-FRET) assay, we tested 49 different types of food extracts, plus 10 concentrated fractions of increasing hydrophobicity from each extract, to find foods containing JAK2 inhibitors. The food extracts tested included grains, meat, fish, shellfish, dairy products, herbs, mushrooms, hops, fruits and vegetables. Several fruits were potent inhibitors of JAK2: blackberry, boysenberry, feijoa, pomegranate, rosehip and strawberry, which all contain ellagitannins, known inhibitors of kinases. These fruits are in the Rosales and Myrtales plant orders. No other foods gave >1% of the maximal JAK2 inhibitory activities of these fruits. AMPK, a sensor and regulator of energy metabolism in cells, is a serine-threonine kinase which is reported to be activated by various flavonoid phytochemicals. Using a TR-FRET assay, we tested various fruit extracts for AMPK activation and inhibition. Ellagitannin containing foods scored highly as AMPK inhibitors. Despite several reports of AMPK activation in whole cells by phytochemicals, no extracts or pure compounds activated AMPK in our assay.

  4. Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro

    SciTech Connect

    Saporito, M.S.; Warwick, R.O. Jr.

    1989-01-01

    We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B decreased K/sup +/ evoked /sup 3/H-5-HT release from superfused HYP slices by 25%. Bacitracin, a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K/sup +/ evoked /sup 3/H-5-HT release. Phosphoramidon (PAN, 10 /mu/M) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K/sup +/ evoked /sup 3/H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 /mu/M), enhanced both BN and NM-C inhibition of /sup 3/H-5-HT release. Bestatin (BST, 10 /mu/M) had no effect on BN or NM-C inhibitory activity on /sup 3/H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of /sup 3/H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit /sup 3/H-5-HT uptake.

  5. A p7 Ion Channel-derived Peptide Inhibits Hepatitis C Virus Infection in Vitro*

    PubMed Central

    Hong, Wei; Lang, Yange; Li, Tian; Zeng, Zhengyang; Song, Yu; Wu, Yingliang; Li, Wenxin; Cao, Zhijian

    2015-01-01

    Viral infection is an early stage of its life cycle and represents a promising target for antiviral drug development. Here we designed and characterized three peptide inhibitors of hepatitis C virus (HCV) infection based on the structural features of the membrane-associated p7 polypeptide of HCV. The three peptides exhibited low toxicity and high stability while potently inhibiting initial HCV infection and suppressed established HCV infection at non-cytotoxic concentrations in vitro. The most efficient peptide (designated H2-3), which is derived from the H2 helical region of HCV p7 ion channel, inhibited HCV infection by inactivating both intracellular and extracellular viral particles. The H2-3 peptide inactivated free HCV with an EC50 (50% effective concentration) of 82.11 nm, which is >1000-fold lower than the CC50 (50% cytotoxic concentration) of Huh7.5.1 cells. H2-3 peptide also bound to cell membrane and protected host cells from viral infection. The peptide H2-3 did not alter the normal electrophysiological profile of the p7 ion channel or block viral release from Huh7.5.1 cells. Our work highlights a new anti-viral peptide design strategy based on ion channel, giving the possibility that ion channels are potential resources to generate antiviral peptides. PMID:26251517

  6. Sildenafil inhibits the growth of human colorectal cancer in vitro and in vivo

    PubMed Central

    Mei, Xiao-Long; Yang, Yang; Zhang, Yao-Jun; Li, Yong; Zhao, Jin-Ming; Qiu, Jian-Ge; Zhang, Wen-Ji; Jiang, Qi-Wei; Xue, You-Qiu; Zheng, Di-Wei; Chen, Yao; Qin, Wu-Ming; Wei, Meng-Ning; Shi, Zhi

    2015-01-01

    Colorectal cancer is the third most common human cancer with frequent overexpression of the cGMP-specific phosphodiesterase 5 (PDE5). In the present study, we investigated that the anticancer effect of sildenafil on human colorectal cancer in vitro and in vivo, which is a potent and selective inhibitor of PDE5 for the treatment of erectile dysfunction and pulmonary arterial hypertension in the clinic. Sildenafil significantly induced cell growth inhibition, cell cycle arrest and apoptosis of human colorectal cancer with increased intracellular reactive oxidative specie (ROS) levels, which were accompanied by obvious alterations of related proteins such as CDKs, Cyclins and PARP etc. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse sildenafil-induced ROS accumulation and cell apoptosis. Inhibition of the activity of protein kinase G with KT-5823 could enhance sildenafil-induced apoptosis. Furthermore, sildenafil caused the reduction of xenograft models of human colorectal cancer in nude mice. Overall, these findings suggest that sildenafil has the potential to be used for treatment of human colorectal cancer. PMID:26807313

  7. CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo.

    PubMed

    Farina, Hernán G; Benavent Acero, Fernando; Perera, Yasser; Rodríguez, Arielis; Perea, Silvio E; Castro, Boris Acevedo; Gomez, Roberto; Alonso, Daniel F; Gomez, Daniel E

    2011-07-15

    We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.

  8. In vitro inhibition of choline acetyltransferase by a series of 2-benzylidene-3-quinuclidinones

    SciTech Connect

    Capacio, B.R.

    1988-01-01

    Ten substituted 2-benzylidene-3-quinuclidinones were synthesized and evaluated for their relative potency as in vitro inhibitors of choline acetyltransferase (ChAT). Acetylcholine (ACh) synthesis was followed radiometrically by the incorporation of labeled acetate originating from {sup 14}C-acetyl-CoA. Woolf-Augustinsson-Hofstee data analysis was used to calculate Vmax, Km, and Ki values. The inhibition was found to be noncompetitive or uncompetitive with respect to choline. Quantitative structure activity relationship correlations demonstrated a primary dependence on {kappa}-{sigma}, as well as steric properties of the substituted benzene ring. Additional radiometric and spectrophotometric were performed with 2-(3{prime}-methyl)-benzylidene-3-quinuclidinone, one of the more potent analogs, to further elucidate the inhibitory mechanism. ChAT-mediated cleavage of ACh was measured spectrophotometrically by following the appearance of NADH at 340 nanometers in an enzyme coupled assay. Lineweaver-Burk analysis indicated mixed or uncompetitive inhibition with respect to both substrates of the forward reaction, suggesting interference with a rate limiting step.

  9. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signalling

    PubMed Central

    Ko, A; Kanehisa, A; Martins, I; Senovilla, L; Chargari, C; Dugue, D; Mariño, G; Kepp, O; Michaud, M; Perfettini, J-L; Kroemer, G; Deutsch, E

    2014-01-01

    Clinical oncology heavily relies on the use of radiotherapy, which often leads to merely transient responses that are followed by local or distant relapse. The molecular mechanisms explaining radioresistance are largely elusive. Here, we identified a dual role of autophagy in the response of cancer cells to ionizing radiation. On one hand, we observed that the depletion of essential autophagy-relevant gene products, such as ATG5 and Beclin 1, increased the sensitivity of human or mouse cancer cell lines to irradiation, both in vitro (where autophagy inhibition increased radiation-induced cell death and decreased clonogenic survival) and in vivo, after transplantation of the cell lines into immunodeficient mice (where autophagy inhibition potentiated the tumour growth-inhibitory effect of radiotherapy). On the other hand, when tumour proficient or deficient for autophagy were implanted in immunocompetent mice, it turned out that defective autophagy reduced the efficacy of radiotherapy. Indeed, radiotherapy elicited an anti-cancer immune response that was dependent on autophagy-induced ATP release from stressed or dying tumour cells and was characterized by dense lymphocyte infiltration of the tumour bed. Intratumoural injection of an ecto-ATPase inhibitor restored the immune infiltration of autophagy-deficient tumours post radiotherapy and improved the growth-inhibitory effect of ionizing irradiation. Altogether, our results reveal that beyond its cytoprotective function, autophagy confers immunogenic properties to tumours, hence amplifying the efficacy of radiotherapy in an immunocompetent context. This has far-reaching implications for the development of pharmacological radiosensitizers. PMID:24037090

  10. 5-aminosalicylic acid in combination with nimesulide inhibits proliferation of colon carcinoma cells in vitro

    PubMed Central

    Fang, Hai-Ming; Mei, Qiao; Xu, Jian-Ming; Ma, Wei-Juan

    2007-01-01

    AIM: To investigate the effects of 5-aminosalicylic acid (5-ASA) in combination with nimesulide on the proliferation of HT-29 colon carcinoma cells and its potential mechanisms. METHODS: Inhibitory effects of drugs (5-ASA, nimesulide and their combination) on HT-29 colon carcinoma cells were investigated by thiazolyl blue tetrazolium bromide (MTT) assay. Cellular apoptosis and proliferation were detected by TUNEL assay and immunocytochemical staining, respectively. RESULTS: Pretreatment with 5-ASA or nimesulide at the concentration of 10-1000 μmol/L inhibited proliferation of HT-29 colon carcinoma cells in a dose-dependent manner in vitro (t = 5.122, P < 0.05; t = 3.086, P < 0.05, respectively). The inhibition rate of HT-29 colon carcinoma cell proliferation was also increased when pretreated with 5-ASA (100 μmol/L) or nimesulide (100 μmol/L) for 12-96 h, which showed an obvious time-effect relationship (t = 6.149, P < 0.05; t = 4.159, P < 0.05, respectively). At the concentration of 10-500 μmol/L, the apoptotic rate of HT-29 colon carcinoma cells significantly increased (t = 18.156, P < 0.001; t = 19.983, P < 0.001, respectively), while expression of proliferating cell nuclear antigen (PCNA) was remarkably decreased (t = 6.828, P < 0.05; t = 14.024, P < 0.05, respectively). 5-ASA in combination with nimesulide suppressed the proliferation of HT-29 colon carcinoma cells more than either of these agents in a dose-dependent and time-dependent manner (t = 5.448, P < 0.05; t = 4.428, P < 0.05, respectively). CONCLUSION: 5-ASA and nimesulide may inhibit the proliferation of HT-29 colon carcinoma cells and coadministration of these agents may have additional chemopreventive potential. PMID:17569127

  11. Inhibition of final maturation of Atlantic croaker oocytes in vitro by organochlorines

    SciTech Connect

    Ghosh, S.; Thomas, P.

    1994-12-31

    Final oocyte maturation (FOM) in teleosts is induced by a maturation inducing steroid (MIS) secreted in response to a surge in maturational gonadotropin secretion. Recently the authors found that a variety of organochlorine pesticides bind to the ovarian plasma membrane receptor for the MIS in the spotted seatrout (Cynoscion nebulosus). In the present study the interaction of various xenobiotics (10{sup {minus}9}--10{sup {minus}4}M) with MIS-induced final maturation of follicle-enclosed oocytes of Atlantic croaker (Micropogonias undulatus), a closely related species, was investigated using an in vitro oocyte maturation bioassay. A wide range of organochlorine compounds (10{sup {minus}9}--10{sup {minus}4}M) inhibited MIS-induced FOM 10%-80% in a concentration-dependent manner. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was the most potent inhibitor among the compounds tested. The minimum concentration of TCDD which inhibited final maturation of 50% of the oocytes (EC{sub 50}) was in the 10{sup {minus}7} M range whereas the EC{sub 50} values for Kepone and naphthalene were in the 10{sup {minus}5}M range. The relative potencies of DDT analogs varied greatly, with EC{sub 50} values ranging from 10{sup {minus}7}--10{sup {minus}4}M (o,p{prime}-DDD > o,p{prime}-DDE > o,p{prime}DDT > p,p{prime}-DDE > p,p{prime}-DDE > p,p{prime}-DDT). PCBs only inhibited FOM at higher concentrations (2--200 ppm); congeners with higher chlorine contents were the most inhibitory. These results indicate that organochlorine compounds at environmentally realistic concentrations can affect reproduction in fish by interfering with FOM, a critical stage of the reproductive cycle.

  12. Novel Triazine JPC-2067-B Inhibits Toxoplasma gondii In Vitro and In Vivo

    PubMed Central

    Mui, Ernest J.; Schiehser, Guy A.; Milhous, Wilbur K.; Hsu, Honghue; Roberts, Craig W.; Kirisits, Michael; Muench, Stephen; Rice, David; Dubey, J. P.; Fowble, Joseph W.; Rathod, Pradipsinh K.; Queener, Sherry F.; Liu, Susan R.; Jacobus, David P.; McLeod, Rima

    2008-01-01

    Background and Methodology Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3′(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3′-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. Principal Findings and Conclusions Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. Significance JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines. PMID:18320016

  13. Novel triazine JPC-2067-B inhibits Toxoplasma gondii in vitro and in vivo.

    PubMed

    Mui, Ernest J; Schiehser, Guy A; Milhous, Wilbur K; Hsu, Honghue; Roberts, Craig W; Kirisits, Michael; Muench, Stephen; Rice, David; Dubey, J P; Fowble, Joseph W; Rathod, Pradipsinh K; Queener, Sherry F; Liu, Susan R; Jacobus, David P; McLeod, Rima

    2008-03-05

    Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3'(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3'-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.

  14. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    SciTech Connect

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B. . E-mail: jbarnett@hsc.wvu.edu

    2007-06-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

  15. Grape seed extract inhibits in vitro and in vivo growth of human colorectal carcinoma cells.

    PubMed

    Kaur, Manjinder; Singh, Rana P; Gu, Mallikarjuna; Agarwal, Rajesh; Agarwal, Chapla

    2006-10-15

    Accumulating evidences suggest the beneficial effects of fruit-and-vegetable consumption in lowering the risk of various cancers, including colorectal cancer. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of grape seed extract (GSE), a rich source of proanthocyanidins, against colorectal cancer. Effects of GSE were examined on human colorectal cancer HT29 and LoVo cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral GSE was examined on HT29 tumor xenograft growth in athymic nude mice. Xenografts were analyzed by immunohistochemistry for proliferation and apoptosis. The molecular changes associated with the biological effects of GSE were analyzed by Western blot analysis. GSE (25-100 microg/mL) causes a significant dose- and time-dependent inhibition of cell growth with concomitant increase in cell death. GSE induced G1 phase cell cycle arrest along with a marked increase in Cip1/p21 protein level and a decrease in G1 phase-associated cyclins and cyclin-dependent kinases. GSE-induced cell death was apoptotic and accompanied by caspase-3 activation. GSE feeding to mice at 200 mg/kg dose showed time-dependent inhibition of tumor growth without any toxicity and accounted for 44% decrease in tumor volume per mouse after 8 weeks of treatment. GSE inhibited cell proliferation but increased apoptotic cell death in tumors. GSE-treated tumors also showed enhanced Cip1/p21 protein levels and poly(ADP-ribose) polymerase cleavage. GSE may be an effective chemopreventive agent against colorectal cancer, and that growth inhibitory and apoptotic effects of GSE against colorectal cancer could be mediated via an up-regulation of Cip1/p21.

  16. Evaluation of In Vitro Cytochrome P450 Inhibition and In Vitro Fate of Structurally Diverse N-Oxide Metabolites: Case Studies with Clozapine, Levofloxacin, Roflumilast, Voriconazole and Zopiclone.

    PubMed

    Giri, Poonam; Naidu, Sneha; Patel, Nirmal; Patel, Harilal; Srinivas, Nuggehally R

    2017-08-01

    The role of metabolite(s) to elicit potential clinical drug-drug interaction (DDI) via cytochrome P450 enzymes (CYP) is gaining momentum. In this context, the role of N-oxides for in vitro CYP inhibition has not been evaluated. The objectives of this study were: (a) to examine in vitro CYP inhibition of N-oxides of clozapine, levofloxacin, roflumilast, voriconazole and zopiclone in a tiered approach and (b) evaluate in vitro fate of aforementioned N-oxides examined in recombinant CYPs, human microsomes and hepatocytes. CYP enzymes evaluated in the work included: CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 using standard procedures for incubation with appropriate probe substrates. The initial cutoff for CYP inhibition was ≥50% using 2 and 10 µM concentrations of various N-oxide metabolites (Tier 1). IC50 values were constructed for the CYP pathway(s) that showed ≥50% inhibition (Tier 2). In addition, co-incubation of N-oxides with parent was performed to evaluate potentiation of CYP inhibition (Tier 3). N-oxides of clozapine (CYP2B6/2C19) and voriconazole (CYP2C9/3A4) showed CYP inhibition ≥50%. Clozapine-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC50 of 8.3 and 8.7 µM, respectively. Voriconazole-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC50 of 10.5 and 11.2 µM, respectively. Co-incubation of clozapine-N-oxide with clozapine potentiated CYP2B6/2C19 pathways; however, incubation of voriconazole-N-oxide with voriconazole did not appear to potentiate the CYP pathways because parent caused an inhibition of almost 80%. None of the N-oxides appeared to further undergo biotransformation as judged by the in vitro metabolic fate experiments (stage 2). Clinical DDI potential of specific CYP enzymes needs to be considered arising due to circulatory concentrations of certain N-oxides depending on the dose size and/or frequency of dosing of the respective parent drugs.

  17. A modified in vitro larvae migration inhibition assay using rumen fluid to evaluate Haemonchus contortus viability.

    PubMed

    Whitney, T R; Lee, A E; Klein, D R; Scott, C B; Craig, T M; Muir, J P

    2011-03-10

    Anthelmintic effects of plant secondary compounds may be occurring in the rumen, but in vitro larvae migration inhibition (LMI) methods using rumen fluid and forage material have not been widely used. Forage material added to an in vitro system can affect rumen pH, ammonia N, and volatile fatty acids, which may affect larvae viability (LV). Validating a LMI assay using rumen fluid and a known anthelmintic drug (Ivermectin) and a known anthelmintic plant extract (Quebracho tannins; QT) is important. Rumen fluid was collected and pooled from 3 goats, mixed with buffer solution and a treatment (1 jar/treatment), and placed into an anaerobic incubator for 16h. Ensheathed larvae (<3 months old) were then anaerobically incubated with treatment rumen fluid for 2, 4, or 16h depending on the trial. Larvae (n=15-45) were then transferred onto a screen (n=4-6 wells/treatment) within a multi-screen 96-well plate that contained treatment rumen fluid. Larvae were incubated overnight and those that passed through the 20-μm screen were considered viable. Adding dry or fresh juniper material reduced (P<0.05) pH, ammonia N, and isobutyric, butyric, isovaleric, and valeric acids, and increased (P<0.001) acetic, propionic, and total VFA. Including 4.5% (w/v) polyethylene glycol (PEG) in rumen fluid mixture with or without forage material reduced (P<0.01) LV. However, LV was similar at all PEG concentrations tested (0-2%, w/v; 89.4, 78.9, 76.5, 75.5, and 77.5% viable). Q. tannin concentrations from 0 to 1.2% (w/v) quadratically reduced (P<0.001) LV; 89.4, 65.5, 22.8, and 9.2%. Ivermectin concentrations from 0 to 15μg/mL quadratically reduced (P<0.001) LV; 90.2, 82.6, 73.6, 66.3, 51.9, 56.5, 43.5, 41.9, 29.3, and 19.9% viable, respectively. Effects of altering in vitro rumen fluid pH, ammonia N, and VFA and using PEG when evaluating LV need to be further investigated. In vitro rumen fluid assays using QT and Ivermectin resulted in decreased LV, validating the efficacy of this

  18. Invasion of erythrocytes in vitro by Plasmodium falciparum can be inhibited by monoclonal antibody directed against an S antigen.

    PubMed

    Saul, A; Cooper, J; Ingram, L; Anders, R F; Brown, G V

    1985-11-01

    A monoclonal antibody has been produced which binds to the heat stable S antigen present in the FCQ-27/PNG isolate of Plasmodium falciparum. This monoclonal antibody also inhibits the invasion in vitro of erythrocytes by malarial merozoites thus demonstrating that the S antigens of Plasmodium falciparum may be a target of protective immune responses.

  19. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

    EPA Science Inventory

    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC.
    SP22, a rat sperm membrane protein that is highly-correlated w...

  20. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

    EPA Science Inventory

    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC.
    SP22, a rat sperm membrane protein that is highly-correlated w...

  1. A model of in vitro UDP-glucuronosyltransferase inhibition by bile acids predicts possible metabolic disorders[S

    PubMed Central

    Fang, Zhong-Ze; He, Rong-Rong; Cao, Yun-Feng; Tanaka, Naoki; Jiang, Changtao; Krausz, Kristopher W.; Qi, Yunpeng; Dong, Pei-Pei; Ai, Chun-Zhi; Sun, Xiao-Yu; Hong, Mo; Ge, Guang-Bo; Gonzalez, Frank J.; Ma, Xiao-Chi; Sun, Hong-Zhi

    2013-01-01

    Increased levels of bile acids (BAs) due to the various hepatic diseases could interfere with the metabolism of xenobiotics, such as drugs, and endobiotics including steroid hormones. UDP-glucuronosyltransferases (UGTs) are involved in the conjugation and elimination of many xenobiotics and endogenous compounds. The present study sought to investigate the potential for inhibition of UGT enzymes by BAs. The results showed that taurolithocholic acid (TLCA) exhibited the strongest inhibition toward UGTs, followed by lithocholic acid. Structure-UGT inhibition relationships of BAs were examined and in vitro-in vivo extrapolation performed by using in vitro inhibition kinetic parameters (Ki) in combination with calculated in vivo levels of TLCA. Substitution of a hydrogen with a hydroxyl group in the R1, R3, R4, R5 sites of BAs significantly weakens their inhibition ability toward most UGTs. The in vivo inhibition by TLCA toward UGT forms was determined with following orders of potency: UGT1A4 > UGT2B7 > UGT1A3 > UGT1A1 ∼ UGT1A7 ∼ UGT1A10 ∼ UGT2B15. In conclusion, these studies suggest that disrupted homeostasis of BAs, notably taurolithocholic acid, found in various diseases such as cholestasis, could lead to altered metabolism of xenobiotics and endobiotics through inhibition of UGT enzymes. PMID:24115227

  2. In vitro inhibition of methadone and oxycodone cytochrome P450-dependent metabolism: reversible inhibition by H2-receptor agonists and proton-pump inhibitors.

    PubMed

    Moody, David E; Liu, Fenyun; Fang, Wenfang B

    2013-10-01

    In vitro inhibition of oxycodone metabolism to noroxycodone and oxymorphone and R- and S-methadone metabolism to R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was measured for four H2-receptor antagonists and five proton-pump inhibitors (PPIs) using human liver microsomes (HLM) and cDNA-expressed human cytochrome P450s (rCYPs). Inhibitors were first incubated with HLM at three concentrations with and without preincubation of inhibitor, enzyme source and reducing equivalents to also screen for time-dependent inhibition (TDI). Cimetidine and famotidine (10-1,000 µM) inhibited all the four pathways >50%. Nizatidine and ranitidine did not. All the five PPIs (1-200 µM) inhibited one or more pathways >50%. Half maximal inhibitory concentrations (IC50s) were then determined using rCYPs. Cimetidine and famotidine both inhibited CYP3A4-mediated formation of noroxycodone and CYP2D6-mediated formation of oxymorphone, and famotidine inhibited CYP3A4-mediated formation of R- and S-EDDP, but IC50s were so high that only >10× therapeutic concentrations may have potential for reversible in vivo inhibition. The PPIs were more potent inhibitors; many have the potential for reversible in vivo inhibition at therapeutic concentrations. Omeprazole, esomeprazole and pantoprazole had greater effects on CYP3A4-mediated reactions, whereas lansoprazole was selective for CYP2D6-mediated formation of oxymorphone. Preincubation enhanced cimetidine inhibition of noroxycodone formation and rabeprazole inhibition of all pathways. Future studies will explore irreversible TDI.

  3. Flavonoids casticin and chrysosplenol D from Artemisia annua L. inhibit inflammation in vitro and in vivo

    SciTech Connect

    Li, Yu-Jie; Guo, Yan; Yang, Qing; Weng, Xiao-Gang; Yang, Lan; Wang, Ya-Jie; Chen, Ying; Zhang, Dong; Li, Qi; Liu, Xu-Cen; Kan, Xiao-Xi; Chen, Xi; Zhu, Xiao-Xin; Kmoníèková, Eva; Zídek, Zdenìk

    2015-08-01

    Background: The aim of our experiments was to investigate the anti-inflammatory properties of casticin and chrysosplenol D, two flavonoids present in Artemisia annua L. Methods: Topical inflammation was induced in ICR mice using croton oil. Mice were then treated with casticin or chrysosplenol D. Cutaneous histological changes and edema were assessed. ICR mice were intragastrically administrated with casticin or chrysosplenol D followed by intraperitoneal injection of lipopolysaccharide (LPS). Mouse Raw264.7 macrophage cells were incubated with casticin or chrysosplenol D. Intracellular phosphorylation was detected, and migration was assessed by trans-well assay. HT-29/NFκB-luc cells were incubated with casticin or chrysosplenol D in the presence or absence of LPS, and NF-κB activation was quantified. Results: In mice, administration of casticin (0.5, 1 and 1.5 μmol/cm{sup 2}) and chrysosplenol D (1 and 1.5 μmol/cm{sup 2}) inhibited croton oil-induced ear edema (casticin: 29.39–64.95%; chrysosplenol D: 37.76–65.89%, all P < 0.05) in a manner similar to indomethacin (0.5, 1 and 1.5 μmol/cm{sup 2}; 55.63–84.58%). Casticin (0.07, 0.13 and 0.27 mmol/kg) and chrysosplenol D (0.07, 0.14 and 0.28 mmol/kg) protected against LPS-induced systemic inflammatory response syndrome (SIRS) in mice (all P < 0.05), in a manner similar to dexamethasone (0.03 mmol/kg). Casticin and chrysosplenol D suppressed LPS-induced release of IL-1 beta, IL-6 and MCP-1, inhibited cell migration, and reduced LPS-induced IκB and c-JUN phosphorylation in Raw264.7 cells. JNK inhibitor SP600125 blocked the inhibitory effect of chrysosplenol D on cytokine release. Conclusions: The flavonoids casticin and chrysosplenol D from A. annua L. inhibited inflammation in vitro and in vivo. - Highlights: • We report a new activity of the flavonoids present in Artemisia annua L. • These flavonoids inhibit croton oil-induced ear edema in mice. • These flavonoids protect against LPS-induced SIRS in

  4. Thrombomodulin Induces a Quiescent Phenotype and Inhibits Migration in Vascular Smooth Muscle Cells In Vitro.

    PubMed

    Bass, Heather M; Beard, Richard S; Cha, Byeong J; Yuan, Sarah Y; Nelson, Peter R

    2016-01-01

    Loss of critical endothelial cell function and subsequent vascular smooth muscle cell (VSMC) migration is central to the pathology of injury-induced neointimal hyperplasia and recurrent stenosis. Thrombomodulin (TM), well known for its function as an endothelial surface anticoagulant, may have an unknown direct effect on VSMC physiology that would be lost after injury. Here, we examined a novel effect of TM on VSMC by testing the hypothesis that direct application of TM induces favorable changes to the morphology of VSMC and inhibits their migration. Primary human VSMC were harvested using the explant technique and used in early passage (1-4) for all experiments. Laser-scanning confocal fluorescent imaging was performed to assess the effect of soluble TM on VSMC morphology. In vitro, migration of VSMC was measured using: (1) a 4-hr modified Boyden chemotaxis assay and (2) a 24-hr electric cell-substrate impedance sensing injury migration assay. Migration experiments were conducted with VSMC exposed to increasing doses of soluble recombinant TM. Recombinant thrombin served as a positive control and serum-free media as a negative control for all experimentation. Data were analyzed using a Student's t-test or repeated measures analysis of variance where appropriate (α < 0.05). VSMC exposed to TM clearly demonstrated a quiescent morphology with organized stress fibers consistent with a quiescent, differentiated, contractile phenotype; whereas, thrombin stimulation led to an activated, dedifferentiated, synthetic phenotype. VSMC demonstrated a low, baseline level of migration in unstimulated serum-free conditions. Thrombin significantly stimulated VSMC migration as expected. TM, independent of thrombin, significantly inhibited baseline VSMC migration in a dose-response fashion. The maximal inhibition was observed at (5 μg/mL) with 70% reduction (56 ± 1.7 vs. 18 ± 3.5 cells/5 high-power fields, P = 0.0005). TM has a direct effect on VSMC resulting in a quiescent

  5. Correlating In Vitro Target-Oriented Screening and Docking: Inhibition of Matrix Metalloproteinases Activities by Flavonoids.

    PubMed

    Crascì, Lucia; Basile, Livia; Panico, Annamaria; Puglia, Carmelo; Bonina, Francesco P; Basile, Pierluigi Maria; Rizza, Luisa; Guccione, Salvatore

    2017-03-13

    Metalloproteases are a family of zinc-containing endopeptidases involved in a variety of pathological disorders. The use of flavonoid derivatives as potential metalloprotease inhibitors has recently increased.Particular plants growing in Sicily are an excellent yielder of the flavonoids luteolin, apigenin, and their respective glycoside derivatives (7-O-rutinoside, 7-O-glucoside, and 7-O-glucuronide).The inhibitory activity of luteolin, apigenin, and their respective glycoside derivatives on the metalloproteases MMP-1, MMP-3, MMP-13, MMP-8, and MMP-9 was assessed and rationalized correlating in vitro target-oriented screening and in silico docking.The flavones apigenin, luteolin, and their respective glucosides have good ability to interact with metalloproteases and can also be lead compounds for further development. Glycones are more active on MMP-1, -3, -8, and -13 than MMP-9. Collagenases MMP-1, MMP-8, and MMP-13 are inhibited by compounds having rutinoside glycones. Apigenin and luteolin are inactive on MMP-1, -3, and -8, which can be interpreted as a better selectivity for both -9 and -13 peptidases. The more active compounds are apigenin-7-O-rutinoside on MMP-1 and luteolin-7-O-rutinoside on MMP-3. The lowest IC50 values were also found for apigenin-7-O-glucuronide, apigenin-7-O-rutinoside, and luteolin-7-O-glucuronide. The glycoside moiety might allow for a better anchoring to the active site of MMP-1, -3, -8, -9, and -13. Overall, the in silico data are substantially in agreement with the in vitro ones (fluorimetric assay).

  6. Selenium and vitamin E inhibit radiogenic and chemically induced transformation in vitro via different mechanisms

    SciTech Connect

    Borek, C.; Ong, A.; Mason, H.; Donahue, L.; Biaglow, J.E.

    1986-03-01

    Results from in vivo and in vitro studies showing that antioxidants may act as anticarcinogens support the role of active oxygen in carcinogenesis and provide impetus for exploring the functions of dietary antioxidants in cancer prevention by using in vitro models. The authors examined the single and combined effects of selenium, a component of glutathione peroxidase, and vitamin E, a known antioxidant, on cell transformation induced in C3H/10T-1/2 cells by x-rays, benzo(a)pyrene, or tryptophan pyrolysate and on the levels of cellular scavenging systems peroxide destruction. Incubation of C3H/10T-1/2 cells with 2.5 ..mu..M Na/sup 2/SeO/sup 3/ (selenium) or with 7 ..mu..M ..cap alpha..-tocopherol succinate (vitamin E) 24 hr prior to exposure to x-rays or the chemical carcinogens resulted in an inhibition of transformation by each of the antioxidants with an additive-inhibitory action when the two nutrients were combined. Cellular pretreatment with selenium resulted in increased levels of cellular glutathione peroxidase, catalase, and nonprotein thiols (glutathione) and in an enhanced destruction of peroxide. The results support our earlier studies showing that free radical-mediated events play a role in radiation and chemically induced transformation. They indicate that selenium and vitamin E act alone and in additive fashion as radioprotecting and chemopreventing agents. The results further suggest that selenium confers protection in part by inducing or activating cellular free-radical scavenging systems and by enhancing peroxide breakdown while vitamin E appears to confer its protection by and alternate complementary mechanism.

  7. Plant polyphenols alter a pathway of energy metabolism by inhibiting fecal Bacteroidetes and Firmicutes in vitro.

    PubMed

    Xue, Bin; Xie, Jinli; Huang, Jiachen; Chen, Long; Gao, Lijuan; Ou, Shiyi; Wang, Yong; Peng, Xichun

    2016-03-01

    The function of plant polyphenols in controlling body weight has been in focus for a long time. The aim of this study was to investigate the effect of plant polyphenols on fecal microbiota utilizing oligosaccharides. Three plant polyphenols, quercetin, catechin and puerarin, were added into liquid media for fermenting for 24 h. The pH values, OD600 of the cultures and the content of carbohydrates at 0, 6, 10, 14, 18 and 24 h were determined. The abundance of Bacteroidetes and Firmicutes in each culture was quantified with qPCR after 10 h of fermentation, and the bacterial composition was analyzed using the software Quantitative Insights Into Microbial Ecology. The results revealed that all three plant polyphenols could significantly inhibit the growth of Bacteroidetes (P < 0.01) and Firmicutes (P < 0.01) while at the same time down-regulate the ratio of Bacteroidetes to Firmicutes (P < 0.01). But the fecal bacteria could maintain the ability to hydrolyze fructo-oligosaccharide (FOS) in vitro. Among the tested polyphenols, catechin presented the most intense inhibitory activity towards the growth of Bacteroidetes and Firmicutes, and quercetin was the second. Only the samples with catechin had a significantly lower energy metabolism (P < 0.05). In conclusion, plant polyphenols can change the pathway of degrading FOS or even energy metabolism in vivo by altering gut microbiota composition. It may be one of the mechanisms in which plant polyphenols can lead to body weight loss. It's the first report to study in vitro gastrointestinal microbiota fermenting dietary fibers under the intervention of plant polyphenols.

  8. Sunlight inhibits growth and induces markers of programmed cell death in Plasmodium falciparum in vitro.

    PubMed

    Engelbrecht, Dewaldt; Coetzer, Thérèsa Louise

    2015-09-29

    Plasmodium falciparum is responsible for the majority of global malaria deaths. During the pathogenic blood stages of infection, a rapid increase in parasitaemia threatens the survival of the host before transmission of slow-maturing sexual parasites to the mosquito vector to continue the life cycle. Programmed cell death (PCD) may provide the parasite with the means to control its burden on the host and thereby ensure its own survival. Various environmental stress factors encountered during malaria may induce PCD in P. falciparum. This study is the first to characterize parasite cell death in response to natural sunlight. The 3D7 strain of P. falciparum was cultured in vitro in donor erythrocytes. Synchronized and mixed-stage parasitized cultures were exposed to sunlight for 1 h and compared to cultures maintained in the dark, 24 h later. Mixed-stage parasites were also subjected to a second one-hour exposure at 24 h and assessed at 48 h. Parasitaemia was measured daily by flow cytometry. Biochemical markers of cell death were assessed, including DNA fragmentation, mitochondrial membrane polarization and phosphatidylserine externalization. Sunlight inhibited P. falciparum growth in vitro. Late-stage parasites were more severely affected than early stages. However, some late-stage parasites survived exposure to sunlight to form new rings 24 h later, as would be expected during PCD whereby only a portion of the population dies. DNA fragmentation was observed at 24 and 48 h and preceded mitochondrial hyperpolarization in mixed-stage parasites at 48 h. Mitochondrial hyperpolarization likely resulted from increased oxidative stress. Although data suggested increased phosphatidylserine externalization in mixed-stage parasites, results were not statistically significant. The combination of biochemical markers and the survival of some parasites, despite exposure to a lethal stimulus, support the occurrence of PCD in P. falciparum.

  9. Endophytic fungi from mangrove inhibit lung cancer cell growth and angiogenesis in vitro.

    PubMed

    Liu, Xin; Wu, Xin; Ma, Yuefan; Zhang, Wenzhang; Hu, Liang; Feng, Xiaowei; Li, Xiangyong; Tang, Xudong

    2017-03-01

    The secondary metabolites of mangrove-derived endophytic fungi contain multiple substances with novel structures and biological activities. In the present study, three types of mangrove plants, namely Kandelia candel, Rhizophora stylosa and Rhizophoraceae from Zhanjiang region including the leaves, roots and stems were collected, and endophytic fungi were isolated, purified and identified from these mangrove plants. MTT assay was used to observe the effects of the isolated endophytic fungi on the growth of A549 and NCI-H460 lung cancer cells. The effect of the endophytic fungi on lung cancer angiogenesis in vitro induced by the HPV-16 E7 oncoprotein was observed. Our results showed that 28 strains of endophytic fungi were isolated, purified and identified from the three types of mangrove plants. Ten strains of endophytic fungi significantly suppressed the growth of A549 and NCI-H460 cells. The average inhibitory rates in the A549 cells were 64.4, 59.5, 81.9, 43.9, 58.3, 56.2, 48.3, 42.4, 93.0 and 49.7%, respectively. The average inhibitory rates in the NCI-H460 cells were 41.2, 49.3, 82.7, 40.7, 53.9, 52.6, 56.8, 64.3, 91.0 and 45.6%, respectively. Particularly, three strains of endophytic fungi markedly inhibited HPV-16 E7 oncoprotein‑induced lung cancer angiogenesis in vitro. These findings contribute to the further screening of potential chemotherapeutic agents from mangrove-derived endophytic fungi.

  10. Raltegravir Has a Low Propensity To Cause Clinical Drug Interactions through Inhibition of Major Drug Transporters: an In Vitro Evaluation

    PubMed Central

    Houle, Robert; Chan, Grace Hoyee; Hafey, Mike; Rhee, Elizabeth G.; Chu, Xiaoyan

    2014-01-01

    Raltegravir (RAL) is a human immunodeficiency virus type 1 (HIV-1) integrase inhibitor approved to treat HIV infection in adults in combination with other antiretrovirals. The potential of RAL to cause transporter-related drug-drug interactions (DDIs) as an inhibitor has not been well described to date. In this study, a series of in vitro experiments were conducted to assess the inhibitory effects of RAL on major human drug transporters known to be involved in clinically relevant drug interactions, including hepatic and renal uptake transporters and efflux transporters. For hepatic uptake transporters, RAL showed no inhibition of organic anion-transporting polypeptide 1B1 (OATP1B1), weak inhibition of OATP1B3 (40% inhibition at 100 μM), and no inhibition of organic cation transporter 1 (OCT1). Studies of renal uptake transporters showed that RAL inhibited organic anion transporters 1 and 3 (OAT1 and OAT3) with 50% inhibitory concentrations (IC50s) (108 μM and 18.8 μM, respectively) well above the maximum concentration of drug in plasma (Cmax) at the clinical 400-mg dose and did not inhibit organic cation transporter 2 (OCT2). As for efflux transporters, RAL did not inhibit breast cancer resistance protein (BCRP) and showed weak inhibition of multidrug and toxin extrusion protein 1 (MATE1) (52% inhibition at 100 μM) and MATE2-K (29% inhibition at 100 μM). These studies indicate that at clinically relevant exposures, RAL does not inhibit or only weakly inhibits hepatic uptake transporters OATP1B1, OATP1B3, and OCT1, renal uptake transporters OCT2, OAT1, and OAT3, as well as efflux transporters BCRP, MATE1, and MATE2-K. The propensity for RAL to cause DDIs via inhibition of these transporters is therefore considered low. PMID:24295974

  11. Revisiting the mechanistic basis of the French Paradox: Red wine inhibits the activity of protein disulfide isomerase in vitro.

    PubMed

    Galinski, Christine N; Zwicker, Jeffrey I; Kennedy, Daniel R

    2016-01-01

    Although epidemiologic evidence points to cardioprotective activity of red wine, the mechanistic basis for antithrombotic activity has not been established. Quercetin and related flavonoids are present in high concentrations in red but not white wine. Quercetin-glycosides were recently shown to prevent thrombosis in animal models through the inhibition of extracellular protein disulfide isomerase (PDI). We evaluated whether red or white wine inhibited PDI activity in vitro. Quercetin levels in red and white wines were measured by HPLC analysis. Inhibition of PDI activity by red and white wines was assessed by an insulin reduction turbidity assay at various concentrations of wine. PDI inhibition was confirmed using a reduced peptide that contained a disulfide containing peptide as a substrate. The inhibition of PDI related thiol isomerases ERp5 and ERp57 was also assessed. We observed a dose-dependent decrease of PDI activity for a variety of red but not white wines. Red wine diluted to 3% final concentration resulted in over 80% inhibition of PDI activity by insulin reductase assay for all varieties tested. This inhibition was also observed in the peptide based assay. Red grape juice yielded similar results but ethanol alone did not affect PDI activity. Interestingly, red wine also inhibited the PDI related thiol isomerases ERp5 and ERp57, albeit to a lesser degree than PDI. PDI activity is inhibited by red wine and grape juice, identifying a potentially novel mechanism underlying the cardiovascular benefits attributed to wine consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

    PubMed Central

    Elliot, B C; Wisnewski, A V; Johnson, J; Fenwick-Smith, D; Wiest, P; Hamer, D; Kresina, T; Flanigan, T P

    1997-01-01

    Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection. PMID:9284173

  13. Overexpression of RUNX3 inhibits malignant behaviour of Eca109 cells in vitro and vivo.

    PubMed

    Chen, Hua-Xia; Wang, Shuai; Wang, Zhou; Zhang, Zhi-Ping; Shi, Shan-Shan

    2014-01-01

    Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may play an important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim of this study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpression on biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues using the SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cell line with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence. CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assay to analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice, tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue to be significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinant lentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpression could inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in mice showed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate that underexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.

  14. Herbacetin inhibits RANKL-mediated osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo.

    PubMed

    Li, Liang; Sapkota, Mahesh; Kim, Se-woong; Soh, Yunjo

    2016-04-15

    Herbacetin is an active flavonol (a type of flavonoid) that has various biologic effects such as antioxidant, antitumor, and anti-inflammatory activities. However, one of its novel effects remains to be investigated, that is, the induction of osteoclastogenesis by the receptor activator of nuclear factor-κB ligand (RANKL). In this study, we examined the effects and mechanisms of action of herbacetin on osteoclastogenesis in RANKL-treated bone marrow-derived macrophages (BMMs) and murine macrophage RAW264.7 cells in vitro and on lipopolysaccharide (LPS)-induced bone destruction in vivo. Herbacetin significantly inhibited RANKL-induced osteoclast formation and differentiation in BMMs and RAW264.7 cells in a dose-dependent manner. Moreover, the suppressive effect of herbacetin resulted in a decrease in osteoclast-related genes, including RANK, tartrate-resistant acid phosphatase, cathepsin K, and matrix metalloproteinase-2 and -9 (MMP-9). Consistent with mRNA results, we confirmed that herbacetin treatment downregulated protein expression of MMP-9 and cathepsin K. Herbacetin also decreased induction of the osteoclastogenic transcription factor c-Fos and nuclear factor of activated T cells c1 (NFATc1) and blocked RANKL-mediated activation of Jun N-terminal kinase (JNK) and nuclear factor-κB. Herbacetin clearly inhibited the bone resorption activity of osteoclasts on plates coated with fluorescein-labeled calcium phosphate. More importantly, the application of herbacetin significantly reduced LPS-induced inflammatory bone loss in mice in vivo. Taken together, our results indicate that herbacetin has potential for use as a therapeutic agent in disorders associated with bone loss.

  15. Inhibition of influenza A virus infection in vitro by peptides designed in silico.

    PubMed

    López-Martínez, Rogelio; Ramírez-Salinas, G Lizbeth; Correa-Basurto, José; Barrón, Blanca L

    2013-01-01

    Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H(+) ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.

  16. Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy

    PubMed Central

    Greene, Whitney A.; Burke, Teresa A.; Wang, Heuy-Ching

    2016-01-01

    Abstract Purpose: Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. Methods: ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFβ2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 μM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and β-catenin expression after TGFβ2 treatment alone or in combination with TSA. Results: TGFβ2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 μM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFβ2-mediated iPS-RPE cell migration. Conclusions: Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFβ2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR. PMID:27494828

  17. Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy.

    PubMed

    Por, Elaine D; Greene, Whitney A; Burke, Teresa A; Wang, Heuy-Ching

    2016-09-01

    Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFβ2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 μM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and β-catenin expression after TGFβ2 treatment alone or in combination with TSA. TGFβ2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 μM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFβ2-mediated iPS-RPE cell migration. Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFβ2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR.

  18. The Novel Ribonucleotide Reductase Inhibitor COH29 Inhibits DNA Repair In Vitro

    PubMed Central

    Chen, Mei-Chuan; Zhou, Bingsen; Zhang, Keqiang; Yuan, Yate-Ching; Un, Frank; Hu, Shuya; Chou, Chih-Ming; Chen, Chun-Han; Wu, Jun; Wang, Yan; Liu, Xiyong; Smith, D. Lynne; Li, Hongzhi; Liu, Zheng; Warden, Charles D.; Su, Leila; Malkas, Linda H.; Chung, Young Min; Hu, Mickey C.-T.

    2015-01-01

    COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1–defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 + BRCA1 cells. By EJ5– and DR–green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 + BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering drug-induced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent. PMID:25814515

  19. Efficacy of eight commercial formulations of lime sulphur on in vitro growth inhibition of Microsporum canis.

    PubMed

    Diesel, Alison; Verbrugge, Maria; Moriello, Karen A

    2011-04-01

    Lime sulphur is a common topical treatment for dermatophytosis in animals. Until recently, a single veterinary lime sulphur formulation was available. The purpose of this study was to compare the efficacy of eight lime sulphur products for in vitro growth inhibition of Microsporum canis using the isolated infected spore model. Infective M. canis spores were isolated from hairs collected from untreated cats. Hairs were macerated in Triton-X solution and isolated according to a previously published protocol. Equal volumes of spore suspension and lime sulphur solutions were incubated for 5 min and plated onto modified BBL™ Mycosel™ agar (Becton, Dickinson and Company; Sparks, MD, USA) plates. Five plates were inoculated for each sample solution. Distilled water and bleach were used as controls. Colony forming units were counted daily for 21 days; positive control plates contained >300 colony forming units/plate. Seven of the products were supplied as concentrates and they were tested at the manufacturer's recommended dilution, twice label concentration and half label concentration. A prediluted product SulfaDip(®) (Trask Research, Inc.; Daluca, GA, USA) was tested at the label and half label concentration. All veterinary products formed recommended treatment dilutions of 3% sulphurated lime solution except one (LymDyp(®), IVX Animal Health Inc.; St Joseph, MO, USA), which formed a 2.4% sulphurated lime solution. Results of the study showed complete growth inhibition of M. canis spores by all products at all dilutions tested. These results indicate that all tested lime sulphur-containing products were equivalent. Field studies are needed to test product equivalency in vivo.

  20. In vitro inhibition of Eimeria tenella invasion of epithelial cells by phytochemicals.

    PubMed

    Burt, S A; Tersteeg-Zijderveld, M H G; Jongerius-Gortemaker, B G M; Vervelde, L; Vernooij, J C M

    2013-01-31

    Resistance to coccidiostats and possible future restrictions on their use raise the need for alternative methods of reducing coccidiosis in poultry. The aim of this study was to evaluate the effect of selected phytochemicals on Eimeria tenella sporozoite invasion in vitro. Four phytochemicals were selected on the basis that they reduce the virulence of Eimeria spp. and/or provide immune modulatory benefits to host cells: betaine, carvacrol, curcumin and Echinacea purpurea extract (EP). Madin-Darby bovine kidney (MDBK) cells were covered by medium containing phytochemicals at the highest concentration which was non-toxic to the cells. Salinomycin 50 μg/ml was positive control; negative control was medium only. E. tenella (Houghton strain) sporozoites were added to wells and after incubation for 2, 4 or 20 h at 37°C, cells were fixed and stained with hematoxylin-eosin. Ten evenly spaced fields per well were photographed and the percentage of cells invaded by sporozoites was calculated and normalized to the control. At 2h, carvacrol, curcumin and EP showed a significantly lower percentage of sporozoite invasion than the untreated control; in contrast, betaine treatment represented a significantly higher invasion percentage. Combining carvacrol with EP inhibited E. tenella invasion more effectively than applying the compounds individually, but the further addition of curcumin did not reduce invasion further. In conclusion, this study shows that invasion of MDBK epithelial cells by E. tenella sporozoites is inhibited in the presence of carvacrol, curcumin, or EP and enhanced by betaine. There may be potential for developing these phytochemicals as anti-coccidial feed or water additives for poultry.

  1. Amygdalin inhibits the growth of renal cell carcinoma cells in vitro.

    PubMed

    Juengel, Eva; Thomas, Anita; Rutz, Jochen; Makarevic, Jasmina; Tsaur, Igor; Nelson, Karen; Haferkamp, Axel; Blaheta, Roman A

    2016-02-01

    Although amygdalin is used by many cancer patients as an antitumor agent, there is a lack of information on the efficacy and toxicity of this natural compound. In the present study, the inhibitory effect of amygdalin on the growth of renal cell carcinoma (RCC) cells was examined. Amygdalin (10 mg/ml) was applied to the RCC cell lines, Caki-1, KTC-26 and A498, for 24 h or 2 weeks. Untreated cells served as controls. Tumor cell growth and proliferation were determined using MTT and BrdU tests, and cell cycle phases were evaluated. Expression of the cell cycle activating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1 and D3 as well as of the cell cycle inhibiting proteins p19 and p27 was examined by western blot analysis. Surface expression of the differentiation markers E- and N-cadherin was also investigated. Functional blockade by siRNA was used to determine the impact of several proteins on tumor cell growth. Amygdalin treatment caused a significant reduction in RCC cell growth and proliferation. This effect was correlated with a reduced percentage of G2/M-phase RCC cells and an increased percentage of cells in the G0/1-phase (Caki-1 and A498) or cell cycle arrest in the S-phase (KTC-26). Furthermore, amygdalin induced a marked decrease in cell cycle activating proteins, in particular cdk1 and cyclin B. Functional blocking of cdk1 and cyclin B resulted in significantly diminished tumor cell growth in all three RCC cell lines. Aside from its inhibitory effects on growth, amygdalin also modulated the differentiation markers, E- and N-cadherin. Hence, exposing RCC cells to amygdalin inhibited cell cycle progression and tumor cell growth by impairing cdk1 and cyclin B expression. Moreover, we noted that amygdalin affected differentiation markers. Thus, we suggest that amygdalin exerted RCC antitumor effects in vitro.

  2. Sertraline inhibits the transport of PAT1 substrates in vivo and in vitro

    PubMed Central

    Nielsen, C U; Frølund, S; Abdulhadi, S; Sari, H; Langthaler, L; Nøhr, M K; Kall, M A; Brodin, B; Holm, R

    2013-01-01

    Background and Purpose Intestinal nutrient transporters may mediate the uptake of drugs. The aim of this study was to investigate whether sertraline interacts with the intestinal proton-coupled amino acid transporter 1 PAT1 (SLC36A1). Experimental Approach In vitro investigations of interactions between sertraline and human (h)PAT1, hSGLT1 (sodium-glucose linked transporter 1) and hPepT1 (proton-coupled di-/tri-peptide transporter 1) were conducted in Caco-2 cells using radiolabelled substrates. In vivo pharmacokinetic investigations were conducted in male Sprague–Dawley rats using gaboxadol (10 mg·kg−1, p.o.) as a PAT1 substrate and sertraline (0–30.6 mg·kg−1). Gaboxadol was quantified by hydrophilic interaction chromatography followed by MS/MS detection. Key Results Sertraline inhibited hPAT1-mediated L-[3H]-Pro uptake in Caco-2 cells. This interaction between sertraline and PAT1 appeared to be non-competitive. The uptake of the hSGLT1 substrate [14C]-α–methyl-D-glycopyranoside and the hPepT1 substrate [14C]-Gly-Sar in Caco-2 cells was also decreased in the presence of 0.3 mM sertraline. In rats, the administration of sertraline (0.1–10 mM, corresponding to 0.3–30.6 mg·kg−1, p.o.) significantly reduced the maximal gaboxadol plasma concentration and AUC after its administration p.o. Conclusions and Implications Sertraline is an apparent non-competitive inhibitor of hPAT1-mediated transport in vitro. This inhibitory effect of sertraline is not specific to hPAT1 as substrate transport via hPepT1 and hSGLT1 was also reduced in the presence of sertraline. In vivo, sertraline reduced the amount of gaboxadol absorbed, suggesting that the inhibitory effect of sertraline on PAT1 occurs both in vitro and in vivo. Hence, sertraline could alter the bioavailability of drugs absorbed via PAT1. PMID:23962042

  3. Sertraline inhibits the transport of PAT1 substrates in vivo and in vitro.

    PubMed

    Nielsen, C U; Frølund, S; Abdulhadi, S; Sari, H; Langthaler, L; Nøhr, M K; Kall, M A; Brodin, B; Holm, R

    2013-11-01

    Intestinal nutrient transporters may mediate the uptake of drugs. The aim of this study was to investigate whether sertraline interacts with the intestinal proton-coupled amino acid transporter 1 PAT1 (SLC36A1). In vitro investigations of interactions between sertraline and human (h)PAT1, hSGLT1 (sodium-glucose linked transporter 1) and hPepT1 (proton-coupled di-/tri-peptide transporter 1) were conducted in Caco-2 cells using radiolabelled substrates. In vivo pharmacokinetic investigations were conducted in male Sprague-Dawley rats using gaboxadol (10 mg·kg(-1), p.o.) as a PAT1 substrate and sertraline (0-30.6 mg·kg(-1)). Gaboxadol was quantified by hydrophilic interaction chromatography followed by MS/MS detection. Sertraline inhibited hPAT1-mediated L-[(3)H]-Pro uptake in Caco-2 cells. This interaction between sertraline and PAT1 appeared to be non-competitive. The uptake of the hSGLT1 substrate [(14)C]-α-methyl-D-glycopyranoside and the hPepT1 substrate [(14)C]-Gly-Sar in Caco-2 cells was also decreased in the presence of 0.3 mM sertraline. In rats, the administration of sertraline (0.1-10 mM, corresponding to 0.3-30.6 mg·kg(-1), p.o.) significantly reduced the maximal gaboxadol plasma concentration and AUC after its administration p.o. Sertraline is an apparent non-competitive inhibitor of hPAT1-mediated transport in vitro. This inhibitory effect of sertraline is not specific to hPAT1 as substrate transport via hPepT1 and hSGLT1 was also reduced in the presence of sertraline. In vivo, sertraline reduced the amount of gaboxadol absorbed, suggesting that the inhibitory effect of sertraline on PAT1 occurs both in vitro and in vivo. Hence, sertraline could alter the bioavailability of drugs absorbed via PAT1. © 2013 The British Pharmacological Society.

  4. In vitro evaluation of single- and multi-strain probiotics: Inter-species inhibition between probiotic strains, and inhibition of pathogens.

    PubMed

    Chapman, C M C; Gibson, G R; Rowland, I

    2012-08-01

    Many studies comparing the effects of single- and multi-strain probiotics on pathogen inhibition compare treatments with different concentrations. They also do not examine the possibility of inhibition between probiotic strains with a mixture. We tested the ability of 14 single-species probiotics to inhibit each other using a cross-streak assay, and agar spot test. We then tested the ability of 15 single-species probiotics and 5 probiotic mixtures to inhibit Clostridium difficile, Escherichia coli and S. typhimurium, using the agar spot test. Testing was done with mixtures created in two ways: one group contained component species incubated together, the other group of mixtures was made using component species which had been incubated separately, equalised to equal optical density, and then mixed in equal volumes. Inhibition was observed for all combinations of probiotics, suggesting that when used as such there may be inhibition between probiotics, potentially reducing efficacy of the mixture. Significant inter-species variation was seen against each pathogen. When single species were tested against mixtures, the multi-species preparations displayed significantly (p < 0.05 or less) greater inhibition of pathogens in 12 out of 24 cases. Despite evidence that probiotic species will inhibit each other when incubated together in vitro, in many cases a probiotic mixture was more effective at inhibiting pathogens than its component species when tested at approximately equal concentrations of biomass. This suggests that using a probiotic mixture might be more effective at reducing gastrointestinal infections, and that creating a mixture using species with different effects against different pathogens may have a broader spectrum of action that a single provided by a single strain.

  5. Celecoxib inhibits invasion and metastasis via a cyclooxygenase 2-independent mechanism in an in vitro model of Ewing sarcoma.

    PubMed

    Barlow, Meade; Edelman, Morris; Glick, Richard D; Steinberg, Bettie M; Soffer, Samuel Z

    2012-06-01

    Previously, we reported that celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, prevented lung metastases but did not affect tumor growth in a model of Ewing sarcoma. Cyclooxygenase-2 inhibition has been proposed as an antimetastatic strategy. The mechanism of action remains unclear. Ewing sarcoma cells were suspended in a soluble basement membrane extract (Cultrex; Trevigen, Inc, Gaithersburg, MD) and supplemented with celecoxib or with rofecoxib, a second COX-2 inhibitor, above a filter. Controls received solvent. After 48 hours, the cells that invaded through the basement membrane and filter were stained and counted. The assay was repeated with the addition of 500-nM prostaglandin E2 (PGE(2)). Invasion was significantly decreased in the celecoxib groups compared with the control. The addition of PGE(2) did not overcome celecoxib inhibition. Rofecoxib did not significantly affect invasion compared with control either with or without PGE(2). Celecoxib significantly inhibits invasion of Ewing sarcoma cells in vitro. Prostaglandin E2, a downstream product of COX-2, did not reverse in vitro inhibition, suggesting that celecoxib acts through a COX-2-independent mechanism. This is further supported by the failure of rofecoxib to inhibit invasion despite more selectively inhibiting COX-2. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. A Novel Antifungal Is Active against Candida albicans Biofilms and Inhibits Mutagenic Acetaldehyde Production In Vitro

    PubMed Central

    Nieminen, Mikko T.; Novak-Frazer, Lily; Rautemaa, Vilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

    2014-01-01

    The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm

  7. Red wine and component flavonoids inhibit UGT2B17 in vitro

    PubMed Central

    2012-01-01

    Background The metabolism and excretion of the anabolic steroid testosterone occurs by glucuronidation to the conjugate testosterone glucuronide which is then excreted in urine. Alterations in UGT glucuronidation enzyme activity could alter the rate of testosterone excretion and thus its bioavailability. The aim of this study is to investigate if red wine, a common dietary substance, has an inhibitory effect on UGT2B17. Methods Testosterone glucuronidation was assayed using human UGT2B17 supersomes with quantification of unglucuronidated testosterone over time using HPLC with DAD detection. The selected red wine was analyzed using HPLC; and the inhibitory effects of the wine and phenolic components were tested independently in a screening assay. Further analyses were conducted for the strongest inhibitors at physiologically relevant concentrations. Control experiments were conducted to determine the effects of the ethanol on UGT2B17. Results Over the concentration range of 2 to 8%, the red wine sample inhibited the glucuronidation of testosterone by up to 70% over 2 hours. The ethanol content had no significant effect. Three red wine phenolics, identified by HPLC analyses, also inhibited the enzyme by varying amounts in the order of quercetin (72%), caffeic acid (22%) and gallic acid (9%); using a ratio of phenolic:testosterone of 1:2.5. In contrast p-coumaric acid and chlorogenic acid had no effect on the UGT2B17. The most active phenolic was selected for a detailed study at physiologically relevant concentrations, and quercetin maintained inhibitory activity of 20% at 2 μM despite a ten-fold excess of testosterone. Conclusion This study reports that in an in vitro supersome-based assay, the key steroid-metabolizing enzyme UGT2B17 is inhibited by a number of phenolic dietary substances and therefore may reduce the rate of testosterone glucuronidation in vivo. These results highlight the potential interactions of a number of common dietary compounds on

  8. Ellagic acid inhibits human glioblastoma growth in vitro and in vivo.

    PubMed

    Wang, Dongliang; Chen, Qianxue; Tan, Yinqiu; Liu, Baohui; Liu, Chao

    2017-02-01

    Ellagic acid (EA) is present in various fruits and plants and has recently been found to possess anticarcinogenic effects. The objective of this study was to investigate the anti‑glioblastoma effect of EA and its mechanisms in vitro and in vivo. We first studied the anticancer activity of EA in U87 and U118 human glioblastoma cell lines. The cell viability and cell proliferation were examined by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine staining respectively. The cell cycle was detected with propidium iodide staining method by flow cytometry and the DNA damage of the cells caused by EA exposure was evaluated by detection of γ-H2AX foci. Then we examined the effect of EA on tumor growth in glioblastoma xenografted mice, and expression of Akt and Notch signaling and their target gene products were detected by immunohistochemistry and western blot analysis. As a result, we found that the cell viability and proliferation of glioblastoma cells treated with EA were significantly suppressed compared with the control; EA significantly increased the proportion of cells in the S phase accompanied by a decrease in the population in the G1 and G2/M phase in both cell lines. Meanwhile, the level of DNA damage in the EA-treated group was significantly higher than that of the control. Treatment of glioblastoma in xenografted mice by EA led to a significant suppression in tumor growth. EA upregulated the expression of E-cadherin and inhibited the expression of Snail, matrix metalloproteinase (MMP)-2 and MMP-9. EA also inhibited the expression of Bcl-2, cyclin D1, cyclin-dependent kinase (CDK)2 and CDK6 in U87 xenograft tissues. In addition, significant suppression of Akt and Notch was found in the xenografts of the tumor-bearing mice treated with EA. These data indicate that EA can suppress glioblastoma proliferation and invasion by inhibiting the Akt and Notch signaling pathways, which suggests that EA may be beneficial for the treatment of

  9. A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

    PubMed

    Nieminen, Mikko T; Novak-Frazer, Lily; Rautemaa, Wilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

    2014-01-01

    The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 μM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm

  10. A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

    PubMed

    Nieminen, Mikko T; Novak-Frazer, Lily; Rautemaa, Vilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

    2014-01-01

    The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm

  11. Flavonoids casticin and chrysosplenol D from Artemisia annua L. inhibit inflammation in vitro and in vivo.

    PubMed

    Li, Yu-Jie; Guo, Yan; Yang, Qing; Weng, Xiao-Gang; Yang, Lan; Wang, Ya-Jie; Chen, Ying; Zhang, Dong; Li, Qi; Liu, Xu-Cen; Kan, Xiao-Xi; Chen, Xi; Zhu, Xiao-Xin; Kmoníèková, Eva; Zídek, Zdenìk

    2015-08-01

    The aim of our experiments was to investigate the anti-inflammatory properties of casticin and chrysosplenol D, two flavonoids present in Artemisia annua L. Topical inflammation was induced in ICR mice using croton oil. Mice were then treated with casticin or chrysosplenol D. Cutaneous histological changes and edema were assessed. ICR mice were intragastrically administrated with casticin or chrysosplenol D followed by intraperitoneal injection of lipopolysaccharide (LPS). Mouse Raw264.7 macrophage cells were incubated with casticin or chrysosplenol D. Intracellular phosphorylation was detected, and migration was assessed by trans-well assay. HT-29/NFκB-luc cells were incubated with casticin or chrysosplenol D in the presence or absence of LPS, and NF-κB activation was quantified. In mice, administration of casticin (0.5, 1 and 1.5μmol/cm(2)) and chrysosplenol D (1 and 1.5μmol/cm(2)) inhibited croton oil-induced ear edema (casticin: 29.39-64.95%; chrysosplenol D: 37.76-65.89%, all P<0.05) in a manner similar to indomethacin (0.5, 1 and 1.5μmol/cm(2); 55.63-84.58%). Casticin (0.07, 0.13 and 0.27mmol/kg) and chrysosplenol D (0.07, 0.14 and 0.28mmol/kg) protected against LPS-induced systemic inflammatory response syndrome (SIRS) in mice (all P<0.05), in a manner similar to dexamethasone (0.03mmol/kg). Casticin and chrysosplenol D suppressed LPS-induced release of IL-1 beta, IL-6 and MCP-1, inhibited cell migration, and reduced LPS-induced IκB and c-JUN phosphorylation in Raw264.7 cells. JNK inhibitor SP600125 blocked the inhibitory effect of chrysosplenol D on cytokine release. The flavonoids casticin and chrysosplenol D from A. annua L. inhibited inflammation in vitro and in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. In vitro inhibition of cytochrome P-450 activities and quantification of constituents in a selection of commercial Rhodiola rosea products.

    PubMed

    Thu, Ole Kristian; Nilsen, Odd Georg; Hellum, Bent

    2016-12-01

    Rhodiola rosea L. (Crassulaceae) products are popular natural remedies with a worldwide distribution. Recent studies have revealed potent CYP inhibition by R. rosea extracts both in vitro and in vivo, but information on in vitro CYP inhibition by commercial products are lacking. Variations in commercial R. rosea product quality have also been published. This study evaluates the variation of in vitro CYP inhibition potential and product quality of six commercially available R. rosea products. Human CYPs isolated from baculovirus-infected cell system were incubated with testosterone (CYP3A4), dextromethorphan (CYP2D6) or phenacetin (CYP1A2). Positive CYP inhibitors ketoconazole (CYP3A4), quinidine (CYP2D6) and β-naphtoflavone (CYP1A2) were used as controls. Quantification of rosavin, rosarin, rosin, tyrosol and salidroside were used to evaluate R. rosea content. IC50 values ranged from 7.2-106.6 μg/mL for CYP3A4, 13.0-186.1 μg/mL for 2D6 and 10.7-116.0 μg/mL for 1A2. The tincture formulation of R. rosea was the strongest inhibitor giving the lowest IC50 values of 7.2 ± 0.7, 13 ± 1.7 and 10.7 ± 5.6 μg/mL, respectively. CYP3A4 was significantly more inhibited by the different products than CYP1A2 (p < .05). One of the six products did not contain any rosavin, rosarin or rosin and is not a R. rosea product. Constituent concentrations were not linked to enzyme inhibition. The present results show a large variation in inhibitory potential between the products. Several of the products demonstrate similar inhibition levels as the product Arctic Root already proven to inhibit CYP enzyme activity in man.

  13. Optimization of in vitro inhibition of HT-29 colon cancer cell cultures by Solanum tuberosum L. extracts.

    PubMed

    Zuber, T; Holm, D; Byrne, P; Ducreux, L; Taylor, M; Kaiser, M; Stushnoff, C

    2015-01-01

    Secondary metabolites in potato have been reported to possess bioactive properties, including growth inhibition of cancer cells. Because potatoes are widely consumed globally, potential health benefits may have broad application. Thus we investigated growth inhibition of HT-29 colon cancer cell cultures by extracts from 13 diverse genetic breeding clones. Extracts from three pigmented selections (CO97226-2R/R, CO97216-1P/P, CO04058-3RW/RW) inhibited growth of in vitro HT-29 cell cultures more effectively than other clones tested. While inhibition was highest from pigmented selections and pigmented tuber tissue sectors, not all pigmented breeding lines tested had appreciable inhibitory properties. Thus, inhibition was not uniquely linked to pigmentation. Immature tubers had the highest inhibitory properties, and in most cases mature tubers retained very low inhibition properties. Flowers and skins inhibited strongly at lower extract concentrations. An extract consisting of 7.2 mg mL⁻¹ cell culture medium was the lowest effective concentration. While raw tuber extracts inhibited most effectively, a few clones at higher concentrations retained inhibition after cooking. Heated whole tubers retained higher inhibition than heated aqueous extracts. While all aqueous extracts from the two tuber selections (CO97216-1P/P and CO97226-2R/R) inhibited HT-29 cell cultures, inhibition was significantly enhanced in purple pigmented tubers of CO97216-1P/P prepared cryogenically as liquid nitrogen powders compared to extracts from freeze dried samples. Upregulation of caspase-3 protease activity, indicative of apoptosis, was highest among the most inhibitory clone samples. The unique sectorial red pigment expressing selection (CO04058-3RW/RW) provided a model system that isolated expression in pigmented sectors, and thus eliminated developmental, environmental and genetic confounding.

  14. In vitro inhibition of fish rhabdoviruses by Japanese flounder, Paralichthys olivaceus Mx.

    PubMed

    Caipang, Christopher Marlowe A; Hirono, Ikuo; Aoki, Takashi

    2003-12-20

    A homologous fish cell line stably expressing the recombinant Japanese flounder Mx (JFMx) was infected with hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), both of which are negative single-stranded RNA viruses belonging to the Rhabdoviridae family. Analysis of primary transcription of the two rhabdoviruses showed that there was lower expression level and copy number of the viral nucleoprotein transcript in the JFMx-transfected cell line than the infected, control cells, although no significant difference was observed. This suggests that JFMx may not be a potent inhibitor of rhabdoviral primary transcription. Kinetics of rhabdovirus expression by RT-PCR and quantitative real-time RT-PCR showed reduced levels of the rhabdoviral glycoprotein and nucleoprotein transcripts over time, indicating the possible role of JFMx in blocking rhabdoviral replication by interfering with the transcription of the viral subgenomic mRNAs. Significant inhibition in rhabdovirus replication consequently resulted in the synthesis of fewer viral particles. This may explain why JFMx-expressing cells are less susceptible to virus-induced cell lysis, and thus, why they would have a significantly higher survival than the infected, control cells. These results provide direct evidence that JFMx has an antiviral effect in vitro.

  15. Inhibition of oxidative DNA damage in vitro by extracts of brussels sprouts.

    PubMed

    Zhu, C; Poulsen, H E; Loft, S

    2000-08-01

    Cruciferous vegetables have cancer preventive effects which may be due to reduction of oxidative DNA damage. We investigated the effect of an aqueous extract of cooked Brussels sprouts on formation of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in calf thymus DNA in vitro. Damage was induced by a Fenton reaction, UVC (254 nm), UVA (365 nm), sunlamp light, and methylene blue with visible light. The extract inhibited 8-oxodG formation in all systems except visible light with methylene blue. The IC50 values were 6-20 microg/ml corresponding to the extract of 5-20 g of Brussels sprouts distributed in a volume of 50 L. The protective effect in the Fenton reaction was unaffected by addition of EDTA. After HPLC separation fractions were identified with similar DNA protective effects. Sinigrin, a glucosinolate abundant in Brussels sprouts, co-eluted with the most effective fraction and had DNA protective effects. In comparison with other antioxidants the patterns of effect of the extract in the five damage systems were more similar to that of sodium azide than to those of dimethylsulfoxide and vitamin C. Constituents of Brussels sprouts can protect DNA by direct scavenging, e.g. hydroxyl radical and other oxidants, without prooxidant effects at concentrations potentially achievable by modest intake of the vegetable.

  16. The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro.

    PubMed

    Golderman, Valery; Shavit-Stein, Efrat; Tamarin, Ilia; Rosman, Yossi; Shrot, Shai; Rosenberg, Nurit; Maggio, Nicola; Chapman, Joab; Eisenkraft, Arik

    Organophosphates (OPs) are potentially able to affect serine proteases by reacting with their active site. The potential effects of OPs on coagulation factors such as thrombin and on coagulation tests have been only partially characterized and potential interactions with OPs antidotes such as oximes and muscarinic blockers have not been addressed. In the current study, we investigated the in vitro interactions between coagulation, thrombin, the OP paraoxon, and its antidotes obidoxime and atropine. The effects of these substances on thrombin activity were measured in a fluorescent substrate and on coagulation by standard tests. Both paraoxon and obidoxime but not atropine significantly inhibited thrombin activity, and prolonged prothrombin time, thrombin time, and partial thromboplastin time. When paraoxon and obidoxime were combined, a significant synergistic effect was found on both thrombin activity and coagulation tests. In conclusion, paraoxon and obidoxime affect thrombin activity and consequently alter the function of the coagulation system. Similar interactions may be clinically relevant for coagulation pathways in the blood and possibly in the brain.

  17. In vitro inhibition of feline leukaemia virus infection by synthetic peptides derived from the transmembrane domain.

    PubMed

    Boenzli, Eva; Robert-Tissot, Céline; Sabatino, Giuseppina; Cattori, Valentino; Meli, Marina Luisa; Gutte, Bernd; Rovero, Paolo; Flynn, Norman; Hofmann-Lehmann, Regina; Lutz, Hans

    2011-01-01

    The feline leukaemia virus (FeLV) is a gammaretrovirus commonly affecting cats. Infection with this virus often leads to fatal outcomes and, so far, no cure is available for this disease. Synthetic peptides with structures mimicking the transmembrane protein of the viral surface proteins hold the potential to effectively interfere with viral entry by hampering the fusion of viral and host cell membranes and constitute a novel approach for the treatment of infections with retroviruses. We identified and synthetically produced 11 FeLV peptides and evaluated their potential to block FeLV infection in vitro. Cell cultures were exposed to FeLV subgroup A prior to the addition of the peptides. The inhibitory effect of the peptides was assessed by measuring FeLV gag protein in the supernatant of peptide versus mock-treated cell cultures using an ELISA. A peptide (EPK364) of 37 amino acids in length, with sequence homology to the HIV fusion inhibitor T-20, significantly suppressed viral replication by 88%, whereas no effects were found for shorter peptides. Two structurally modified variants of EPK364 also inhibited viral replication by up to 58% (EPK397) and 27% (EPK398). Our data support the identification of synthetic FeLV peptides that have the potential for a curative short-term therapy of viraemic cats. In addition, these peptides might become an important tool in xenotransplantation, where endogenous gammaretroviruses of the donor species might be able to infect the host. © 2011 International Medical Press

  18. In vitro antileishmanial activity and iron superoxide dismutase inhibition of arylamine Mannich base derivatives.

    PubMed

    Martin-Montes, Alvaro; Santivañez-Veliz, Mery; Moreno-Viguri, Elsa; Martín-Escolano, Rubén; Jiménez-Montes, Carmen; Lopez-Gonzalez, Catalina; Marín, Clotilde; Sanmartín, Carmen; Gutiérrez Sánchez, Ramón; Sánchez-Moreno, Manuel; Pérez-Silanes, Silvia

    2017-08-09

    Leishmaniasis is one of the world's most neglected diseases, and it has a worldwide prevalence of 12 million. There are no effective human vaccines for its prevention, and treatment is hampered by outdated drugs. Therefore, research aiming at the development of new therapeutic tools to fight leishmaniasis remains a crucial goal today. With this purpose in mind, we present 20 arylaminoketone derivatives with a very interesting in vitro and in vivo efficacy against Trypanosoma cruzi that have now been studied against promastigote and amastigote forms of Leishmania infantum, Leishmania donovani and Leishmania braziliensis strains. Six out of the 20 Mannich base-type derivatives showed Selectivity Index between 39 and 2337 times higher in the amastigote form than the reference drug glucantime. These six derivatives affected the parasite infectivity rates; the result was lower parasite infectivity rates than glucantime tested at an IC25 dose. In addition, these derivatives were substantially more active against the three Leishmania species tested than glucantime. The mechanism of action of these compounds has been studied, showing a greater alteration in glucose catabolism and leading to greater levels of iron superoxide dismutase inhibition. These molecules could be potential candidates for leishmaniasis chemotherapy.

  19. Cimetidine induces apoptosis in gastric cancer cells in vitro and inhibits tumor growth in vivo.

    PubMed

    Jiang, Cheng-Gang; Liu, Fu-Rong; Yu, Miao; Li, Jia-Bin; Xu, Hui-Mian

    2010-03-01

    Cimetidine, a histamine-2 (H2) receptor antagonist, has been demonstrated to have anticancer effects on various types of malignancies. However, the mechanisms of its action on gastric cancer are not completely understood. This study was designed to investigate its antitumor effect and underlying mechanisms in human gastric cancer SGC-7901 and MGC-803 cells. The MTT assay was used to evaluate cell viability, and flow cytometry, acridine orange staining and transmission electron microscopy were used to detect apoptosis, for cultured cells. The protein expression in cells was evaluated by Western blot analysis and colorimetric assay. Gastric tumors were established by subcutaneous injection of SGC-7901 cells in nude BALB/c mice, and cimetidine was administered to the mice. The size of tumors was monitored and the weight of tumors was examined. The exposure of gastric cancer cells to cimetidine resulted in growth inhibition and the induction of apoptosis in a dose-dependent manner. Activation of the caspase cascade for both the extrinsic and intrinsic pathways were demonstrated in vitro, including caspase-8, -9 and -3. We also found that the expression of Bcl-2 protein decreased and the expression of Bax protein increased which lead to an increase of the Bax/Bcl-2 ratio. In mice bearing SGC-7901 xenograft tumors, administration of cimetidine showed a significant decrease of tumor volumes and tumor weight compared with the control. Our results showed that cimetidine exhibited antitumor effects in gastric cancer cells with an induction of apoptosis.

  20. Inhibition dominates in shaping spontaneous CA3 hippocampal network activities in vitro.

    PubMed

    Ho, Ernest C Y; Zhang, Liang; Skinner, Frances K

    2009-02-01

    We have assessed the balance of excitation and inhibition in in vitro rodent hippocampal slices exhibiting spontaneous, basal sharp waves (bSPWs). A defining signature of a network exhibiting bSPWs is the rise and fall in local field activities with frequencies ranging from 0.5 to 4.5 Hz. This variation of extracellular local field activities manifests at the intracellular level as postsynaptic potentials (PSPs). In correspondence with the local field bSPWs, we consider "sparse" and "synchronous" parts of bSPWs at the intracellular level. We have used intracellular data of bSPW-associated PSPs together with mathematical extraction techniques to quantify the mean and variance of synaptic conductances that a neuron experiences during bSPW episodes. We find that inhibitory conductances dominate in pyramidal cells and in a putative interneuron, and that inhibitory variances are much greater than excitatory ones during synchronous parts of bSPWs. Specifically, we find that there is at least a twofold increase in inhibitory conductance dominance from "sparse" to "synchronous" bSPW states and that this transition is associated with inhibitory fluctuations of greater than 10% of the change in mean inhibitory conductance. On the basis of our findings, we suggest that such inhibitory fluctuations during transition may be a physiological feature of systems expressing such population activities. In summary, our results provide a quantified basis for understanding the interaction of excitatory and inhibitory neuronal subpopulations in bSPW activities.

  1. Boric acid inhibits germination and colonization of Saprolegnia spores in vitro and in vivo.

    PubMed

    Ali, Shimaa E; Thoen, Even; Evensen, Øystein; Skaar, Ida

    2014-01-01

    Saprolegnia infections cause severe economic losses among freshwater fish and their eggs. The banning of malachite green increased the demand for finding effective alternative treatments to control the disease. In the present study, we investigated the ability of boric acid to control saprolegniosis in salmon eggs and yolk sac fry. Under in vitro conditions, boric acid was able to decrease Saprolegnia spore activity and mycelial growth in all tested concentrations above 0.2 g/L, while complete inhibition of germination and growth was observed at a concentration of 0.8 g/L. In in vivo experiments using Atlantic salmon eyed eggs, saprolegniosis was controlled by boric acid at concentrations ranging from 0.2-1.4 g/L during continuous exposure, and at 1.0-4.0 g/L during intermittent exposure. The same effect was observed on salmon yolk sac fry exposed continuously to 0.5 g/L boric acid during the natural outbreak of saprolegniosis. During the experiments no negative impact with regard to hatchability and viability was observed in either eggs or fry, which indicate safety of use at all tested concentrations. The high hatchability and survival rates recorded following the in vivo testing suggest that boric acid is a candidate for prophylaxis and control of saprolegniosis.

  2. Boric Acid Inhibits Germination and Colonization of Saprolegnia Spores In Vitro and In Vivo

    PubMed Central

    Ali, Shimaa E.; Thoen, Even; Evensen, Øystein; Skaar, Ida

    2014-01-01

    Saprolegnia infections cause severe economic losses among freshwater fish and their eggs. The banning of malachite green increased the demand for finding effective alternative treatments to control the disease. In the present study, we investigated the ability of boric acid to control saprolegniosis in salmon eggs and yolk sac fry. Under in vitro conditions, boric acid was able to decrease Saprolegnia spore activity and mycelial growth in all tested concentrations above 0.2 g/L, while complete inhibition of germination and growth was observed at a concentration of 0.8 g/L. In in vivo experiments using Atlantic salmon eyed eggs, saprolegniosis was controlled by boric acid at concentrations ranging from 0.2–1.4 g/L during continuous exposure, and at 1.0–4.0 g/L during intermittent exposure. The same effect was observed on salmon yolk sac fry exposed continuously to 0.5 g/L boric acid during the natural outbreak of saprolegniosis. During the experiments no negative impact with regard to hatchability and viability was observed in either eggs or fry, which indicate safety of use at all tested concentrations. The high hatchability and survival rates recorded following the in vivo testing suggest that boric acid is a candidate for prophylaxis and control of saprolegniosis. PMID:24699283

  3. Salubrinal inhibits the expression of proteoglycans and favors neurite outgrowth from cortical neurons in vitro.

    PubMed

    Barreda-Manso, M Asunción; Yanguas-Casás, Natalia; Nieto-Sampedro, Manuel; Romero-Ramírez, Lorenzo

    2015-07-01

    After CNS injury, astrocytes and mesenchymal cells attempt to restore the disrupted glia limitans by secreting proteoglycans and extracellular matrix proteins (ECMs), forming the so-called glial scar. Although the glial scar is important in sealing the lesion, it is also a physical and functional barrier that prevents axonal regeneration. The synthesis of secretory proteins in the RER is under the control of the initiation factor of translation eIF2α. Inhibiting the synthesis of secretory proteins by increasing the phosphorylation of eIF2α, might be a pharmacologically efficient way of reducing proteoglycans and other profibrotic proteins present in the glial scar. Salubrinal, a neuroprotective drug, decreased the expression and secretion of proteoglycans and other profibrotic proteins induced by EGF or TGFβ, maintaining eIF2α phosphorylated. Besides, Salubrinal also reduced the transcription of proteoglycans and other profibrotic proteins, suggesting that it induced the degradation of non-translated mRNA. In a model in vitro of the glial scar, cortical neurons grown on cocultures of astrocytes and fibroblasts with TGFβ treated with Salubrinal, showed increased neurite outgrowth compared to untreated cells. Our results suggest that Salubrinal may be considered of therapeutic value facilitating axonal regeneration, by reducing overproduction and secretion of proteoglycans and profibrotic protein inhibitors of axonal growth. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. In vitro inhibition of enamel demineralization by a polymerizable amphiphilic film.

    PubMed

    Tantbirojn, D; Rozzi, S M; Mitra, S B; Kedrowski, B L; Douglas, W H

    2000-12-01

    An amphiphilic coating is configured as a substantive film that has a tendency for an in-plane two-dimensional polymerization. This coating is hypothesized to protect enamel from in vitro acid decalcification, assessed through the following artificial caries model. Three regions on labial enamel of eight bovine incisors were treated with an acid resistant varnish (A), the amphiphilic coating (B), or left undisturbed (C), and the teeth were immersed for 3 wk in lactic acid gel. Mineral loss (deltaZ-value) was determined by a cross-sectional microhardness technique. deltaZ-values (mean +/- SD; volume percent mineral-microm) were: -4 +/- 24 (A), 29 +/- 69 (B), and 7,372 +/- 1,766 (C). deltaZ-value of the uncoated enamel (C) was significantly different from the other groups. Scanning electron microscopy showed enamel etched pattern from citric acid, and the coating firmly attached on enamel surface. This amphiphilic coating can inhibit enamel decalcification under the present experimental condition.

  5. Inhibition of Matriptase Activity Results in Decreased Intestinal Epithelial Monolayer Integrity In Vitro

    PubMed Central

    Pászti-Gere, E.; McManus, S.; Meggyesházi, N.; Balla, P.; Gálfi, P.; Steinmetzer, T.

    2015-01-01

    Barrier dysfunction in inflammatory bowel diseases implies enhanced paracellular flux and lowered transepithelial electrical resistance (TER) causing effective invasion of enteropathogens or altered intestinal absorption of toxins and drug compounds. To elucidate the role of matriptase-driven cell surface proteolysis in the maintenance of intestinal barrier function, the 3-amidinophenylalanine-derived matriptase inhibitor, MI-432 was used on porcine IPEC-J2 cell monolayer. Studies with two fluorescent probes revealed that short (2 h) treatment with MI-432 caused an altered distribution of oxidative species between intracellular and extracellular spaces in IPEC-J2 cells. This perturbation was partially compensated when administration of inhibitor continued for up to 48 h. Significant decrease in TER between apical and basolateral compartments of MI-432-treated IPEC-J2 cell monolayers proved that matriptase is one of the key effectors in the maintenance of barrier integrity. Changes in staining pattern of matriptase and in localization of the junctional protein occludin were observed suggesting that inhibition of matriptase by MI-432 can also exert an effect on paracellular gate opening via modulation of tight junctional protein assembly. This study confirms that non-tumorigenic IPEC-J2 cells can be used as an appropriate small intestinal model for the in vitro characterization of matriptase-related effects on intestinal epithelium. These findings demonstrate indirectly that matriptase plays a pivotal role in the development of barrier integrity; thus matriptase dysfunction can facilitate the occurence of leaky gut syndrome observed in intestinal inflammatory diseases. PMID:26488575

  6. In vitro antioxidant and H+, K+-ATPase inhibition activities of Acalypha wilkesiana foliage extract

    PubMed Central

    Prakash Gupta, Rajesh Kashi; Pradeepa; Hanumanthappa, Manjunatha

    2013-01-01

    Aims: The aim of this study was to evaluate the antioxidant activty and anti-acid property of Acalypha wilkesiana foliage extract. Materials and Methods: Hot and cold aqueous extracts were prepared from healthy leaves of A. wilkesiana. Free radical scavenging activity and H+, K+-ATPase inhibition activities of aqueous foliage extracts was screened by in vitro models. Statistical Analysis Used: All experiments were performed in triplicate and results are expressed as mean ± SEM. Results: A. wilkesiana hot aqueous extract (AWHE) showed significant antioxidants and free radical scavenging activity. Further, AWHE has shown a potent H+, K+-ATPase inhibitory activity (IC50: 51.5 ± 0.28 μg/ml) when compare to standard proton pump inhibitor omeprazole (56.2 ± 0.64 μg/ml); however, latter activity is equal to A. wilkesiana cold aqueous extract (AWCE). Quantitative analysis of AWHE has revealed more content of phenols and flavonoids; this is found to be the reason for good antioxidant activity over AWCE. Molecular docking was carried out against H+, K+-ATPase enzyme crystal structure to validate the anti-acid activity of A. wilkesiana major phytochemicals. Conclusions: The present study indicates that the constituents of AWHE and AWCE have good antacid and free radical scavenging activity. PMID:24082698

  7. The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro

    PubMed Central

    Golderman, Valery; Shavit-Stein, Efrat; Tamarin, Ilia; Rosman, Yossi; Shrot, Shai; Rosenberg, Nurit

    2016-01-01

    Organophosphates (OPs) are potentially able to affect serine proteases by reacting with their active site. The potential effects of OPs on coagulation factors such as thrombin and on coagulation tests have been only partially characterized and potential interactions with OPs antidotes such as oximes and muscarinic blockers have not been addressed. In the current study, we investigated the in vitro interactions between coagulation, thrombin, the OP paraoxon, and its antidotes obidoxime and atropine. The effects of these substances on thrombin activity were measured in a fluorescent substrate and on coagulation by standard tests. Both paraoxon and obidoxime but not atropine significantly inhibited thrombin activity, and prolonged prothrombin time, thrombin time, and partial thromboplastin time. When paraoxon and obidoxime were combined, a significant synergistic effect was found on both thrombin activity and coagulation tests. In conclusion, paraoxon and obidoxime affect thrombin activity and consequently alter the function of the coagulation system. Similar interactions may be clinically relevant for coagulation pathways in the blood and possibly in the brain. PMID:27689805

  8. In vitro inhibition of blood cholinesterase activities from horse, cow, and rat by tetrachlorvinphos.

    PubMed

    Karanth, Subramanya; Pope, Carey

    2003-01-01

    The organophosphorus insecticide tetrachlorvinphos (TCVP) is commonly used as a feed-through larvicide in many livestock species, including cattle and horses. Cholinesterase (ChE) activity in blood (generally plasma or whole blood) is often employed to assess organophosphorus insecticide intoxication in animals as well as humans. In many species, including horse and man, plasma contains predominantly butyrylcholinesterase whereas red blood cells in all species express exclusively acetylcholinesterase. To evalulate the comparative interaction of TCVP with blood ChEs in different species, we compared the in vitro sensitivity of ChE activity in plasma and erythrocytes from horse, cow, and rat. Horse plasma ChE was most sensitive (IC(50), 30 minutes, 30 degrees C = 97 nM), whereas horse erythrocyte ChE activity was least sensitive (IC(50) > 1 mM). In contrast, cow plasma ChE showed lower sensitivity (IC(50) = 784 microM) to inhibition by TCVP than erythrocyte ChE (IC(50) = 216 microM). Rat plasma and erythrocyte ChE activities had relatively similar sensitivity to TCVP (IC(50) = 54 microM and 78 microM, respectively). The results suggest that plasma and erythrocyte ChE from horse, cow, and rat show marked species- and blood fraction-dependent differences in sensitivity to TCVP. Knowledge of such differences in sensitivity of blood ChE activities to TCVP may be important in the clinical interpretation of intoxication with this pesticide across species.

  9. Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.

    PubMed

    Liu, Bo; Yu, Zhuoteng; Chen, Ceng; Kling, David E; Newburg, David S

    2012-08-01

    Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate-labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

  10. Analysis of inhibition of DNA replication in irradiated cells using the SV40 based in vitro assay of DNA replication.

    PubMed

    Iliakis, George; Mladenov, Emil; Wang, Ya; Wang, Hong Yan

    2012-01-01

    The deleterious effects of DNA damage on DNA replication have been demonstrated in many model systems and the mechanisms of the resulting inhibition have been a research focus for at least 40 years. Moreover, recent studies have identified several major components of the S-phase checkpoint, providing a mechanistic background for understanding the basis of inhibition of the initiation and elongation steps of DNA replication after DNA damage. Yet several aspects of the underlying biochemical mechanisms remain unresolved including the characterization of the enzymatic activities involved in checkpoint activation and the coordination of this process with DNA repair. Helpful for the delineation of the mechanism of the S-phase checkpoint is the observation that factors inhibiting DNA replication in vivo can be found in active form in extracts prepared from irradiated cells, when these are tested using the simian virus 40 (SV40) assay for in vitro DNA replication. In this assay, replication of plasmid DNA carrying the minimal origin of SV40 DNA replication is achieved in vitro using cytoplasmic cell extracts and SV40 large tumor antigen (TAg) as the only noncellular protein. Here, we describe protocols developed to measure in vitro DNA replication with the purpose of analyzing its regulation after exposure to DNA damage. The procedures include the preparation of components of the in vitro DNA replication reaction including cytoplasmic extracts from cells that have sustained DNA damage. The assay provides a powerful tool for investigating the effect of distinct agents acting either by inducing lesions in the DNA, or by inhibiting the functions of checkpoint proteins. Nevertheless, the fact that several steps of DNA replication initiation are carried out in this in vitro assay by TAg and not the corresponding cellular factors, might be considered as a limitation of the approach.

  11. Overexpression of Bcl-2–Associated Death Inhibits A549 Cell Growth In Vitro and In Vivo

    PubMed Central

    Huang, Na; Zhu, Jing; Liu, Dan; Li, Ya-Lun; Chen, Bo-Jiang; He, Yan-Qi; Liu, Kun; Mo, Xian-Ming

    2012-01-01

    Abstract The importance of apoptosis during the process of inhibiting tumorigenesis has been recognized. The role of BH3-only proapoptotic protein Bcl-2–associated death (BAD) in tumor growth remains controversial. The aim of this study was to explore the role of BAD in lung cancer cells. Our study showed that expression of BAD was upregulated in A549 cells by a recombinant lentivirus overexpressing BAD. In vitro, BAD overexpression significantly inhibited A549 cell proliferation and induced apoptosis in cell proliferation and apoptosis assays, respectively. The effect of BAD on A549 cells was studied in tumor xenograft of nude mice and the results showed that the tumor volume in the experimental group was smaller than the control groups. Further, immunohistochemical technique was used to determine the cell proliferation and apoptosis status of the lung tumor xenograft cells. This demonstrated that the in vivo and in vitro results were consistent. Taken together, our results indicate that overexpression of BAD inhibits the growth of A549 cells in vitro and in vivo, through inhibiting cell proliferation and inducing apoptosis. Thus, BAD could be a potential therapeutic target. PMID:22011203

  12. Pomegranate ellagitannins inhibit α-glucosidase activity in vitro and reduce starch digestibility under simulated gastro-intestinal conditions.

    PubMed

    Bellesia, Andrea; Verzelloni, Elena; Tagliazucchi, Davide

    2015-02-01

    Pomegranate extract was tested for its ability to inhibit α-amylase and α-glucosidase activity. Pomegranate extract strongly inhibited rat intestinal α-glucosidase in vitro whereas it was a weak inhibitor of porcine α-amylase. The inhibitory activity was recovered in an ellagitannins-enriched fraction and punicalagin, punicalin, and ellagic acid were identified as α-glucosidase inhibitors (IC(50) of 140.2, 191.4, and 380.9 μmol/L, respectively). Kinetic analysis suggested that the pomegranate extract and ellagitannins inhibited α-glucosidase activity in a mixed mode. The inhibitory activity was demonstrated using an in vitro digestion system, mimicking the physiological gastro-intestinal condition, and potatoes as food rich in starch. Pre-incubation between ellagitannins and α-glucosidase increased the inhibitory activity, suggesting that they acted by binding to α-glucosidase. During digestion punicalin and punicalagin concentration decreased. Despite this loss, the pomegranate extract retained high inhibitory activity. This study suggests that pomegranate ellagitannins may inhibit α-glucosidase activity in vitro possibly affecting in vivo starch digestion.

  13. Studies on the Carbon Dioxide Promotion and Ethylene Inhibition of Tuberization in Potato Explants Cultured in Vitro1

    PubMed Central

    Mingo-Castel, Angel M.; Smith, Orrin E.; Kumamoto, Junji

    1976-01-01

    Ethylene inhibited the tuberization of etiolated potato (Solanum tuberosum L. var. Red La Soda) sprout sections cultured in vitro. Carbon dioxide did not overcome the C2H4 inhibition but it was required for normal tuberization. Ethylene totally prevented root formation and development. It inhibited stolon elongation, and caused thickening and diageotropical growth of the stolon. In addition, C2H4 prevented the accumulation of both starch and red anthocyanin which are always present in a tuber. Ethylene also inhibited the kinetin-increased tuberization of sprout sections. Three to five days of exposure to CO2 were required to obtain promotion of tuberization of stolons cultured in vitro. Bicarbonate ion did not affect starch synthetase activity isolated from potato tubers in vitro. The evidence presented suggests that CO2 gas rather than HCO−3 or CO2−3 ions in equilibrium with dissolved CO2 was probably responsible for the stimulation. Morphological changes elicited by CO2 and C2H4 are described and the mechanism of action of both on tuberization is discussed. Images PMID:16659510

  14. In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways

    PubMed Central

    Lei, Xueping; Chen, Minfeng; Nie, Qiulin; Hu, Jianyang; Zhuo, Zhenjian; Yiu, Anita; Chen, Heru; Xu, Nanhui; Huang, Maohua; Ye, Kaihe; Bai, Liangliang; Ye, Wencai; Zhang, Dongmei

    2016-01-01

    Tumor angiogenic process is regulated by multiple proangiogenic pathways, such as vascular endothelial growth factor receptor 2 (VEGFR2) and Axl receptor tyrosine kinase (Axl). Axl is one of many important factors involved in anti-VEGF resistance. Inhibition of VEGF/VEGFR2 signaling pathway alone fails to block tumor neovascularization. Therefore, discovery of novel agents targeting multiple angiogenesis pathways is in demand. Desacetylvinblastine monohydrazide (DAVLBH), a derivative of vinblastine (VLB), has been reported exhibit an anticancer activity via its cytotoxic effect. However, little attention has been paid to the antiangiogenic properties of DAVLBH. Here, we firstly reported that DAVLBH exerted a more potent antiangiogenic effect than VLB in vitro and in vivo, which was associated with inactivation of VEGF/VEGFR2 and Gas6/Axl signaling pathways. We found that DAVLBH inhibited VEGF- and Gas6-induced HUVECs proliferation, migration, tube formation and vessel sprouts formation in vitro and ex vivo. It significantly inhibited in vivo tumor angiogenesis and tumor growth in HeLa xenografts. It also inhibited Gas6-induced pericytes recruitment to endothelial tubes accompanied with a decrease in expression and activation of Axl. Besides, it could block the compensatory up-regulating expression and activation of Axl in response to bevacizumab treatment in HUVECs. Taken together, our results suggest that DAVLBH potently inhibits angiogenesis-mediated tumor growth through blockage of the activation of VEGF/VEGFR2 and Gas6/Axl pathways and it might serve as a promising antiangiogenic agent for the cancer therapy. PMID:27186435

  15. Inhibition of Streptococcus pneumoniae adherence to human epithelial cells in vitro by the probiotic Lactobacillus rhamnosus GG

    PubMed Central

    2013-01-01

    Background Colonization of the nasopharynx by Streptococcus pneumoniae is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. Probiotic bacteria can influence disease outcomes through various mechanisms, including inhibition of pathogen colonization. Here, we examine the effect of the probiotic Lactobacillus rhamnosus GG (LGG) on S. pneumoniae colonization of human epithelial cells using an in vitro model. We investigated the effects of LGG administered before, at the same time as, or after the addition of S. pneumoniae on the adherence of four pneumococcal isolates. Results LGG significantly inhibited the adherence of all the pneumococcal isolates tested. The magnitude of inhibition varied with LGG dose, time of administration, and the pneumococcal isolate used. Inhibition was most effective when a higher dose of LGG was administered prior to establishment of pneumococcal colonization. Mechanistic studies showed that LGG binds to epithelial cells but does not affect pneumococcal growth or viability. Administration of LGG did not lead to any significant changes in host cytokine responses. Conclusions These findings demonstrate that LGG can inhibit pneumococcal colonization of human epithelial cells in vitro and suggest that probiotics could be used clinically to prevent the establishment of pneumococcal carriage. PMID:23561014

  16. Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

    PubMed Central

    Said, Harun M; Polat, Buelent; Stein, Susanne; Guckenberger, Mathias; Hagemann, Carsten; Staab, Adrian; Katzer, Astrid; Anacker, Jelena; Flentje, Michael; Vordermark, Dirk

    2012-01-01

    AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5’-GCATTATTGGCATGGGAAC-3’ and 5’-ATGCAGAGTAACGTGGAAG-3’. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor (HIF)-1α mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human

  17. Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo‡

    PubMed Central

    Sadhukha, Tanmoy; Prabha, Swayam; Sanaú, Mercedes; Rotenberg, Susan A.

    2016-01-01

    Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO−) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds. PMID

  18. SILIBININ INHIBITS ETHANOL METABOLISM AND ETHANOL-DEPENDENT CELL PROLIFERATION IN AN IN VITRO MODEL OF HEPATOCELLULAR CARCINOMA

    PubMed Central

    Brandon-Warner, Elizabeth; Sugg, James A.; Schrum, Laura W.; McKillop, Iain H.

    2009-01-01

    Chronic ethanol consumption is a known risk factor for developing hepatocellular carcinoma (HCC). The use of plant-derived antioxidants is gaining increasing clinical prominence as a potential therapy to ameliorate the effects of ethanol on hepatic disease development and progression. This study demonstrates silibinin, a biologically active flavanoid derived from milk thistle, inhibits cytochrome p4502E1 induction, ethanol metabolism and reactive oxygen species generation in HCC cells in vitro. These silibinin-mediated effects also inhibit ethanol-dependent increases in HCC cell proliferation in culture. PMID:19900758

  19. Screening the Brazilian flora for antihypertensive plant species for in vitro angiotensin-I-converting enzyme inhibiting activity.

    PubMed

    Castro Braga, F; Wagner, H; Lombardi, J A; de Oliveira, A B

    2000-06-01

    The evaluation of several antihypertensive activity of Brazilian plant species was performed using in vitro inhibition of the angiotensin I-converting enzyme (ACE). Nineteen species belonging to 13 families were investigated. Plants were selected based on their use as diuretics and on a chemosystematic consideration. Extracts of the following species presented the highest ACE inhibition rate, at concentrations of 0.33 mg/ml: Ouratea semiserrata (Mart. & Nees) Engl. stems (68%), Cuphea cartagenesis (Jacq.) Macbride leaves (50%) and Mansoa hirsuta DC. leaves (54%). Some hypotheses about the nature of the compounds that may be responsible for the activity of these species are discussed in the paper.

  20. [Inhibition of human breast cancer cell line BCap-37 by flavonoid extract of wheat germ in vitro].

    PubMed

    Xu, G; Zhao, X; Zhao, L; Xu, H

    1999-05-30

    Cell growth and proliferation were measured by microculture tetrazolium(MTT) assay, cell colony-forming assay and the synthesis of DNA by 3H-thymidine incorporation. Flavonoid extract of wheat germ resulted to a dose-dependent, time-dependent growth inhibition, reduction of colony and 3H-thymiding incorporation in DNA of human breast cancer cell BCap-37. These findings indicated that the flavonoid extract of wheat germ can inhibit tumor cell growth and proliferation by blocking DNA synthesis in vitro.

  1. Parallel in vivo and in vitro melanoma RNAi dropout screens reveal synthetic lethality between hypoxia and DNA damage response inhibition.

    PubMed

    Possik, Patricia A; Müller, Judith; Gerlach, Carmen; Kenski, Juliana C N; Huang, Xinyao; Shahrabi, Aida; Krijgsman, Oscar; Song, Ji-Ying; Smit, Marjon A; Gerritsen, Bram; Lieftink, Cor; Kemper, Kristel; Michaut, Magali; Beijersbergen, Roderick L; Wessels, Lodewyk; Schumacher, Ton N; Peeper, Daniel S

    2014-11-20

    To identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. The Polyphenol Altenusin Inhibits in Vitro Fibrillization of Tau and Reduces Induced Tau Pathology in Primary Neurons.

    PubMed

    Chua, Sook Wern; Cornejo, Alberto; van Eersel, Janet; Stevens, Claire H; Vaca, Inmaculada; Cueto, Mercedes; Kassiou, Michael; Gladbach, Amadeus; Macmillan, Alex; Lewis, Lev; Whan, Renee; Ittner, Lars M

    2017-04-19

    In Alzheimer's disease, the microtubule-associated protein tau forms intracellular neurofibrillary tangles (NFTs). A critical step in the formation of NFTs is the conversion of soluble tau into insoluble filaments. Accordingly, a current therapeutic strategy in clinical trials is aimed at preventing tau aggregation. Here, we assessed altenusin, a bioactive polyphenolic compound, for its potential to inhibit tau aggregation. Altenusin inhibits aggregation of tau protein into paired helical filaments in vitro. This was associated with stabilization of tau dimers and other oligomers into globular structures as revealed by atomic force microscopy. Moreover, altenusin reduced tau phosphorylation in cells expressing pathogenic tau, and prevented neuritic tau pathology induced by incubation of primary neurons with tau fibrils. However, treatment of tau transgenic mice did not improve neuropathology and functional deficits. Taken together, altenusin prevents tau fibrillization in vitro and induced tau pathology in neurons.

  3. Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse.

    PubMed

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2014-10-01

    Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Tissue distribution, characterization and in vitro inhibition of B-esterases in the earwig Forficula auricularia.

    PubMed

    Malagnoux, Laure; Capowiez, Yvan; Rault, Magali

    2014-10-01

    Earwigs are important natural enemies of numerous pests in pome fruit orchards worldwide. Studying the effects of agricultural practices on these biological control agents is important for understanding its vulnerability in the field. The aim of this study was to characterize the B-esterase activities in the European earwig Forficula auricularia and to evaluate in vitro its sensitivity to organophosphate and carbamate pesticides. Acetylcholinesterase (AChE) activity was mainly measured with 1.5 mM acetylthiocholine as the substrate in the microsomal fraction of earwig heads (70% of total AChE activity). Carboxylesterase (CbE) activities were measured with three substrates [5 mM 4-nitrophenyl acetate (4-NPA), 1mM 4-nitrophenyl valerate (4-NPV), and 2 mM α-naphtyl acetate (α-NA)] to examine different isoenzymes, which were present mainly in the cytosolic fraction (about 70-88% of total activities) of all earwig tissues. CbE activity was higher than AChE activity, especially with α-NA, then 4-NPA and lastly 4-NPV. Chlorpyrifos-oxon an organophosphate, and carbaryl a carbamate pesticide, inhibited AChE and CbE activities in a concentration-dependent manner. Earwig CbE activities showed a stronger sensitivity to organophosphate than AChE, with the strongest effect for chlorpyrifos-oxon on male carboxylesterase activities. CbE and AChE showed about the same sensitivity to carbamate pesticides regardless of sex. These results suggest that B-type esterases in the European earwig F.auricularia are suitable biomarkers of pesticide exposure.

  5. Guava leaves polyphenolics-rich extract inhibits vital enzymes implicated in gout and hypertension in vitro

    PubMed Central

    Irondi, Emmanuel Anyachukwu; Agboola, Samson Olalekan; Oboh, Ganiyu; Boligon, Aline Augusti; Athayde, Margareth Linde; Shode, Francis O.

    2016-01-01

    Background/Aim: Elevated uric acid level, an index of gout resulting from the over-activity of xanthine oxidase (XO), increases the risk of developing hypertension. However, research has shown that plant-derived inhibitors of XO and angiotensin 1-converting enzyme (ACE), two enzymes implicated in gout and hypertension, respectively, can prevent or ameliorate both diseases, without noticeable side effects. Hence, this study characterized the polyphenolics composition of guava leaves extract and evaluated its inhibitory effect on XO and ACE in vitro. Materials and Methods: The polyphenolics (flavonoids and phenolic acids) were characterized using high-performance liquid chromatography (HPLC) coupled with diode array detection (DAD). The XO, ACE, and Fe2+-induced lipid peroxidation inhibitory activities, and free radicals (2,2-diphenylpicrylhydrazyl [DPPH]* and 2,2´-azino-bis-3-ethylbenzthiazoline-6-sulphonic [ABTS]*+) scavenging activities of the extract were determined using spectrophotometric methods. Results: Flavonoids were present in the extract in the order of quercetin > kaempferol > catechin > quercitrin > rutin > luteolin > epicatechin; while phenolic acids were in the order of caffeic acid > chlorogenic acid > gallic acids. The extract effectively inhibited XO, ACE and Fe2+-induced lipid peroxidation in a dose-dependent manner; having half-maximal inhibitory concentrations (IC50) of 38.24 ± 2.32 μg/mL, 21.06 ± 2.04 μg/mL and 27.52 ± 1.72 μg/mL against XO, ACE and Fe2+-induced lipid peroxidation, respectively. The extract also strongly scavenged DPPH* and ABTS*+. Conclusion: Guava leaves extract could serve as functional food for managing gout and hypertension and attenuating the oxidative stress associated with both diseases. PMID:27104032

  6. Knockdown of lymphoid enhancer factor 1 inhibits colon cancer progression in vitro and in vivo.

    PubMed

    Wang, Wen-Juan; Yao, Yu; Jiang, Li-Li; Hu, Ting-Hua; Ma, Jie-Qun; Liao, Zi-Jun; Yao, Jun-Tao; Li, Dong-Fan; Wang, Shu-Hong; Nan, Ke-Jun

    2013-01-01

    Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

  7. Knockdown of Lymphoid Enhancer factor 1 Inhibits Colon Cancer Progression In Vitro and In Vivo

    PubMed Central

    Jiang, Li-Li; Hu, Ting-Hua; Ma, Jie-Qun; Liao, Zi-Jun; Yao, Jun-Tao; Li, Dong-Fan; Wang, Shu-Hong; Nan, Ke-Jun

    2013-01-01

    Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy. PMID:24098538

  8. Staphylococcus aureus enterotoxins A and B inhibit human and mice eosinophil chemotaxis and adhesion in vitro.

    PubMed

    Squebola-Cola, Dalize M; De Mello, Glaucia C; Anhê, Gabriel F; Condino-Neto, Antonio; DeSouza, Ivani A; Antunes, Edson

    2014-12-01

    Staphylococcus aureus aggravates the allergic eosinophilic inflammation. We hypothesized that Staphylococcus aureus-derived enterotoxins directly affect eosinophil functions. Therefore, this study investigated the effects of Staphylococcal enterotoxins A and B (SEA and SEB) on human and mice eosinophil chemotaxis and adhesion in vitro, focusing on p38 MAPK phosphorylation and intracellular Ca(2+) mobilization. Eosinophil chemotaxis was evaluated using a microchemotaxis chamber, whereas adhesion was performed in VCAM-1 and ICAM-1-coated plates. Measurement of p38 MAPK phosphorylation and intracellular Ca(2+) levels were monitored by flow cytometry and fluorogenic calcium-binding dye, respectively. Prior incubation (30 to 240 min) of human blood eosinophils with SEA (0.5 to 3 ng/ml) significantly reduced eotaxin-, PAF- and RANTES-induced chemotaxis (P<0.05). Likewise, SEB (1 ng/ml, 30 min) significantly reduced eotaxin-induced human eosinophil chemotaxis (P<0.05). The reduction of eotaxin-induced human eosinophil chemotaxis by SEA and SEB was prevented by anti-MHC monoclonal antibody (1 μg/ml). In addition, SEA and SEB nearly suppressed the eotaxin-induced human eosinophil adhesion in ICAM-1- and VCAM-1-coated plates. SEA and SEB prevented the increases of p38 MAPK phosphorylation and Ca(2+) levels in eotaxin-activated human eosinophils. In separate protocols, we evaluated the effects of SEA on chemotaxis and adhesion of eosinophils obtained from mice bone marrow. SEA (10 ng/ml) significantly reduced the eotaxin-induced chemotaxis along with cell adhesion to both ICAM-1 and VCAM-1-coated plates (P<0.05). In conclusion, the inhibition by SEA and SEB of eosinophil functions (chemotaxis and adhesion) are associated with reductions of p38 MAPK phosphorylation and intracellular Ca(2+) mobilization.

  9. Polarized currents inhibit in vitro growth of bacteria colonizing cutaneous ulcers.

    PubMed

    Gomes, Rosana Caetano; Brandino, Hugo Evangelista; de Sousa, Natanael Teixeira Alves; Santos, Marcos Ferracioli; Martinez, Roberto; Guirro, Rinaldo Roberto de Jesus

    2015-01-01

    Infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli are among the microorganisms that often lead to infection in pressure ulcers. Polarized current has emerged as a possible intervention to limit bacterial proliferation. We analyzed the effect of fixed diphasic - Bernard (FD-B) and high voltage monophasic pulsed (HVMP) currents on bacteria S. aureus ATCC 25923 (Gram +), P. aeruginosa ATCC 27853 (Gram -), and E. coli ATCC 25922 (Gram -). After the bacterial strains were activated the bacteria were suspended in physiological solution (0.9%) and the concentration adjusted to 1.5 × 10(3) CFU/mL. The cultures were stimulated with FD-B current at (3, 6, and 9 mA, 100 Hz, 15 and 30 minutes) and HVMP (32, 64, and 95 V, 100 Hz, 30 and 60 min) while monitoring the pH and temperature. After the stimulation, the suspensions were plated and incubated for 24 hours at 37°C. Then the counts were made of colony forming units (CFU). Data were submitted to normality Shapiro-Wilk test followed by nonparametric ANOVA test and post hoc Tukey test with p < 0.05. There was a decrease in the CFU for the two currents, but the most effective reduction was in FD-B. The temperature remained constant and the pH measured alkaline at the negative pole and acid at the positive pole during stimulation. The application of FD-B and HVMP currents promoted inhibition of bacterial proliferation when stimulated in vitro, acting as an adjuvant resource in the healing process. © 2015 by the Wound Healing Society.

  10. Antibodies against MAEBL Ligand Domains M1 and M2 Inhibit Sporozoite Development In Vitro

    PubMed Central

    Preiser, Peter; Rénia, Laurent; Singh, Naresh; Balu, Bharath; Jarra, William; Voza, Tatiana; Kaneko, Osamu; Blair, Peter; Torii, Motomi; Landau, Irène; Adams, John H.

    2004-01-01

    MAEBL is a type 1 membrane protein that is implicated in the merozoite invasion of erythrocytes and sporozoite invasion of mosquito salivary glands. This apical organelle protein is structurally similar to the ebl erythrocyte binding proteins, such as EBA-175, except that the tandem ligand domains of MAEBL are similar to part of the extracellular domain of apical membrane antigen 1 and not the Duffy binding-like domain. Although midgut and salivary gland sporozoites are morphologically similar, salivary gland sporozoites undergo a period of new gene expression after infecting the salivary glands, display distinct phenotypic differences, and are more infectious for the mammalian host. The objectives of this project were to determine the molecular form of MAEBL in the infectious salivary gland sporozoites and whether the ligand has a role in the sporozoite development to exoerythrocytic stages in hepatocytes. We determined that MAEBL is newly expressed in salivary gland sporozoites and in a form distinct from what is present in the midgut sporozoites or present in erythrocytic stages. Both ligand domains (M1 and M2) were expressed as part of a full-length membrane form of MAEBL in the salivary gland sporozoites in contrast to the other stages that retain only the M2 ligand domain as part of the membrane form of the protein. Antisera developed against the cysteine-rich regions of the extracellular portion of MAEBL inhibited sporozoite development to exoerythrocytic forms in vitro. Together these data indicate that MAEBL has a role in this third developmental stage in the life cycle of the malaria parasite. Thus, MAEBL is another target for pre-erythrocytic-stage vaccine development against malaria parasites. PMID:15155670

  11. A Furan-Based Lewis-Y-(CD174)-Saccharide Mimetic Inhibits Endothelial Functions and In Vitro Angiogenesis.

    PubMed

    Sauer, Sandra; Meissner, Tobias; Moehler, Thomas

    2015-01-01

    Angiogenesis is a fundamental process underlying cancer progression and autoimmune disease. Lewis Y is known as a regulated glycan-structure supporting human endothelial function and angiogenesis. We hypothesize that Lewis Y based analogues interfere with Lewis Y mediated endothelial functions and angiogenesis. We therefore evaluated the ability of 3, 4-bis [(b-D-galactopyranosyl)osy]-methyl-furan (BGF) a furan-based Lewis-Y saccharide mimetic to inhibit human endothelial adhesion, migration and in vitro angiogenesis. The ability of BGF and additional furan-based saccharide-mimetics was investigated to inhibit adhesion and migration of human bone marrow endothelial cells (HBMEC). Influence of BGF was tested on a multicelluar in vitro - angiogenesis assay in the presence of VEGF. BGF significantly inhibited HBMEC adhesion and migration stimulated by TNF-alpha by up to 70%. The anti-adhesive effect of BGF was particularly evident when HBMEC adhesion and migration was tested on collagen as extracellular matrix with weaker effect when laminin and fibronectin were used as an extracellular matrix. BGF was ineffective when HBMEC were stimulated with VEGF. The inhibition of endothelial function translated into a significant inhibitory effect of BGF in the multicellular in vitro angiogenesis-assay. BGF reduced the angiogenesis index compared to the positive controls by 32%. We identified the ability of the furan-based Lewis Y saccharide mimetic BGF as a specific modulator of TNF-alpha activated endothelial function and in vitro angiogenesis. BGF and other related glycan analogues should further be explored for their ability to down modulate endothelial activation in TNF-alpha driven pathophysiologic conditions in autoimmune disease and cancer indications.

  12. Bayesian models trained with HTS data for predicting β-haematin inhibition and in vitro antimalarial activity.

    PubMed

    Wicht, Kathryn J; Combrinck, Jill M; Smith, Peter J; Egan, Timothy J

    2015-08-15

    A large quantity of high throughput screening (HTS) data for antimalarial activity has become available in recent years. This includes both phenotypic and target-based activity. Realising the maximum value of these data remains a challenge. In this respect, methods that allow such data to be used for virtual screening maximise efficiency and reduce costs. In this study both in vitro antimalarial activity and inhibitory data for β-haematin formation, largely obtained from publically available sources, has been used to develop Bayesian models for inhibitors of β-haematin formation and in vitro antimalarial activity. These models were used to screen two in silico compound libraries. In the first, the 1510 U.S. Food and Drug Administration approved drugs available on PubChem were ranked from highest to lowest Bayesian score based on a training set of β-haematin inhibiting compounds active against Plasmodium falciparum that did not include any of the clinical antimalarials or close analogues. The six known clinical antimalarials that inhibit β-haematin formation were ranked in the top 2.1% of compounds. Furthermore, the in vitro antimalarial hit-rate for this prioritised set of compounds was found to be 81% in the case of the subset where activity data are available in PubChem. In the second, a library of about 5000 commercially available compounds (Aldrich(CPR)) was virtually screened for ability to inhibit β-haematin formation and then for in vitro antimalarial activity. A selection of 34 compounds was purchased and tested, of which 24 were predicted to be β-haematin inhibitors. The hit rate for inhibition of β-haematin formation was found to be 25% and a third of these were active against P. falciparum, corresponding to enrichments estimated at about 25- and 140-fold relative to random screening, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Bayesian models trained with HTS data for predicting β-haematin inhibition and in vitro antimalarial activity

    PubMed Central

    Wicht, Kathryn J.; Combrinck, Jill M.; Smith, Peter J.; Egan, Timothy J.

    2015-01-01

    A large quantity of high throughput screening (HTS) data for antimalarial activity has become available in recent years. This includes both phenotypic and target-based activity. Realising the maximum value of these data remains a challenge. In this respect, methods that allow such data to be used for virtual screening maximise efficiency and reduce costs. In this study both in vitro antimalarial activity and inhibitory data for β-haematin formation, largely obtained from publically available sources, has been used to develop Bayesian models for inhibitors of β-haematin formation and in vitro antimalarial activity. These models were used to screen two in silico compound libraries. In the first, the 1510 U.S. Food and Drug Administration approved drugs available on PubChem were ranked from highest to lowest Bayesian score based on a training set of β-haematin inhibiting compounds active against P. falciparum that did not include any of the clinical antimalarials or close analogues. The six known clinical antimalarials that inhibit β-haematin formation were ranked in the top 2.1% of compounds. Furthermore, the in vitro antimalarial hit-rate for this prioritised set of compounds was found to be 81% in the case of the subset where activity data are available in PubChem. In the second, a library of about 5,000 commercially available compounds (AldrichCPR) was virtually screened for ability to inhibit β-haematin formation and then for in vitro antimalarial activity. A selection of 34 compounds was purchased and tested, of which 24 were predicted to be β-haematin inhibitors. The hit rate for inhibition of β-haematin formation was found to be 25% and a third of these were active against P. falciparum, corresponding to enrichments estimated at about 25- and 140-fold relative to random screening, respectively. PMID:25573118

  14. Kinase Inhibition-Related Adverse Events Predicted from in vitro Kinome and Clinical Trial Data

    PubMed Central

    Yang, Xinan; Huang, Yong; Crowson, Matthew; Li, Jianrong; Maitland, Michael L.; Lussier, Yves A.

    2010-01-01

    Background Kinase inhibition is an increasingly popular strategy for pharmacotherapy of human diseases. Although many of these agents have been described as “targeted therapy”, they will typically inhibit multiple kinases with varying potency. Pre-clinical model testing has not predicted the numerous significant toxicities identified during clinical development. The purpose of this study was to develop a bioinformatics-based method to predict specific adverse events (AEs) in humans associated with the inhibition of particular kinase targets (KTs). Methods The AE frequencies of protein kinase inhibitors (PKIs) were curated from three sources (PubMed, Thompson Physician Desk Reference and PharmGKB), and affinities of 38 PKIs for 317 kinases, representing > 50% of the predicted human kinome, were collected from published in vitro assay results. A novel quantitative computational method was developed to predict associations between KTs and AEs that included a whole panel of 71 AEs and 20 PKIs targeting 266 distinct kinases with Kd < 10uM. The method calculated an unbiased, kinome-wide association score via linear algebra on (i) the normalized frequencies of AEs associated with 20 PKIs and (ii) the negative log-transformed dissociation constant of kinases targeted by these PKIs. Finally, a reference standard was calculated by applying Fisher’s exact test to the co-occurrence of indexed Pubmed terms (p≤0.05, and manually verified) for AE and associated kinase targets (AE-KT) pairs from standard literature search techniques. We also evaluated the enrichment of predictions between the quantitative method and the literature search by Fisher’s Exact testing. Results We identified significant associations among already empirically well established pairs of AEs (e.g. diarrhea and rash) and KTs (e.g. EGFR). The following less well recognized AE-KT pairs had similar association scores: diarrhea-(DDR1; ERBB4), rash-ERBB4, and fatigue-(CSF1R; KIT). With no filtering, the

  15. Azole antifungal inhibition of buprenorphine, methadone and oxycodone in vitro metabolism.

    PubMed

    Moody, David E; Liu, Fenyun; Fang, Wenfang B

    2015-06-01

    Opioid-related mortality rates have escalated. Drug interactions may increase blood concentrations of the opioid. We therefore used human liver microsomes (HLMs) and cDNA-expressed human cytochrome P450s (rCYPs) to study in vitro inhibition of buprenorphine metabolism to norbuprenorphine (CYP3A4 and 2C8), oxycodone metabolism to noroxycodone (CYP3A4 and 2C18) and oxymorphone (CYP2D6), and methadone metabolism to R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP; CYP3A4 and 2B6). In this study, we have examined the inhibitory effect of 12 (mostly antifungal) azoles. These compounds have a wide range of solubility; to keep organic solvent ≤1%, there was an equally wide range of highest concentration tested (e.g., itraconazole 5 µM to fluconazole 1000 µM). Inhibitors were first incubated with HLMs at three concentrations with or without preincubation of inhibitor with reducing equivalents to also screen for time-dependent inhibition (TDI). Posaconazole displayed evidence of TDI; metronidazole and albendazole had no significant effect. Azoles were next screened at the highest achievable concentration for non-CYP3A4 pathways. IC50 values (µM) were determined for most CYP3A4 pathways (ranges) and other pathways as dictated by screen results: clotrimazole (0.30 - 0.35; others >30 µM); econazole (2.2 - 4.9; 2B6 R-EDDP - 9.5, S-EDDP - 6.8; 2C8 - 6.0; 2C18 - 1.0; 2D6 - 1.2); fluconazole (7.7 - 66; 2B6 - 313, 361; 2C8 - 1240; 2C18 - 17; 2D6 - 1000); itraconazole (2.5 to >5; others >5); ketoconazole (0.032 - 0.094; 2B6 - 12, 31; 2C8 - 78; 2C18 - 0.98; 2D6 - 182); miconazole (2.3 - 7.6; 2B6 - 2.8, 2.8; 2C8 - 5.3; 2C18 - 3.1; 2D6 - 5.9); posaconazole (3.4 - 20; 2C18 - 3.8; others >30); terconazole (0.48 to >10; 2C18 - 8.1; others >10) and voriconazole (0.40 - 15; 2B6 - 2.4, 2.5; 2C8 - 170; 2C18 - 13; 2D6 >300). Modeling based on estimated Ki values and plasma concentrations from the literature suggest that the orally administered azoles, particularly

  16. Trans-3,4,5,4′-tetramethoxystilbene, a resveratrol analog, potently inhibits angiogenesis in vitro and in vivo

    PubMed Central

    Chen, Liang-ke; Qiang, Peng-fei; Xu, Qi-ping; Zhao, Yi-hua; Dai, Fang; Zhang, Lu

    2013-01-01

    Aim: Trans-3,4,5,4′-tetramethoxystilbene (DMU-212) has shown strong antiproliferative activities against a variety of cancer cells. The aim of this study was to investigate the anti-angiogenic effects of DMU-212 in vitro and in vivo. Methods: Human umbilical vein endothelial cells (HUVECs) were used in this study. Cell viability was studied with MTT assay, and cell apoptosis was evaluated using TUNEL assay and morphological observation. The expression of the related genes and proteins was analyzed with qRT-PCR and Western blot, respectively. Angiogenesis of HUVECs were studied using cell migration and capillary-like tube formation assays in vitro, and mouse Matrigel plug assay and chick chorioallantoic membrane (CAM) assay in vivo. The tyrosine kinase activities of VEGFR1 and VEGFR2 were measured using commercial kits. Results: DMU-212 (5–80 μmol/L) significantly inhibited VEGF-stimulated proliferation of HUVECs (IC50 value was approximately 20 μmol/L), and induced apoptosis. Furthermore, DMU-212 concentration-dependently inhibited VEGF-induced migration of HUVECs and capillary-like structure formation in vitro. DMU-212 also inhibited VEGF-induced generation of new vasculature in Matrigel plugs in vivo with significantly decreased area of infiltrating CD31-positive endothelial cells, and inhibited newly formed microvessels in chick CAMs. Moreover, DMU-212 concentration-dependently suppressed VEGF-induced phosphorylation of VEGFR2, and inhibited phosphorylation of multiple downstream signaling components in the VEGFR2 pathway, including c-Src, FAK, Erk1/2, Akt, mTOR, and p70S6K in HUVECs. DMU-212 had no effect on VEGF-induced phosphorylation of VEGFR1. Conclusion: DMU-212 is a potent inhibitor of angiogenesis that exerts anti-angiogenic activity at least in part through the VEGFR2 signaling pathway. PMID:23770989

  17. Inhibition of the angiogenesis and growth of Aloin in human colorectal cancer in vitro and in vivo

    PubMed Central

    2013-01-01

    Background Angiogenesis has been an attractive target for drug therapy. Aloin (AL), an natural compound derived from Aloe barbadensis Miller leaves, has been shown to possess anti-cancer potential activities. However, its roles in tumor angiogenesis and the involved molecular mechanism are unknown. Method To evaluate the antiangiogenic and anticancer activities of AL, endothelial cell scratch, modified Boyden chamber inserts and tube formation assays were done in HUVECs, and MTT and Live-Dead assays were used to determine the proliferation inhibition and apoptosis induction of colorectal cancer cells in vitro. The inhibition effects of AL were further confirmed by a mouse xenograft model in vivo. The expression levels of STAT3 signaling pathway and that mediated-target genes were measured in HUVECs and SW620 cells by Western blots. Results Here, we demonstrated that AL significantly inhibited HUVECs proliferation, migration and tube formation in vitro. Western blotting showed that AL suppressed activation of VEGF receptor (VEGFR) 2 and STAT3 phosphorylation in endothelial cells. In addition, the constitutively activated STAT3 protein, and the expression of STAT3-regulated antiapoptotic (Bcl-xL), proliferative (c-Myc), and angiogenic (VEGF) proteins were also down-regulated in response to AL in human SW620 cancer cells. Consistent with the above findings, AL inhibited tumor cell viability and induced cell apoptosis in vitro, and substantially reduced tumor volumes and weight in vivo mouse xenografts, without obviously toxicity. Conclusion Our studies provided the first evidence that AL may inhibit tumor angiogenesis and growth via blocking STAT3 activation, with the potential of a drug candidate for cancer therapy. PMID:23848964

  18. ROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPARα -mediated inhibition of Glioma cell motility in vitro

    PubMed Central

    2010-01-01

    Background Glioblastomas are characterized by rapid cell growth, aggressive CNS infiltration, and are resistant to all known anticancer regimens. Recent studies indicate that fibrates and statins possess anticancer potential. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that can switch energy metabolism from glycolysis to fatty acid β-oxidation, and has low systemic toxicity. Fenofibrate also attenuates IGF-I-mediated cellular responses, which could be relevant in the process of glioblastoma cell dispersal. Methods The effects of fenofibrate on Glioma cell motility, IGF-I receptor (IGF-IR) signaling, PPARα activity, reactive oxygen species (ROS) metabolism, mitochondrial potential, and ATP production were analyzed in human glioma cell lines. Results Fenofibrate treatment attenuated IGF-I signaling responses and repressed cell motility of LN-229 and T98G Glioma cell lines. In the absence of fenofibrate, specific inhibition of the IGF-IR had only modest effects on Glioma cell motility. Further experiments revealed that PPARα-dependent accumulation of ROS is a strong contributing factor in Glioma cell lines responses to fenofibrate. The ROS scavenger, N-acetyl-cysteine (NAC), restored cell motility, improved mitochondrial potential, and increased ATP levels in fenofibrate treated Glioma cell lines. Conclusions Our results indicate that although fenofibrate-mediated inhibition of the IGF-IR may not be sufficient in counteracting Glioma cell dispersal, PPARα-dependent metabolic switch and the resulting ROS accumulation strongly contribute to the inhibition of these devastating brain tumor cells. PMID:20569465

  19. AGE-RELATED BRAIN CHOLINESTERASE INHIBITION KINETICS FOLLOWING IN VITRO INCUBATION WITH CHLORPYRIFOS-OXON AND DIAZINON-OXON

    SciTech Connect

    Kousba, Ahmed A.; Poet, Torka S.; Timchalk, Chuck

    2007-01-01

    Chlorpyrifos and diazinon are two commonly used organophosphorus (OP) insecticides, and their primary mechanism of action involves the inhibition of acetylcholinesterase (AChE) by their metabolites chlorpyrifos-oxon (CPO) and diazinon-oxon (DZO), respectively. The study objectives were to assess the in vitro age-related inhibition kinetics of neonatal rat brain cholinesterase (ChE) by estimating the bimolecular inhibitory rate constant (ki) values for CPO and DZO. Brain ChE inhibition and ki values following CPO and DZO incubation with neonatal Sprague-Dawley rats rat brain homogenates were determined at post natal day (PND) -5, -12 and -17 and compared with the corresponding inhibition and ki values obtained in the adult rat. A modified Ellman method was utilized for measuring the ChE activity. Chlorpyrifos-oxon resulted in greater ChE inhibition than DZO consistent with the estimated ki values of both compounds. Neonatal brain ChE inhibition kinetics exhibited a marked age-related sensitivity to CPO, where the order of ChE inhibition was PND-5 > PND-7 > PND-17 with ki values of 0.95, 0.50 and 0.22 nM-1hr-1, respectively. In contrast, DZO did not exhibit an age-related inhibition of neonatal brain ChE, and the estimated ki value at all PND ages was 0.02 nM-1hr-1. These results demonstrated an age- and chemical-related OP-selective inhibition of rat brain ChE which may be critically important in understanding the potential sensitivity of juvenile humans to specific OP exposures.

  20. Juice of eclipta prostrata inhibits cell migration in vitro and exhibits anti-angiogenic activity in vivo.

    PubMed

    Lirdprapamongkol, Kriengsak; Kramb, Jan-Peter; Chokchaichamnankit, Daranee; Srisomsap, Chantragan; Surarit, Rudee; Sila-Asna, Monnipha; Bunyaratvej, Ahnond; Dannhardt, Gerd; Svasti, Jisnuson

    2008-01-01

    The invasion of cancer cells is critical for metastasis. The effects of Eclipta prostrata, a Thai medicinal plant, on invasion, migration and adhesion of cancer cells were investigated and the anti-angiogenic activity in vivo was evaluated. In vitro invasion and migration assays were performed in modified Boyden chambers. In vivo anti-angiogenic activity was determined using the chick chorioallantoic membrane (CAM) assay. E. prostrata juice inhibited cancer invasion and migration, without affecting cell adhesion. Cell migration was inhibited in a variety of cancer cell types and in endothelial cells, with IC50 values of 31-70 microg/ml, much lower than the IC50 values for cytotoxicity of 203-1,217 microg/ml for cancer cells and >4,000 microg/ml for endothelial cells. Fifty percent inhibition of angiogenesis by E. prostrata juice was observed at 200 microg/egg. E. prostrata juice inhibited cancer and endothelial cell migration in vitro and also showed in vivo anti-angiogenic activity.

  1. Alginate as a protease inhibitor in vitro and in a model gut system; selective inhibition of pepsin but not trypsin.

    PubMed

    Chater, Peter Ian; Wilcox, Mathew D; Brownlee, Iain A; Pearson, Jeffrey P

    2015-10-20

    Alginates are widely used in the food and medical industries, including as a Gastro-Oesophagul Reflux treatment. This work investigates the inhibitory effects of alginate on the reflux aggressors trypsin and pepsin and the role of alginate-substrate binding, pH and alginate structure on inhibition. Alginates were shown to reduce pepsin activity by up to 53.9% (±9.5SD) in vitro. Strong positive correlation between alginate mannuronate residue frequency and levels of pepsin inhibition was observed. Limited inhibition of trypsin was shown. Viscometric observations of pH dependent interactions between alginate and protein suggest a mechanism whereby pH dependent ionic interactions reduce substrate availability to enzyme at acidic pH. To understand how dietary protein digestion is affected by alginate, proteolytic digestion was investigated in an in vitro model of the upper digestive tract. Significant inhibition of proteolysis was shown in the gastric phase of digestion, but not the small intestinal phase. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. GSK-3 inhibition in vitro and in vivo enhances antitumor effect of sorafenib in renal cell carcinoma (RCC)

    SciTech Connect

    Kawazoe, Hisashi; Bilim, Vladimir N.; Ugolkov, Andrey V.; Yuuki, Kaori; Naito, Sei; Nagaoka, Akira; Kato, Tomoyuki; Tomita, Yoshihiko

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Sorafenib treatment upregulated GSK-3{beta} levels in RCC cells. Black-Right-Pointing-Pointer Pharmacologic inhibition of GSK-3 suppressed xenograft RCC tumor growth. Black-Right-Pointing-Pointer Inhibition of GSK-3 enhanced antitumor effect of sorafenib in vitro and in vivo. -- Abstract: Sorafenib is a multikinase inhibitor approved for the systemic treatment of renal cell carcinoma (RCC). However, sorafenib treatment has a limited effect due to acquired chemoresistance of RCC. Previously, we identified glycogen synthase kinase-3 (GSK-3) as a new therapeutic target in RCC. Here, we observed that sorafenib inhibits proliferation and survival of RCC cells. Significantly, we revealed that sorafenib enhances GSK-3 activity in RCC cells, which could be a potential mechanism of acquired chemoresistance. We found that pharmacological inhibition of GSK-3 potentiates sorafenib antitumor effect in vitro and in vivo. Our results suggest that combining GSK-3 inhibitor and sorafenib might be a potential new therapeutic approach for RCC treatment.

  3. Alginate as a protease inhibitor in vitro and in a model gut system; selective inhibition of pepsin but not trypsin

    PubMed Central

    Chater, Peter Ian; Wilcox, Mathew D.; Brownlee, Iain A.; Pearson, Jeffrey P.

    2015-01-01

    Alginates are widely used in the food and medical industries, including as a Gastro-Oesophagul Reflux treatment. This work investigates the inhibitory effects of alginate on the reflux aggressors trypsin and pepsin and the role of alginate-substrate binding, pH and alginate structure on inhibition. Alginates were shown to reduce pepsin activity by up to 53.9% (±9.5SD) in vitro. Strong positive correlation between alginate mannuronate residue frequency and levels of pepsin inhibition was observed. Limited inhibition of trypsin was shown. Viscometric observations of pH dependent interactions between alginate and protein suggest a mechanism whereby pH dependent ionic interactions reduce substrate availability to enzyme at acidic pH. To understand how dietary protein digestion is affected by alginate, proteolytic digestion was investigated in an in vitro model of the upper digestive tract. Significant inhibition of proteolysis was shown in the gastric phase of digestion, but not the small intestinal phase. PMID:26256170

  4. AAV-Mediated angiotensin 1-7 overexpression inhibits tumor growth of lung cancer in vitro and in vivo

    PubMed Central

    Mao, Yingying; Wang, Shengyao; Yan, Renhe; Bai, Na; Li, Andrew; Zhang, Yanling; Du, Hongyan; Chen, Baihong; Sumners, Colin; Li, Jinlong; Li, Hongwei

    2017-01-01

    Ang-(1-7) inhibits lung cancer cell growth both in vitro and in vivo. However, the molecular mechanism of action is unclear and also the rapid degradation of Ang-(1-7) in vivo limits its clinical application. Here, we have demonstrated that Ang- (1-7) inhibits lung cancer cell growth by interrupting pre-replicative complex assembly and restrains epithelial-mesenchymal transition via Cdc6 inhibition. Furthermore, we constructed a mutant adeno-associated viral vector AAV8 (Y733F) that produced stable and high efficient Ang-(1-7) expression in a xenograft tumor model. The results show that AAV8-mediated Ang-(1-7) over-expression can remarkably suppress tumor growth in vivo by down-regulating Cdc6 and anti-angiogenesis. Ang-(1-7) over-expression via the AAV8 method may be a promising strategy for lung cancer treatment. PMID:27861149

  5. Rhodacyanine dye MKT-077 inhibits in vitro telomerase assay but has no detectable effects on telomerase activity in vivo.

    PubMed

    Wadhwa, Renu; Colgin, Lorel; Yaguchi, Tomoko; Taira, Kazunari; Reddel, Roger R; Kaul, Sunil C

    2002-08-01

    MKT-077, a cationic rhodacyanine dye analogue, causes selective toxicity to cancer cells. Its cellular targets elucidated thus far include oncogenic Ras, F-actin, mortalin (hmot-2)/mthsp70, and telomerase. Here we report that MKT-077 causes growth arrest of cancer cells in culture independent of their Ras, p53, or telomerase status. Telomerase activity is inhibited in vitro by MKT-077 in the telomerase assay used. However, the in vivo toxicity seen in telomerase-positive cancer cells was not accompanied by inhibition of telomerase activity or telomere shortening. Furthermore, cells with an alternative mechanism for lengthening of telomeres were also sensitive to MKT-077 and showed enhanced formation of alternative mechanism for lengthening of telomeres-associated PML bodies in their nuclei. The data suggested that inhibition of telomerase activity is unlikely to be a prime cause of MKT-077-induced toxicity in cancer cells.

  6. In vitro monoamine oxidase inhibition potential of alpha-methyltryptamine analog new psychoactive substances for assessing possible toxic risks.

    PubMed

    Wagmann, Lea; Brandt, Simon D; Kavanagh, Pierce V; Maurer, Hans H; Meyer, Markus R

    2017-03-14

    Tryptamines have emerged as new psychoactive substances (NPS), which are distributed and consumed recreationally without preclinical studies or safety tests. Within the alpha-methylated tryptamines, some of the psychoactive effects of the prototypical alpha-methyltryptamine (AMT) have been described decades ago and a contributing factor of its acute toxicity appears to involve the inhibition of monoamine oxidase (MAO). However, detailed information about analogs is scarce. Therefore, thirteen AMT analogs were investigated for their potential to inhibit MAO. An in vitro assay analyzed using hydrophilic interaction liquid chromatography-high resolution-tandem mass spectrometry was developed and validated. The AMT analogs were incubated with recombinant human MAO-A or B and kynuramine, a non-selective MAO substrate to determine the IC50 values. The known MAO-A inhibitors 5-(2-aminopropyl)indole (5-IT), harmine, harmaline, yohimbine, and the MAO-B inhibitor selegiline were tested for comparison. AMT and all analogs showed MAO-A inhibition properties with IC50 values between 0.049 and 166μM, whereas four analogs inhibited also MAO-B with IC50 values between 82 and 376μM. 7-Me-AMT provided the lowest IC50 value against MAO-A comparable to harmine and harmaline and was identified as a competitive MAO-A inhibitor. Furthermore, AMT, 7-Me-AMT, and nine further analogs inhibited MAO activity in human hepatic S9 fraction used as model for the human liver which expresses both isoforms. The obtained results suggested that MAO inhibition induced by alpha-methylated tryptamines might be clinically relevant concerning possible serotonergic and adrenergic effects and interactions with drugs (of abuse) particularly acting as monoamine reuptake inhibitors. However, as in vitro assays have only limited conclusiveness, further studies are needed.

  7. Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression

    PubMed Central

    QI, Xiaonan; LI, Huatao; CONG, Xia; WANG, Xin; JIANG, Zhongling; CAO, Rongfeng; TIAN, Wenru

    2016-01-01

    Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

  8. TGFβ1 Inhibition Increases the Radiosensitivity of Breast Cancer Cells In Vitro and Promotes Tumor Control by Radiation In Vivo

    PubMed Central

    Bouquet, Fanny; Pal, Anupama; Pilones, Karsten A.; Demaria, Sandra; Hann, Byron; Akhurst, Rosemary J.; Babb, Jim S.; Lonning, Scott M.; DeWyngaert, J. Keith; Formenti, Silvia C.; Barcellos-Hoff, Mary Helen

    2013-01-01

    Purpose To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. Experimental Design TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. Results Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radio-sensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFfS type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. Conclusion These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy. PMID:22028490

  9. Growth-Inhibiting Activity of Resveratrol Imine Analogs on Tumor Cells In Vitro

    PubMed Central

    Wang, Shan; Willenberg, Ina; Krohn, Michael; Hecker, Tanja; Meckelmann, Sven; Li, Chang; Pan, Yuanjiang; Schebb, Nils Helge; Steinberg, Pablo; Empl, Michael Telamon

    2017-01-01

    Although resveratrol exerts manifold antitumorigenic effects in vitro, its efficacy against malignancies in vivo seems limited. This has been increasingly recognized in recent years and has prompted scientists to search for structurally related compounds with more promising anticarcinogenic and/or pharmacokinetic properties. A class of structurally modified resveratrol derivatives, so-called resveratrol imine analogs (IRA’s), might meet these requirements. Therefore, the biological activity of five of these compounds was examined and compared to that of resveratrol. Firstly, the antiproliferative potency of all five IRA’s was investigated using the p53 wildtype-carrying colorectal carcinoma cell line HCT-116wt. Then, using the former and a panel of various other tumor cell lines (including the p53 knockout variant HCT-116p53-/-), the growth-inhibiting and cell cycle-disturbing effects of the most potent IRA (IRA 5, 2-[[(2-hydroxyphenyl)methylene]amino]-phenol) were studied as was its influence on cyclooxygenase-2 expression and activity. Finally, rat liver microsomes were used to determine the metabolic stability of that compound. IRA 5 was clearly the most potent compound in HCT-116wt cells, with an unusually high IC50-value of 0.6 μM. However, in the other five cell lines used, the antiproliferative activity was mostly similar to resveratrol and the effects on the cell cycle were heterogeneous. Although all cell lines were affected by treatment with IRA 5, cells expressing functional p53 seemed to react more sensitively, suggesting that this protein plays a modulating role in the induction of IRA 5-mediated biological effects. Lastly, IRA 5 led to contradictory effects on cyclooxygenase-2 expression and activity and was less glucuronidated than resveratrol. As IRA 5 is approximately 50 times more toxic towards HCT-116wt cells, exerts different effects on the cyclooxygenase-2 and is metabolized to a lesser extent, it shows certain advantages over resveratrol

  10. In vitro cytochrome P450 inhibition potential of methylenedioxy-derived designer drugs studied with a two-cocktail approach.

    PubMed

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2016-02-01

    In vitro cytochrome P450 (CYP) inhibition assays are common approaches for testing the inhibition potential of drugs for predicting potential interactions. In contrast to marketed medicaments, drugs of abuse, particularly the so-called novel psychoactive substances, were not tested before distribution and consumption. Therefore, the inhibition potential of methylenedioxy-derived designer drugs (MDD) of different drug classes such as aminoindanes, amphetamines, benzofurans, cathinones, piperazines, pyrrolidinophenones, and tryptamines should be elucidated. The FDA-preferred test substrates, split in two cocktails, were incubated with pooled human liver microsomes and analysed after protein precipitation using LC-high-resolution-MS/MS. IC50 values were determined of MDD showing more than 50 % inhibition in the prescreening. Values were calculated by plotting the relative metabolite concentration formed over the logarithm of the inhibitor concentration. All MDD showed inhibition against CYP2D6 activity and most of them in the range of the clinically relevant CYP2D6 inhibitors quinidine and fluoxetine. In addition, the beta-keto compounds showed inhibition of the activity of CYP2B6, 5,6-MD-DALT of CYP1A2 and CYP3A, and MDAI of CYP2A6, all in the range of clinically relevant inhibitors. In summary, all MDD showed inhibition of the activity of CYP2D6, six of CYP1A2, three of CYP2A6, 13 of CYP2B6, two of CYP2C9, six of CYP2C19, one of CYP2E1, and six of CYP3A. These results showed that the CYP inhibition by MDD might be clinically relevant, but further studies are needed for final conclusions.

  11. Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

    PubMed

    Romero-Aguirregomezcorta, Jon; Santa, Ángela Patricia; García-Vázquez, Francisco Alberto; Coy, Pilar; Matás, Carmen

    2014-01-01

    Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

  12. INHIBITION OF IN VITRO LIPID PEROXIDATION (LPO) EVOKED BY CALOCYBE INDICA (MILKY MUSHROOM)

    PubMed Central

    Selvi, S; Umadevi, P.; Suja, S.; Sridhar, K.; Chinnaswamy, P.

    2006-01-01

    The present study was designed with an objective to assess the inhibition of lipid peroxidation (LPO) by the aqueous extract of Calocybe indica (milky mushroom) using an invitro model of goat liver homogenate and RBC ghosts. The invitro LPO was inhibited to a good extent by the aqueous extract of milky mushroom and the extent of inhibition being higher in the RBC membrane model when compared with liver homogenate model. PMID:22557223

  13. Inhibition of in vitro lipid peroxidation (lpo) evoked by calocybe indica (milky mushroom).

    PubMed

    Selvi, S; Umadevi, P; Suja, S; Sridhar, K; Chinnaswamy, P

    2006-07-01

    The present study was designed with an objective to assess the inhibition of lipid peroxidation (LPO) by the aqueous extract of Calocybe indica (milky mushroom) using an invitro model of goat liver homogenate and RBC ghosts. The invitro LPO was inhibited to a good extent by the aqueous extract of milky mushroom and the extent of inhibition being higher in the RBC membrane model when compared with liver homogenate model.

  14. Xylitol does not inhibit xylose fermentation by engineered Saccharomyces cerevisiae expressing xylA as severely as it inhibits xylose isomerase reaction in vitro.

    PubMed

    Ha, Suk-Jin; Kim, Soo Rin; Choi, Jin-Ho; Park, Myeong Soo; Jin, Yong-Su

    2011-10-01

    Efficient fermentation of xylose, which is abundant in hydrolysates of lignocellulosic biomass, is essential for producing cellulosic biofuels economically. While heterologous expression of xylose isomerase in Saccharomyces cerevisiae has been proposed as a strategy to engineer this yeast for xylose fermentation, only a few xylose isomerase genes from fungi and bacteria have been functionally expressed in S. cerevisiae. We cloned two bacterial xylose isomerase genes from anaerobic bacteria (Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1) and introduced them into S. cerevisiae. While the transformant with xylA from B. longum could not assimilate xylose, the transformant with xylA from B. stercoris was able to grow on xylose. This result suggests that the xylose isomerase (BsXI) from B. stercoris is functionally expressed in S. cerevisiae. The engineered S. cerevisiae strain with BsXI consumed xylose and produced ethanol with a good yield (0.31 g/g) under anaerobic conditions. Interestingly, significant amounts of xylitol (0.23 g xylitol/g xylose) were still accumulated during xylose fermentation even though the introduced BsXI might not cause redox imbalance. We investigated the potential inhibitory effects of the accumulated xylitol on xylose fermentation. Although xylitol inhibited in vitro BsXI activity significantly (K(I) = 5.1 ± 1.15 mM), only small decreases (less than 10%) in xylose consumption and ethanol production rates were observed when xylitol was added into the fermentation medium. These results suggest that xylitol accumulation does not inhibit xylose fermentation by engineered S. cerevisiae expressing xylA as severely as it inhibits the xylose isomerase reaction in vitro.

  15. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    PubMed Central

    Feng, Qin; Gou, Xiao-jun; Meng, Sheng-xi; Huang, Cheng; Zhang, Yu-quan; Tang, Ya-jun; Wang, Wen-jing; Xu, Lin; Peng, Jing-hua; Hu, Yi-yang

    2013-01-01

    Qushi Huayu Decoction (QHD), a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD) in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK) in vivo and in vitro. Nonalcoholic fatty liver (NAFL) model was duplicated with high-fat diet in rats and with free fatty acid (FFA) in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC) and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-responsive element-binding protein (ChREBP) in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro. PMID:23573117

  16. Sodium orthovanadate inhibits growth of human hepatocellular carcinoma cells in vitro and in an orthotopic model in vivo.

    PubMed

    Wu, Yaohua; Ma, Yong; Xu, Zhilin; Wang, Dawei; Zhao, Baolei; Pan, Huayang; Wang, Jizhou; Xu, Dongsheng; Zhao, Xiaoyang; Pan, Shangha; Liu, Lianxin; Dai, Wenjie; Jiang, Hongchi

    2014-08-28

    The transition metal vanadium is widely distributed in the environment and exhibits various biological and physiological effects in the human body. As a well known vanadium compound, sodium orthovanadate (SOV) has shown promising antineoplastic activity in several human cancers. However, the effects of SOV on liver cancer are still unknown. In this study, for the first time, we showed that SOV could effectively suppress proliferation, induce G2/M cell cycle arrest and apoptosis, and diminish the mitochondrial membrane potential (MMP) of HCC cells in vitro. In addition, our in vitro results were recapitulated in vivo, showing that SOV exhibited a dose-dependent inhibition of growth of human HCC in an orthotopic model, evidenced by the reduction in tumor size, proliferation index and microvessel density, and increase in cell apoptosis. Most important, we found that SOV could inhibit autophagy in HCC cells in vitro and in vivo, which plays a prodeath role. Thus, our findings suggest that SOV could effectively suppress the growth of human HCC through the regulations of proliferation, cell cycle, apoptosis and autophagy, and thus may act as a potential therapeutic agent in HCC treatment.

  17. Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models

    NASA Astrophysics Data System (ADS)

    Basheer, Loai; Schultz, Keren; Kerem, Zohar

    2016-08-01

    Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an in silico and in vitro approaches and synthetic model compounds. Mono, di, and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4, and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs.

  18. Inhibition of multixenobiotic resistance transporters (MXR) by silver nanoparticles and ions in vitro and in Daphnia magna.

    PubMed

    Georgantzopoulou, Anastasia; Cambier, Sébastien; Serchi, Tommaso; Kruszewski, Marcin; Balachandran, Yekkuni L; Grysan, Patrick; Audinot, Jean-Nicolas; Ziebel, Johanna; Guignard, Cédric; Gutleb, Arno C; Murk, AlberTinka J

    2016-11-01

    The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATP-binding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MXR). In this study we first evaluated, in vitro, the interaction of silver nanoparticles (Ag NPs, 20, 23 and 27nm), Ag 200nm particles and Ag ions (AgNO3) with MXR efflux transporters using MDCKII and the P-gp over-expressing MDCKII-MDR1 cells and calcein-AM as a substrate of the transporters. Next the in vivo modulation of MXR activity was studied in Daphnia magna juveniles with the model P-gp and MRP1 inhibitors verapamil-HCl and MK571, respectively. The common environmental contaminants perfluorooctane sulfonate and bisphenol A, previously observed to interfere with the P-gp in vitro, also inhibited the efflux of calcein in vivo. Small-sized Ag NPs (with biomolecules present on the surface) and AgNO3 inhibited the MXR activity in daphnids and MDCKII-MDR1 cells, but abcb1 gene expression remained unchanged. Both Ag NPs and dissolved ions contributed to the effects. This study provides evidence of the interference of Ag NPs and AgNO3 with the MXR activity both in vitro and in D. magna, and should be taken into account when Ag NP toxicity is assessed.

  19. Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models

    PubMed Central

    Basheer, Loai; Schultz, Keren; Kerem, Zohar

    2016-01-01

    Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an in silico and in vitro approaches and synthetic model compounds. Mono, di, and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4, and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs. PMID:27530542

  20. In vitro inhibition of growth of Neisseria gonorrhoeae by Neisseria meningitidis isolated from the pharynx of homosexual men.

    PubMed

    Bisaillon, J G; Turgeon, P; Dubreuil, D; Beaudet, R; Sylvestre, M; Ashton, F E

    1984-01-01

    Despite the high prevalence of pharyngeal gonorrhea and of meningococcal carriage among homosexual men, Neisseria gonorrhoeae and Neisseria meningitidis are rarely co-isolated from the throat. Forty-seven meningococcal isolates from the pharynx of homosexual men were examined, by a lawn-spotting method, for their ability to inhibit N. gonorrhoeae in vitro. Eight (17%) of the meningococcal isolates were inhibitory when tested against gonococci from the same patient, while 31 (66%) were inhibitory when tested against N. gonorrhoeae strain 650 (T1). The colonial type T1 of a given strain was, in all cases tested, more sensitive to the inhibitory activities than the corresponding T4 type. Since the meningococci co-isolated from the throat with gonococci were at least as inhibitory in vitro as those isolated without gonococci, the natural resistance to gonococcal pharyngitis cannot be explained on the basis of the inhibitory activities produced by the meningococci in vitro. The inhibitory strains of N. meningitidis were identified in decreasing importance as: nonserogroupable, W135, C, B, 29E, and X. The addition of trypsin to the solid medium removed the inhibition produced by the meningococci, an observation suggesting the involvement of protein inhibitors.

  1. A numerical method for analysis of in vitro time-dependent inhibition data. Part 2. Application to experimental data.

    PubMed

    Korzekwa, Ken; Tweedie, Donald; Argikar, Upendra A; Whitcher-Johnstone, Andrea; Bell, Leslie; Bickford, Shari; Nagar, Swati

    2014-09-01

    Time-dependent inhibition (TDI) of cytochrome P450 enzymes is an important cause of drug-drug interactions. The standard approach to characterize the kinetics of TDI is to determine the rate of enzyme loss, kobs, at various inhibitor concentrations, [I], and replot the kobs versus [I] to obtain the key kinetic parameters, KI and kinact. In our companion manuscript (Part 1; Nagar et al., 2014) in this issue of Drug Metabolism and Disposition, we used simulated datasets to develop and test a new numerical method to analyze in vitro TDI data. Here, we have applied this numerical method to five TDI datasets. Experimental datasets include the inactivation of CYP2B6, CYP2C8, and CYP3A4. None of the datasets exhibited Michaelis-Menten-only kinetics, and the numerical method allowed use of more complex models to fit each dataset. Quasi-irreversible as well as partial inhibition kinetics were observed and parameterized. Three datasets required the use of a multiple-inhibitor binding model. The mechanistic and clinical implications provided by these analyses are discussed. Together with the results in Part 1, we have developed and applied a new numerical method for analysis of in vitro TDI data. This method appears to be generally applicable to model in vitro TDI data with atypical and complex kinetic schemes.

  2. Salinomycin inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cell in vitro and suppresses tumor growth in vivo

    SciTech Connect

    Wu, Danxin; Zhang, Yu; Huang, Jie; Fan, Zirong; Shi, Fengrong; Wang, Senming

    2014-01-10

    Highlight: •We first evaluated the effect of salinomycin on nasopharyngeal carcinoma (NPC). •Salinomycin could inhibit Wnt/β-catenin signaling and induce apoptosis in NPC. •So salinomycin may be a good potential candidate for the chemotherapy of NPC. -- Abstract: Salinomycin (Sal) is a polyether ionophore antibiotic that has recently been shown to induce cell death in various human cancer cells. However, whether salinomycin plays a functional role in nasopharyngeal carcinoma (NPC) has not been determined to date. The present study investigated the chemotherapeutic efficacy of salinomycin and its molecular mechanisms of action in NPC cells. Salinomycin efficiently inhibited proliferation and invasion of 3 NPC cell lines (CNE-1, CNE-2, and CNE-2/DDP) and activated a extensive apoptotic process that is accompanied by activation of caspase-3 and caspase-9, and decreased mitochondrial membrane potential. Meanwhile, the protein expression level of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and β-catenin was down-regulated, which showed that the Wnt/β-catenin signaling was involved in salinomycin-induced apoptosis of NPC cells. In a nude mouse NPC xenograft model, the anti-tumor effect of salinomycin was associated with the downregulation of β-catenin expression. The present study demonstrated that salinomycin can effectively inhibit proliferation and invasion, and induce apoptosis of NPC cells in vitro and inhibit tumor growth in vivo, probably via the inhibition of Wnt/β-catenin signaling, suggesting salinomycin as a potential candidate for the chemotherapy of NPC.

  3. Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops

    PubMed Central

    Li, Jing; Zeng, Li; Xie, Juan; Yue, Zhiying; Deng, Huayun; Ma, Xueyun; Zheng, Chunbing; Wu, Xiushan; Luo, Jian; Liu, Mingyao

    2015-01-01

    Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca2+/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis. PMID:26620037

  4. Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

    PubMed

    Li, Jing; Zeng, Li; Xie, Juan; Yue, Zhiying; Deng, Huayun; Ma, Xueyun; Zheng, Chunbing; Wu, Xiushan; Luo, Jian; Liu, Mingyao

    2015-12-01

    Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

  5. Isoliquiritigenin Attenuates Adipose Tissue Inflammation in vitro and Adipose Tissue Fibrosis through Inhibition of Innate Immune Responses in Mice

    PubMed Central

    Watanabe, Yasuharu; Nagai, Yoshinori; Honda, Hiroe; Okamoto, Naoki; Yamamoto, Seiji; Hamashima, Takeru; Ishii, Yoko; Tanaka, Miyako; Suganami, Takayoshi; Sasahara, Masakiyo; Miyake, Kensuke; Takatsu, Kiyoshi

    2016-01-01

    Isoliquiritigenin (ILG) is a flavonoid derived from Glycyrrhiza uralensis and potently suppresses NLRP3 inflammasome activation resulting in the improvement of diet-induced adipose tissue inflammation. However, whether ILG affects other pathways besides the inflammasome in adipose tissue inflammation is unknown. We here show that ILG suppresses adipose tissue inflammation by affecting the paracrine loop containing saturated fatty acids and TNF-α by using a co-culture composed of adipocytes and macrophages. ILG suppressed inflammatory changes induced by the co-culture through inhibition of NF-κB activation. This effect was independent of either inhibition of inflammasome activation or activation of peroxisome proliferator-activated receptor-γ. Moreover, ILG suppressed TNF-α-induced activation of adipocytes, coincident with inhibition of IκBα phosphorylation. Additionally, TNF-α-mediated inhibition of Akt phosphorylation under insulin signaling was alleviated by ILG in adipocytes. ILG suppressed palmitic acid-induced activation of macrophages, with decreasing the level of phosphorylated Jnk expression. Intriguingly, ILG improved high fat diet-induced fibrosis in adipose tissue in vivo. Finally, ILG inhibited TLR4- or Mincle-stimulated expression of fibrosis-related genes in stromal vascular fraction from obese adipose tissue and macrophages in vitro. Thus, ILG can suppress adipose tissue inflammation by both inflammasome-dependent and -independent manners and attenuate adipose tissue fibrosis by targeting innate immune sensors. PMID:26975571

  6. Celastrol inhibits prostaglandin E2-induced proliferation and osteogenic differentiation of fibroblasts isolated from ankylosing spondylitis hip tissues in vitro

    PubMed Central

    Zou, Yu-Cong; Yang, Xian-Wen; Yuan, Shi-Guo; Zhang, Pei; Li, Yi-Kai

    2016-01-01

    Background Heterotopic ossification on the enthesis, which develops after subsequent inflammation, is one of the most distinctive features in ankylosing spondylitis (AS). Prostaglandin E2 (PGE-2) serves as a key mediator of inflammation and bone remodeling in AS. Celastrol, a well-known Chinese medicinal herb isolated from Tripterygium wilfordii, is widely used in treating inflammatory diseases, including AS. It has been proven that it can inhibit lipopolysac-charide-induced expression of various inflammation mediators, such as PGE-2. However, the mechanism by which celastrol inhibits inflammation-induced bone forming in AS is unclear. Objective To investigate whether celastrol could inhibit isolated AS fibroblast osteogenesis induced by PGE-2. Methods Hip synovial tissues were obtained from six AS patients undergoing total hip replacement in our hospital. Fibroblasts were isolated, primarily cultured, and then treated with PGE-2 for osteogenic induction. Different doses of celastrol and indometacin were added to observe their effects on osteogenic differentiation. Cell proliferation, osteogenic markers, alizarin red staining as well as the activity of alkaline phosphatase were examined in our study. Results Celastrol significantly inhibits cell proliferation of isolated AS fibroblasts and in vitro osteogenic differentiation compared with control groups in a time- and dose-dependent manner. Conclusion Our results demonstrated that celastrol could inhibit isolated AS fibroblast proliferation and in vitro osteogenic differentiation. The interaction of PI3K/AKT signaling and Wnt protein may be involved in the process. Further studies should be performed in vivo and animal models to identify the potential effect of celastrol on the bone metabolism of AS patients. PMID:27022241

  7. Ellagic acid inhibits the proliferation of human pancreatic carcinoma PANC-1 cells in vitro and in vivo.

    PubMed

    Cheng, Hao; Lu, Chenglin; Tang, Ribo; Pan, Yiming; Bao, Shanhua; Qiu, Yudong; Xie, Min

    2017-01-25

    Ellagic aicd (EA), a dietary polyphenolic compound found in plants and fruits, possesses various pharmacological activities. This study investigated the effect of EA on human pancreatic carcinoma PANC-1 cells both in vitro and in vivo; and defined the associated molecular mechanisms. In vitro, the cell growth and repairing ability were assessed by CCK-8 assay and wound healing assay. The cell migration and invasion activity was evaluated by Tanswell assay. In vivo, PANC-1 cell tumor-bearing mice were treated with different concentrations of EA. We found that EA significantly inhibited cell growth, cell repairing activity, and cell migration and invasion in a dose-dependent manner. Treatment of PANC-1 xenografted mice with EA resulted in significant inhibition in tumor growth and prolong mice survival rate. Furthermore, flow cytometric analysis showed that EA increased the percentage of cells in the G1 phase of cell cycle. Western blot analysis revealed that EA inhibited the expression of COX-2 and NF-κB. In addition, EA reversed epithelial to mesenchymal transition by up-regulating E-cadherin and down-regulating Vimentin. In summary, the present study demonstrated that EA inhibited cell growth, cell repairing activity, cell migration and invasion in a dose-dependent manner. EA also effectively inhibit human pancreatic cancer growth in mice. The anti-tumor effect of EA might be related to cell cycle arrest, down-regulating the expression of COX-2 and NF-κB, reversing epithelial to mesenchymal transition by up-regulating E-cadherin and down-regulating Vimentin. Our findings suggest that the use of EA would be beneficial for the management of pancreatic cancer.

  8. Beta-conglycinin embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro.

    PubMed

    Martinez-Villaluenga, Cristina; Bringe, Neal A; Berhow, Mark A; Gonzalez de Mejia, Elvira

    2008-11-26

    Obesity is a worldwide health concern because it is a well-recognized predictor of premature mortality. The objective was to identify soybean varieties that have improved potential to inhibit fat accumulation in adipocytes by testing the effects of soy hydrolysates having a range of protein subunit compositions on lipid accumulation and adiponectin expression in 3T3-L1 adipocytes. The results showed that differences in the protein distribution of 15 soy genotypes led to different potentials for the reduction of fat accumulation. The inhibition of lipid accumulation of soy alcalase hydrolysates in 3T3-L1 adipocytes ranged from 29 to 46%. Soy hydrolysates made from genotypes with 45.3 +/- 3.3% of total protein as beta-conglycinin, on average, showed significantly higher inhibition of lipid accumulation compared to those with 24.7 +/- 1.5% of extracted total protein as beta-conglycinin. Moreover, after in vitro simulated digestion with pepsin-pancreatin of the soy alcalase hydrolysates, 86% of the original activity remained. Adiponectin expression was induced in 3T3-L1 adipocytes treated with 15 soy hydrolysates up to 2.49- and 2.63-fold for high and low molecular weight adiponectin, respectively. The inhibition of lipid accumulation calculated from a partial least squares (PLS) analysis model correlated well with experimental data (R(2) = 0.91). In conclusion, it was feasible to differentiate soy varieties on the basis of the potential of their proteins to reduce fat accumulation using a statistical model and a cell-based assay in vitro. Furthermore, beta-conglycinin embeds more peptides than glycinin subunits that inhibit lipid accumulation and induce adiponectin in 3T3-L1 adipocytes. Therefore, soy ingredients containing beta-conglycinin may be important food components for the control of lipid accumulation in adipose tissue.

  9. The fungicide Pristine® inhibits mitochondrial function in vitro but not flight metabolic rates in honey bees.

    PubMed

    Campbell, Jacob B; Nath, Rachna; Gadau, Juergen; Fox, Trevor; DeGrandi-Hoffman, Gloria; Harrison, Jon F

    2016-03-01

    Honey bees and other pollinators are exposed to fungicides that act by inhibiting fungal mitochondria. Here we test whether a common fungicide (Pristine®) inhibits the function of mitochondria of honeybees, and whether consumption of ecologically-realistic concentrations can cause negative effects on the mitochondria of flight muscles, or the capability for flight, as judged by CO2 emission rates and thorax temperatures during flight. Direct exposure of mitochondria to Pristine® levels above 5 ppm strongly inhibited mitochondrial oxidation rates in vitro. However, bees that consumed pollen containing Pristine® at ecologically-realistic concentrations (≈ 1 ppm) had normal flight CO2 emission rates and thorax temperatures. Mitochondria isolated from the flight muscles of the Pristine®-consuming bees had higher state 3 oxygen consumption rates than control bees, suggesting that possibly Pristine®-consumption caused compensatory changes in mitochondria. It is likely that the lack of a strong functional effect of Pristine®-consumption on flight performance and the in vitro function of flight muscle mitochondria results from maintenance of Pristine® levels in the flight muscles at much lower levels than occur in the food, probably due to metabolism and detoxification. As Pristine® has been shown to negatively affect feeding rates and protein digestion of honey bees, it is plausible that Pristine® consumption negatively affects gut wall function (where mitochondria may be exposed to higher concentrations of Pristine®).

  10. Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies

    SciTech Connect

    Fisone, G.; Wu, C.F.; Consolo, S.; Nordstroem, O.; Brynne, N.; Bartfai, T.; Melander, T.; Hoekfelt, T.

    1987-10-01

    A high density of galanin binding sites was found by using /sup 125/I-labeled galanin, iodinated by chloramine-T, followed by autoradiography in the ventral, but not in the dorsal, hippocampus of the rat. Lesions of the fimbria and of the septum caused disappearance of a major population of these binding sites, suggesting that a large proportion of them is localized on cholinergic nerve terminals of septal afferents. As a functional correlate to these putative galanin receptor sites, it was shown, both in vivo and in vitro, that galanin, in a concentration-dependent manner, inhibited the evoked release of acetylcholine in the ventral, but not in the dorsal, hippocampus. Intracerebroventricularly applied galanin fully inhibited the scopolamine stimulated release of acetylcholine in the ventral, but not in the dorsal, hippocampus, as measured by the microdialysis technique. In vitro, galanin inhibited the 25 mM K/sup +/-evoked release of (/sup 3/H)acetylcholine from slices of the ventral hippocampus, with an IC/sub 50/ value of approx. = 50 nM. These results are discussed with respect to the colocalization of galanin- and choline acetyltransferase-like immunoreactivity in septal somata projecting to the hippocampus.

  11. In vitro growth of multidrug-resistant Neisseria gonorrhoeae isolates is inhibited by ETX0914, a novel spiropyrimidinetrione.

    PubMed

    Papp, John R; Lawrence, Kenneth; Sharpe, Samera; Mueller, John; Kirkcaldy, Robert D

    2016-09-01

    Antimicrobial resistance in Neisseria gonorrhoeae has severely limited the number of treatment options, and the emergence of extended-spectrum cephalosporin resistance threatens the effectiveness of the last remaining recommended treatment regimen. This study assessed the in vitro susceptibility of N. gonorrhoeae to ETX0914, a novel spiropyrimidinetrione that inhibits DNA biosynthesis. In vitro activity was determined by agar dilution against 100 N. gonorrhoeae isolates collected from men presenting with urethritis in the USA during 2012-2013 through the Gonococcal Isolate Surveillance Project. The minimum inhibitory concentration (MIC) that inhibited growth in 50% (MIC50) and 90% (MIC90) of isolates was calculated for each antimicrobial agent. ETX0914 demonstrated a high level of antimicrobial activity against N. gonorrhoeae, including isolates with decreased susceptibility or resistance to currently available agents. The ability of ETX0914 to inhibit the growth of N. gonorrhoeae was similar to ceftriaxone, which is currently recommended in combination with azithromycin to treat gonorrhoea. The data presented in this study strongly suggest that ETX0914 should be evaluated in a clinical trial for the treatment of N. gonorrhoeae.

  12. In vitro and in vivo inhibition of Daphnia magna acetylcholinesterase by surfactant agents: possible implications for contamination biomonitoring.

    PubMed

    Guilhermino, L; Lacerda, M N; Nogueira, A J; Soares, A M

    2000-03-20

    This study was designed to investigate the effect of two surfactants, dodecyl benzyl sulfonate (DBS), sodium dodecyl sulfate (SDS), and of a domestic detergent (Y) on the AChE activity of the crustacean cladoceran Daphnia magna. All the chemicals significantly inhibit the activity of the enzyme, both in vitro and in vivo conditions. In vitro lowest observed effect concentration (LOEC) values ranged from 12.5 to 100 mg/l and correspondent IC50 (50% inhibition concentration) values ranged from 6.6 to 58.5 mg/l. In vivo LOEC values ranged from 2 to 11.9 mg/l, while EC50 (50% effect concentration) values ranged from 11.4 to 56.7 mg/l. AChE inhibition by environmental contaminants such as surfactants, detergents and metals may lead to false diagnostics and even wrong conclusions in biomonitoring studies based on the use of AChE as a specific biomarker for organophosphorous and carbamate compounds.

  13. Coherence between Cellular Responses and in Vitro Splicing Inhibition for the Anti-tumor Drug Pladienolide B and Its Analogs*

    PubMed Central

    Effenberger, Kerstin A.; Anderson, David D.; Bray, Walter M.; Prichard, Beth E.; Ma, Nianchun; Adams, Matthew S.; Ghosh, Arun K.; Jurica, Melissa S.

    2014-01-01

    Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible. PMID:24302718

  14. Comparison of in vitro and ex vivo thyroid hormone synthesis inhibition results and in vivo outcomes for a series of benzothiazoles

    EPA Science Inventory

    Assessing how in vitro data may be used to predict adverse effects in vivo is critical as efforts are advanced to incorporate in vitro assays into a risk assessment framework. Within the context of a thyroid hormone (TH) synthesis inhibition adverse outcome pathway (AOP), in vitr...

  15. Comparison of in vitro and ex vivo thyroid hormone synthesis inhibition results and in vivo outcomes for a series of benzothiazoles

    EPA Science Inventory

    Assessing how in vitro data may be used to predict adverse effects in vivo is critical as efforts are advanced to incorporate in vitro assays into a risk assessment framework. Within the context of a thyroid hormone (TH) synthesis inhibition adverse outcome pathway (AOP), in vitr...

  16. Silibinin and indocyanine green-loaded nanoparticles inhibit the growth and metastasis of mammalian breast cancer cells in vitro.

    PubMed

    Sun, Hui-Ping; Su, Jing-Han; Meng, Qing-Shuo; Yin, Qi; Zhang, Zhi-Wen; Yu, Hai-Jun; Zhang, Peng-Cheng; Wang, Si-Ling; Li, Ya-Ping

    2016-07-01

    To improve the therapeutic efficacy of cancer treatments, combinational therapies based on nanosized drug delivery system (NDDS) has been developed recently. In this study we designed a new NDDS loaded with an anti-metastatic drug silibinin and a photothermal agent indocyanine green (ICG), and investigated its effects on the growth and metastasis of breast cancer cells in vitro. Silibinin and ICG were self-assembled into PCL lipid nanoparticles (SIPNs). Their physical characteristics including the particle size, zeta potential, morphology and in vitro drug release were examined. 4T1 mammalian breast cancer cells were used to evaluate their cellular internalization, cytotoxicity, and their influences on wound healing, in vitro cell migration and invasion. SIPNs showed a well-defined spherical shape with averaged size of 126.3±0.4 nm and zeta potential of -10.3±0.2 mV. NIR laser irradiation substantially increased the in vitro release of silibinin from the SIPNs (58.3% at the first 8 h, and 97.8% for the total release). Furthermore, NIR laser irradiation markedly increased the uptake of SIPNs into 4T1 cells. Under the NIR laser irradiation, both SIPNs and IPNs (PCL lipid nanoparticles loaded with ICG alone) caused dose-dependent ablation of 4T1 cells. The wound healing, migration and invasion experiments showed that SIPNs exposed to NIR laser irradiation exhibited dramatic in vitro anti-metastasis effects. SIPNs show temperature-sensitive drug release following NIR laser irradiation, which can inhibit the growth and metastasis of breast cancer cells in vitro.

  17. Vasoactive Intestinal Peptide Inhibits Human Small-Cell Lung Cancer Proliferation in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Maruno, Kaname; Absood, Afaf; Said, Sami I.

    1998-11-01

    Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.

  18. Acetylcholinesterase inhibition and in vitro and in vivo antioxidant activities of Ganoderma lucidum grown on germinated brown rice.

    PubMed

    Hasnat, Abul; Pervin, Mehnaz; Lim, Beong Ou

    2013-06-07

    In this study, the acetylcholinesterase inhibition and in vitro and in vivo antioxidant activities of Ganoderma lucidum grown on germinated brown rice (GLBR) were evaluated. In antioxidant assays in vitro, GLBR was found to have strong metal chelating activity, DPPH, ABTS, hydroxyl and superoxide radical scavenging activity. Cell-based antioxidant methods were used, including lipid peroxidation on brain homogenate and AAPH-induced erythrocyte haemolysis. In antioxidant assays in vivo, mice were administered with GLBR and this significantly enhanced the activities of antioxidant enzymes in the mice sera, livers and brains. The amount of total phenolic and flavonoid compounds were 43.14 mg GAE/g and 13.36 mg CE/g dry mass, respectively. GLBR also exhibited acetylcholinesterase inhibitory activity. In addition, HPLC analyses of GLBR extract revealed the presence of different phenolic compounds. These findings demonstrate the remarkable potential of GLBR extract as valuable source of antioxidants which exhibit interesting acetylcholinesterase inhibitory activity.

  19. Inhibition of the entomopathogenic fungus Metarhizium anisopliae in vitro by the bed bug defensive secretions (E)-2-hexenal and (E)-2-octenal

    USDA-ARS?s Scientific Manuscript database

    The two major aldehydes (E)-2-hexenal and (E)-2-octenal emitted as defensive secretions by bed bugs Cimex lectularius L. (Hemiptera: Cimicidae), inhibit the in vitro growth of Metarhizium anisopliae (Metsch.) Sokorin (Hypocreales: Clavicipitaceae). These chemicals inhibit fungal growth by direct con...

  20. Monothiocarbamates Strongly Inhibit Carbonic Anhydrases in Vitro and Possess Intraocular Pressure Lowering Activity in an Animal Model of Glaucoma.

    PubMed

    Vullo, Daniela; Durante, Mariaconcetta; Di Leva, Francesco Saverio; Cosconati, Sandro; Masini, Emanuela; Scozzafava, Andrea; Novellino, Ettore; Supuran, Claudiu T; Carta, Fabrizio

    2016-06-23

    A series of monothiocarbamates (MTCs) were prepared from primary/secondary amines and COS as potential carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, using the dithiocarbamates, the xanthates, and the trithiocarbonates as lead compounds. The MTCs effectively inhibited the pharmacologically relevant human (h) hCAs isoforms I, II, IX, and XII in vitro and showed KIs spanning between the low and medium nanomolar range. By means of a computational study, the MTC moiety binding mode on the CAs was explained. Furthermore, a selection of MTCs were evaluated in a normotensive glaucoma rabbit model for their intraocular pressure (IOP) lowering effects and showed interesting activity.

  1. Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro

    PubMed Central

    1991-01-01

    Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony- stimulating factor-driven granulocyte colony formation in vitro. PMID:1719120

  2. Diazepam inhibits phagocytosis and killing exerted by polymorphonuclear cells and monocytes from healthy donors. In vitro studies.

    PubMed

    Covelli, V; Decandia, P; Altamura, M; Jirillo, E

    1989-01-01

    The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte phagocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10(-5) and 10(-6) M concentrations/ 4 x 10(6) phagocytes. 10(-7) M concentration was not effective in all the instances. These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.

  3. In vitro inhibition effect and structure-activity relationships of some saccharin derivatives on erythrocyte carbonic anhydrase I and II.

    PubMed

    Sonmez, Fatih; Bilen, Cigdem; Sumersan, Sinem; Gencer, Nahit; Isik, Semra; Arslan, Oktay; Kucukislamoglu, Mustafa

    2014-02-01

    In this study, in vitro inhibitory effects of some saccharin derivatives on purified carbonic anhydrase I and II were investigated using CO2 as a substrate. The results showed that all compounds inhibited the hCA I and hCA II enzyme activities. Among the compounds, 6-(p-tolylthiourenyl) saccharin (6m) was found to be the most active one for hCA I activity (IC50=13.67 μM) and 6-(m-methoxyphenylurenyl) saccharin (6b) was found to be the most active one for hCA II activity (IC50=6.54 μM). Structure-activity relationships (SARs) study showed that, generally, thiourea derivatives (6l--v) inhibited more hCA I and hCA II than urea derivatives (6a-k). All compounds (excluding 6c and 6r) have higher inhibitory activity on hCA II than on hCA I.

  4. Cirsium maritimum Makino Inhibits the Antigen/Immunoglobulin-E-Mediated Allergic Response In Vitro and In Vivo.

    PubMed

    Tanaka, Mamoru; Suzuki, Masanobu; Takei, Yuichiro; Okamoto, Takeaki; Watanabe, Hiroyuki

    2017-09-27

    We investigated whether Cirsium maritimum Makino can inhibit immunoglobulin-E-mediated allergic response in rat basophilic leukemia (RBL-2H3) cells and passive cutaneous anaphylaxis (PCA) in BALB/c mice. In vitro, the ethyl acetate extract of C. maritimum Makino (ECMM) significantly inhibited β-hexosaminidase release and decreased intracellular Ca(2+) levels in RBL-2H3 cells. Moreover, ECMM leaves more strongly suppressed the release of β-hexosaminidase than ECMM flowers. ECMM leaves also significantly suppressed the PCA reaction in the murine model. High-performance liquid chromatography and (1)H and (13)C nuclear magnetic resonance indicated that cirsimaritin, a flavonoid, was concentrated in active fractions of the extract. Our findings suggest that ECMM leaves have a potential regulatory effect on allergic reactions that may be mediated by mast cells. Furthermore, cirsimaritin may be the active anti-allergic component in C. maritimum Makino.

  5. Synthesis of novel substituted pyrimidine derivatives bearing a sulfamide group and their in vitro cancer growth inhibition activity.

    PubMed

    Lefebvre, Carole-Anne; Forcellini, Elsa; Boutin, Sophie; Côté, Marie-France; C-Gaudreault, René; Mathieu, Patrick; Lagüe, Patrick; Paquin, Jean-François

    2017-01-15

    The synthesis of two series of novel substituted pyrimidine derivatives bearing a sulfamide group have been described and their in vitro cancer growth inhibition activities have been evaluated against three human tumour cell lines (HT-29, M21, and MCF7). In general, growth inhibition activity has been enhanced by the introduction of a bulky substituent on the aromatic ring with the best compound having GI50<6μM for all the human tumour cell lines. The MCF7 selective compounds were evaluated on four additional human invasive breast ductal carcinoma cell lines (MDA-MB-231, MDA-MB-468, SKBR3, and T47D) and were selective against T47D cell line in all cases except one, suggesting a potential antiestrogen activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Hysterosalpingography contrast media and chromotubation dye inhibit peritoneal lymphocyte and macrophage function in vitro: a potential mechanism for fertility enhancement.

    PubMed

    Goodman, S B; Rein, M S; Hill, J A

    1993-05-01

    To determine the effects of hysterosalpingography (HSG) contrast media (CM) and chromotubation dye on peritoneal lymphocyte proliferation and macrophage phagocytosis in vitro. Peritoneal fluid (PF) lymphocytes and macrophages were isolated from 40 subfertile women undergoing diagnostic laparoscopy and 12 fertile women having laparoscopic tubal ligation. Dilutions of renografin, ethiodol, methylene blue, and indigo carmine were added to peritoneal lymphocyte and macrophage cultures. Tissue culture media alone served as control. Lymphocyte proliferation was assessed by hemocytometer counts and 3H-thymidine incorporation. Macrophage function was determined by phagocytosis of fluorescent microspheres. Peritoneal lymphocyte proliferation and macrophage phagocytosis were significantly inhibited by renografin, ethiodol, methylene blue, and indigo carmine in a dose-dependent manner. Inhibition of PF immune cell function by HSG CM and chromotubation dye may provide a potential mechanism for fertility enhancement after these diagnostic procedures.

  7. Monohaloacetic acid drinking water disinfection by-products inhibit follicle growth and steroidogenesis in mouse ovarian antral follicles in vitro

    PubMed Central

    Jeong, Clara H.; Gao, Liying; Dettro, Tyler; Wagner, Elizabeth D.; Ricke, William A.; Plewa, Michael J.; Flaws, Jodi A.

    2016-01-01

    Water disinfection greatly reduced the incidence of waterborne diseases, but the reaction between disinfectants and natural organic matter in water leads to the formation of drinking water disinfection by-products (DBPs). DBPs have been shown to be toxic, but their effects on the ovary are not well defined. This study tested the hypothesis that monohalogenated DBPs (chloroacetic acid, CAA; bromoacetic acid, BAA; iodoacetic acid, IAA) inhibit antral follicle growth and steroidogenesis in mouse ovarian follicles. Antral follicles were isolated and cultured with either vehicle or DBPs (0.25–1.00 mM of CAA; 2–15 µM of BAA or IAA) for 48 and 96 h. Follicle growth was measured every 24 h and the media were analyzed for estradiol levels at 96 h. Exposure to DBPs significantly inhibited antral follicle growth and reduced estradiol levels compared to controls. These data demonstrate that DBP exposure caused ovarian toxicity in vitro. PMID:27151372

  8. Monohaloacetic acid drinking water disinfection by-products inhibit follicle growth and steroidogenesis in mouse ovarian antral follicles in vitro.

    PubMed

    Jeong, Clara H; Gao, Liying; Dettro, Tyler; Wagner, Elizabeth D; Ricke, William A; Plewa, Michael J; Flaws, Jodi A

    2016-07-01

    Water disinfection greatly reduced the incidence of waterborne diseases, but the reaction between disinfectants and natural organic matter in water leads to the formation of drinking water disinfection by-products (DBPs). DBPs have been shown to be toxic, but their effects on the ovary are not well defined. This study tested the hypothesis that monohalogenated DBPs (chloroacetic acid, CAA; bromoacetic acid, BAA; iodoacetic acid, IAA) inhibit antral follicle growth and steroidogenesis in mouse ovarian follicles. Antral follicles were isolated and cultured with either vehicle or DBPs (0.25-1.00mM of CAA; 2-15μM of BAA or IAA) for 48 and 96h. Follicle growth was measured every 24h and the media were analyzed for estradiol levels at 96h. Exposure to DBPs significantly inhibited antral follicle growth and reduced estradiol levels compared to controls. These data demonstrate that DBP exposure caused ovarian toxicity in vitro.

  9. Cereal alkylresorcinols elevate gamma-tocopherol levels in rats and inhibit gamma-tocopherol metabolism in vitro.

    PubMed

    Ross, Alastair B; Chen, Yan; Frank, Jan; Swanson, Joy E; Parker, Robert S; Kozubek, Arkadiusz; Lundh, Torbjörn; Vessby, Bengt; Aman, Per; Kamal-Eldin, Afaf

    2004-03-01

    Alkylresorcinols (AR) are a class of amphiphilic phenolic lipids present in high amounts in wheat and rye bran. They have been reported to be both growth retarding and innocuous when fed to rats, and to have a broad range of bioactivities in vitro, suggested to be related to their ability to bind to proteins and modify membranes. This study was designed to test the effects of AR (purified from rye bran) on growth, tocopherol levels, and cholesterol levels in rats. Rats were fed 1 of 4 different levels of AR for 4 wk: 0 (control), 1, 2, and 4 g/kg diet. AR did not affect final body, liver, or lung weights. The AR diets increased the levels of gamma-tocopherol in liver and lungs (P < 0.05). To investigate whether AR could have increased gamma-tocopherol levels via inhibition of tocopherol-omega-hydroxylase, HepG2 cells were incubated with AR and the metabolism of gamma-tocopherol measured. AR significantly inhibited the conversion of gamma-tocopherol to its water-soluble hydroxychroman metabolite in vitro, indicating that AR may increase gamma-tocopherol levels via inhibition of tocopherol metabolism in vivo. The 4 g AR/kg diet decreased liver cholesterol (P < 0.001), but did not affect plasma lipids. AR were detected in the perirenal adipose tissue samples of rats fed AR, indicating that they can accumulate in the fatty tissues of rats. High levels of dietary AR moderately affect gamma-tocopherol, possibly via inhibition of tocopherol metabolism, and decrease liver cholesterol in rats.

  10. MARK2 Rescues Nogo-66-Induced Inhibition of Neurite Outgrowth via Regulating Microtubule-Associated Proteins in Neurons In Vitro.

    PubMed

    Zuo, Yu-Chao; Xiong, Nan-Xiang; Shen, Jian-Ying; Yu, Hua; Huang, Yi-Zhi; Zhao, Hong-Yang

    2016-11-01

    The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.

  11. Inhibition of the MurA enzyme in Fusobacterium nucleatum by potential inhibitors identified through computational and in vitro approaches.

    PubMed

    Kumar, Amit; Saranathan, Rajagopalan; Prashanth, K; Tiwary, Basant K; Krishna, Ramadas

    2017-03-30

    Fusobacterium nucleatum plays a key role in several diseases such as periodontitis, gingivitis, appendicitis, and inflammatory bowel disease (IBD). The development of antibiotic resistance by this bacterium demands novel therapeutic intervention. Our recent study has reported UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) as one of the potential target proteins in F. nucleatum. In this study, we proposed two novel MurA inhibitors through in silico screening and evaluated their mode of inhibition by in vitro experiments. It was found that MurA structural arrangement (inside-out α/β barrel) was stabilized by L/FXXXG(A) motif-based interactions. The protein was maintained in an open or substrate-free conformation due to repulsive forces between two parallelly arranged positively charged residues of domain I and II. In this conformation, we identified six best compounds that held key interactions with the substrate-binding pocket via a structure-based virtual screening of natural and chemical compound libraries. However, among these, only orientin and quercetin-3-O-d-glucuronide (Q3G) showed better interaction capability through consistent H-bond occupancy and lowest binding free energy during molecular dynamic simulations. In vitro inhibition studies evidenced the mixed and uncompetitive mode of inhibition by orientin and Q3G, respectively, with purified MurA protein. This explains the binding of orientin in both open and closed (substrate-bound) conformations of MurA, and Q3G binding in only closed conformation. Therefore, the Q3G binding mode was predicted on a MurA-substrate complex, which highlighted its constant H-bond with Cys118, a phosphoenolpyruvate (PEP) interacting residue. This suggests that Q3G may interrupt the PEP binding, thereby inhibiting the MurA activity. Thus, the current study discusses the structure of MurA and demonstrates the inhibitory action of two novel compounds.

  12. Solid lipid nanoparticles of cholesteryl butyrate inhibit the proliferation of cancer cells in vitro and in vivo models

    PubMed Central

    Minelli, R; Occhipinti, S; Gigliotti, C L; Barrera, G; Gasco, P; Conti, L; Chiocchetti, A; Zara, G P; Fantozzi, R; Giovarelli, M; Dianzani, U; Dianzani, C

    2013-01-01

    BACKGROUND AND PURPOSE Solid lipid nanoparticles containing cholesteryl butyrate (cholbut SLN) can be a delivery system for the anti-cancer drug butyrate. These nanoparticles inhibit adhesion of polymorphonuclear and tumour cells to endothelial cells and migration of tumour cells, suggesting that they may act as anti-inflammatory and anti-tumour agents. Here we have evaluated the effects of cholbut SLN on tumour cell growth using in vitro and in vivo models. EXPERIMENTAL APPROACH Cholbut SLNs were incubated with cultures of four tumour cell lines, and cell growth was analysed by assessing viability, clonogenic capacity and cell cycle. Effects on intracellular signalling was assessed by Western blot analysis of Akt expression. The in vivo anti-tumour activity was measured in two models of PC-3 cell xenografts in SCID/Beige mice. KEY RESULTS Cholbut SLN inhibited tumour cell line viability, clonogenic activity, Akt phosphorylation and cell cycle progression. In mice injected i.v. with PC3-Luc cells and treated with cholbut SLN, . in vivo optical imaging and histological analysis showed no metastases in the lungs of the treated mice. In another set of mice injected s.c. with PC-3 cells and treated with cholbut SLN when the tumour diameter reached 2 mm, analysis of the tumour dimensions showed that treatment with cholbut SLN substantially delayed tumour growth. CONCLUSION AND IMPLICATIONS Cholbut SLN were effective in inhibiting tumour growth in vitro and in vivo. These effects may involve, in part, inhibition of Akt phosphorylation, which adds another mechanism to the activity of this multipotent drug. PMID:23713413

  13. Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors.

    PubMed

    Strålberg, Fredrik; Kassem, Ali; Kasprzykowski, Franciszek; Abrahamson, Magnus; Grubb, Anders; Lindholm, Catharina; Lerner, Ulf H

    2017-05-01

    Inflammation-induced bone destruction is a major treatment target in many inflammatory skeletal diseases. The aim of this study was to investigate if the cysteine proteinase inhibitors cystatin C, fungal cysteine proteinase inhibitor (E-64), and N-benzyloxycarbonyl-arginyl-leucyl-valyl-glycyl-diazomethane acetate (Z-RLVG-CHN2) can inhibit LPS-induced osteoclast formation. Mouse bone marrow macrophages (BMMs) were isolated and primed with receptor activator of NF-κB ligand (RANKL) for 24 h, followed by stimulation with LPS, with and without inhibitors. Adult mice were injected locally with LPS and then treated with E-64 and osteoclast formation assessed by the number of cathepsin K(+) multinucleated cells. Cystatin C inhibited LPS-induced osteoclast formation time and concentration dependently (IC50 = 0.3 μM). The effect was associated with decreased mRNA and protein expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and of the osteoclastogenic transcription factors c-Fos and NFATc1. LPS-induced osteoclast formation on bone slices was also inhibited by cystatin C, resulting in decreased pit formation and release of bone matrix proteins. Similar data were obtained with E-64 and Z-RLVG-CHN2 Cystatin C was internalized in BMMs stimulated by LPS but not in unstimulated BMMs. Osteoclast formation induced by LPS was dependent on TNF-α, and the 3 inhibitors abolished LPS-induced TNF superfamily 2 (gene encoding TNF-α; Tnfsf2) mRNA expression without affecting Il1b, Il6, or oncostatin M (Osm) expression. Formation of osteoclasts in the skull bones after local LPS stimulation was inhibited by E-64. It is concluded that cysteine proteinase inhibitors effectively inhibit LPS-induced osteoclast formation in vivo and in vitro by inhibition of TNF-α expression. The targeting of cysteine proteinases might represent a novel treatment modality for prevention of inflammatory bone loss. © Society for Leukocyte Biology.

  14. Interleukin-1 Inhibits Putative Cholinergic Neurons in Vitro and REM Sleep when Microinjected into the Rat Laterodorsal Tegmental Nucleus

    PubMed Central

    Brambilla, Dario; Barajon, Isabella; Bianchi, Susanna; Opp, Mark R.; Imeri, Luca

    2010-01-01

    Study Objectives: REM sleep is suppressed during infection, an effect mimicked by the administration of cytokines such as interleukin-1 (IL-1). In spite of this observation, brain sites and neurochemical systems mediating IL-1-induced suppression of REM sleep have not been identified. Cholinergic neurons in the brainstem laterodorsal tegmental nucleus (LDT) are part of the neuronal circuitry responsible for REM sleep generation. Since IL-1 inhibits acetylcholine synthesis and release, the aim of this study was to test the two different, but related hypotheses. We hypothesized that IL-1 inhibits LDT cholinergic neurons, and that, as a result of this inhibition, IL-1 suppresses REM sleep. Design, Measurement, and Results: To test these hypotheses, the electrophysiological activity of putative cholinergic LDT neurons was recorded in a rat brainstem slice preparation. Interleukin-1 significantly inhibited the firing rate of 76% of recorded putative cholinergic LDT neurons and reduced the amplitude of glutamatergic evoked potentials in 60% of recorded neurons. When IL-1 (1 ng) was microinjected into the LDT of freely behaving rats, REM sleep was reduced by about 50% (from 12.7% ± 1.5% of recording time [after vehicle] to 6.1% ± 1.4% following IL-1 administration) during post-injection hours 3-4. Conclusions: Results of this study support the hypothesis that IL-1 can suppress REM sleep by acting at the level of the LDT nucleus. Furthermore this effect may result from the inhibition of evoked glutamatergic responses and of spontaneous firing of putative cholinergic LDT neurons. Citation: Brambilla D; Barajon I; Bianchi S; Opp MR; Imeri L. Interleukin-1 inhibits putative cholinergic neurons in vitro and REM sleep when microinjected into the rat laterodorsal tegmental nucleus. SLEEP 2010;33(7):919-929. PMID:20614852

  15. Herbal extracts of Tribulus terrestris and Bergenia ligulata inhibit growth of calcium oxalate monohydrate crystals in vitro

    NASA Astrophysics Data System (ADS)

    Joshi, V. S.; Parekh, B. B.; Joshi, M. J.; Vaidya, A. B.

    2005-02-01

    A large number of people in this world are suffering from urinary stone problem. Calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) containing stones (calculi) are commonly found. In the present study, COM crystals were grown by a double diffusion gel growth technique using U-tubes. The gel was prepared from hydrated sodium metasilicate solution. The gel framework acts like a three-dimensional crucible in which the crystal nuclei are delicately held in the position of their formation, and nutrients are supplied for the growth. This technique can be utilized as a simplified screening static model to study the growth, inhibition and dissolution of urinary stones in vitro. The action of putative litholytic medicinal plants, Tribulus terrestris Linn. ( T.t) and Bergenia ligulata Linn. ( B.l.), has been studied in the growth of COM crystals. Tribulus terrestris and Bergenia ligulata are commonly used as herbal medicines for urinary calculi in India. To verify the inhibitive effect, aqueous extracts of Tribulus terrestris and Bergenia ligulata were added along with the supernatant solutions. The growth was measured and compared, with and without the aqueous extracts. Inhibition of COM crystal growth was observed in the herbal extracts. Maximum inhibition was observed in Bergenia ligulata followed by Tribulus terrestris. The results are discussed.

  16. Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro

    SciTech Connect

    Herkert, N.M.; Schulz, S.; Wille, T.; Thiermann, H.; Hatz, R.A.; Worek, F.

    2011-05-15

    Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by decarbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.

  17. Patrinia scabiosaefolia inhibits the proliferation of colorectal cancer in vitro and in vivo via G1/S cell cycle arrest.

    PubMed

    Zhang, Mingyue; Sun, Guodong; Shen, Aling; Liu, Liya; Ding, Jingzhen; Peng, Jun

    2015-02-01

    Patrinia scabiosaefolia (PS) has long been used as an important component in traditional Chinese medicine formulas to treat gastrointestinal malignancies including colorectal cancer (CRC). We recently reported that PS can inhibit CRC growth through induction of apoptosis and inhibition of tumor angiogenesis. To further elucidate the mode of action of PS, in the present study, we used a CRC mouse xenograft model and a human CRC cell line HT-29 to evaluate the effect of the ethanol extract of PS (EEPS) on cancer cell proliferation and investigated the underlying molecular mechanisms. We found that EEPS inhibited CRC growth both in vivo and in vitro, which was associated with the inhibitory effects of EEPS on cancer cell proliferation. In addition, EEPS treatment significantly blocked G1 to S phase cell cycle progression in HT-29 cells. Moreover, EEPS treatment decreased the expression of pro-proliferative CyclinD1 and CDK4, at both the mRNA and protein levels. Thus, inhibition of cell proliferation via G1/S cell cycle arrest might be a potential mechanism whereby PS effectively treats cancers.

  18. Metabolic interactions between acetaminophen (paracetamol) and two flavonoids, luteolin and quercetin, through in-vitro inhibition studies.

    PubMed

    Cao, Lei; Kwara, Awewura; Greenblatt, David J

    2017-09-05

    Excessive exposure to acetaminophen (APAP, paracetamol) can cause liver injury through formation of a reactive metabolite that depletes hepatic glutathione and causes hepatocellular oxidative stress and damage. Generation of this metabolite is mediated by Cytochrome-P450 (CYP) isoforms, mainly CYP2E1. A number of naturally occurring flavonoids can mitigate APAP-induced hepatotoxicity in experimental animal models. Our objective was to determine the mechanism of these protective effects and to evaluate possible human applicability. Two flavonoids, luteolin and quercetin, were evaluated as potential inhibitors of eight human CYP isoforms, of six UDP-glucuronosyltransferase (UGT) isoforms and of APAP glucuronidation and sulfation. The experimental model was based on in-vitro metabolism by human liver microsomes, using isoform-specific substrates. Luteolin and quercetin inhibited human CYP isoforms to varying degrees, with greatest potency towards CYP1A2 and CYP2C8. However, 50% inhibitory concentrations (IC50 values) were generally in the micromolar range. UGT isoforms were minimally inhibited. Both luteolin and quercetin inhibited APAP sulfation but not glucuronidation. Inhibition of human CYP activity by luteolin and quercetin occurred with IC50 values exceeding customary in-vivo human exposure with tolerable supplemental doses of these compounds. The findings indicate that luteolin and quercetin are not likely to be of clinical value for preventing or treating APAP-induced hepatotoxicity. © 2017 Royal Pharmaceutical Society.

  19. Pharmacological studies of FUT-175, nafamstat mesilate. I. Inhibition of protease activity in in vitro and in vivo experiments.

    PubMed

    Aoyama, T; Ino, Y; Ozeki, M; Oda, M; Sato, T; Koshiyama, Y; Suzuki, S; Fujita, M

    1984-07-01

    FUT-175, 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate (nafamstat mesilate), a novel synthetic protease-inhibiting agent, was studied to determine its in vitro effects against various proteases and other enzymes, as well as to determine its in vivo protease inhibitory effects. FUT-175 was found to inhibit, in an intense, specific and reversible way, the enzyme activities of trypsin, C1r, C1s, thrombin, kallikrein and plasmin with IC50 values of the order of 10(-6)-10(-8) M. FUT-175 also inhibited complement-mediated hemolysis, including both classical and alternative pathways, sites of inhibition being on C1r and C1s as evidenced by the intermediate-cell technique. In animal model reactions in which the complement system is known to be involved as pathogenetic factors, e.g., Forssman shock, Forssman cutaneous vasculitis, zymosan-induced paw edema, endotoxin shock and local Shwartzman reaction, FUT-175 was highly effective in that, for example, intravenous dosing at 3 mg/kg could completely protect guinea pigs from the lethal Forssman shock. FUT-175 was also found to be effective in trypsin-induced shock in mice, in lethality due to thrombin-thrombosis in mice and in kinin formation in the inflammatory process in rats.

  20. A novel in vitro approach for simultaneous evaluation of CYP3A4 inhibition and kinetic aqueous solubility.

    PubMed

    Pérez, José; Díaz, Caridad; Asensio, Francisco; Palafox, Alexandra; Genilloud, Olga; Vicente, Francisca

    2015-02-01

    In the early stages of the drug discovery process, evaluation of the drug metabolism and physicochemical properties of new chemical entities is crucial to prioritize those candidates displaying a better profile for further development. In terms of metabolism, drug-drug interactions mediated through CYP450 inhibition are a significant safety concern, and therefore the effect of new candidate drugs on CYP450 activity should be screened early. In the initial stages of drug discovery, when physicochemical properties such as aqueous solubility have not been optimized yet, there might be a large number of candidate compounds showing artificially low CYP450 inhibition, and consequently potential drug-drug interaction toxicity might be overlooked. In this work, we present a novel in vitro approach for simultaneous evaluation of CYP3A4 inhibition potential and kinetic aqueous solubility (NIVA-CYPI-KS). This new methodology is based on fluorogenic CYP450 activities and turbidimetric measurements for compound solubility, and it provides a significant improvement in the use of resources and a better understanding of CYP450 inhibition data.

  1. Salinomycin inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cell in vitro and suppresses tumor growth in vivo.

    PubMed

    Wu, Danxin; Zhang, Yu; Huang, Jie; Fan, Zirong; Shi, Fengrong; Wang, Senming

    2014-01-10

    Salinomycin (Sal) is a polyether ionophore antibiotic that has recently been shown to induce cell death in various human cancer cells. However, whether salinomycin plays a functional role in nasopharyngeal carcinoma (NPC) has not been determined to date. The present study investigated the chemotherapeutic efficacy of salinomycin and its molecular mechanisms of action in NPC cells. Salinomycin efficiently inhibited proliferation and invasion of 3 NPC cell lines (CNE-1, CNE-2, and CNE-2/DDP) and activated a extensive apoptotic process that is accompanied by activation of caspase-3 and caspase-9, and decreased mitochondrial membrane potential. Meanwhile, the protein expression level of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and β-catenin was down-regulated, which showed that the Wnt/β-catenin signaling was involved in salinomycin-induced apoptosis of NPC cells. In a nude mouse NPC xenograft model, the anti-tumor effect of salinomycin was associated with the downregulation of β-catenin expression. The present study demonstrated that salinomycin can effectively inhibit proliferation and invasion, and induce apoptosis of NPC cells in vitro and inhibit tumor growth in vivo, probably via the inhibition of Wnt/β-catenin signaling, suggesting salinomycin as a potential candidate for the chemotherapy of NPC.

  2. In vitro evaluation of bis-pyridinium oximes bearing methoxy alkane linker as reactivators of sarin inhibited human acetylcholinesterase.

    PubMed

    Acharya, Jyotiranjan; Dubey, Devendra Kumar; Raza, Syed Kalbey

    2010-09-01

    A series of bis-pyridinium oximes connected by methoxy alkane linkers were synthesized and their in vitro reactivation efficacy was evaluated against sarin-inhibited human AChE, and data were compared with 2-PAM and obidoxime. Among the synthesized compounds, 1,2-dimethoxy ethylene bis-[4,4'-(hydroxyiminomethyl) pyridinium] dichloride (4P-2) and 1,2-dimethoxy ethylene bis-[3,3'-(hydroxyiminomethyl) pyridinium] dichloride (3P-2) were found to be the most potent reactivators of human AChE inhibited by nerve agent sarin. The oximes 4P-2 and 3P-2 exhibited 41% and 36% regeneration of sarin-inhibited AChE, respectively, whereas 2-PAM showed 32% regeneration. The higher reactivation efficacy of the oximes was attributed to their acid dissociation constants (pK(a)). The pK(a) values of all the oximes were determined by UV-vis spectrophotometric method and correlated with their observed reactivation potential. Overall, the study reveals that the oxime 4P-2 may have therapeutic potential in the reactivation of human AChE inhibited by sarin.

  3. In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives

    SciTech Connect

    Herraez, Elisa; Macias, Rocio I.R.; Vazquez-Tato, Jose; Vicens, Marta; Monte, Maria J.; Marin, Jose J.G.

    2009-08-15

    Hepatocyte uptake of phalloidin is carried out mainly by OATP1B1. We have used this compound as a prototypic substrate and assayed the ability to inhibit OATP-mediated phalloidin transport of four bile acid derivatives (BALU-1, BALU-2, BALU-3 and BALU-4) that showed positive results in preliminary screening. Using Xenopus laevis oocytes for heterologous expression of transporters, BALUs were found to inhibit taurocholic acid (TCA) transport by OATP1B1 (but not OATP1B3) as well as by rat Oatp1a1, Oatp1a4 and Oatp1b2. The study of their ability to inhibit sodium-dependent bile acid transporters revealed that the four BALUs induced an inhibition of rat Asbt-mediated TCA transport, which was similar to TCA-induced self-inhibition. Regarding human NTCP and rat Ntcp, BALU-1 differs from the other three BALUS in its lack of effect on TCA transport by these proteins. Using HPLC-MS/MS and CHO cells stably expressing OATP1B1 the ability of BALU-1 to inhibit the uptake of phalloidin itself by this transporter was confirmed. Kinetic analysis using X. laevis oocytes revealed that BALU-1-induced inhibition of OATP1B1 was mainly due to a competitive mechanism (Ki = 8 {mu}M). In conclusion, BALU-1 may be useful as a pharmacological tool to inhibit the uptake of compounds mainly taken up by OATP1B1 presumably without impairing bile acid uptake by the major carrier accounting for this process, i.e., NTCP.

  4. Nicotine, cotinine, and anabasine inhibit aromatase in human trophoblast in vitro.

    PubMed Central

    Barbieri, R L; Gochberg, J; Ryan, K J

    1986-01-01

    Epidemiologic studies suggest that women who smoke have lower endogenous estrogen than nonsmokers. To explore the possible link between cigarette smoking and decreased endogenous estrogens, we have examined the effects of constituents of tobacco on estrogen production in human choriocarcinoma cells and term placental microsomes. In choriocarcinoma cell cultures, nicotine, cotinine (a major metabolite of nicotine), and anabasine (a minor component of cigarette tobacco) all inhibited androstenedione conversion to estrogen in a dose-dependent fashion. Removal of nicotine, cotinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of aromatase. In the choriocarcinoma cell cultures, a supraphysiologic concentration of androstenedione (73 microM) in the culture medium blocked the inhibition of aromatase caused by nicotine, cotinine, and anabasine. In preparations of term placental microsomes, nicotine, cotinine, and anabasine inhibited the conversion of testosterone to estrogen. Kinetic analysis demonstrated the inhibition to be competitive with respect to the substrate. These findings suggest that some nicotinic alkaloids directly inhibit aromatase. This mechanism may explain, in part, the decreased estrogen observed in women who smoke. PMID:3711333

  5. Malathion, carbofuran and paraquat inhibit Bungarus sindanus (krait) venom acetylcholinesterase and human serum butyrylcholinesterase in vitro.

    PubMed

    Ahmed, Mushtaq; Rocha, João Batista T; Mazzanti, Cinthia M; Morsch, André L B; Cargnelutti, Denise; Corrêa, Maísa; Loro, Vânia; Morsch, Vera Maria; Schetinger, Maria R C

    2007-05-01

    Carbofuran and malathion, well known pesticides, and paraquat, a world widely used herbicide, were tested on acetylcholinesterase (AChE) from Bungarus sindanus venom and butyrylcholinesterase (BChE) from human serum. The calculated IC(50 )values for inhibition of venom enzyme by malathion, carbofuran and paraquat were 2.5, 0.14, and 0.16 microM, respectively. The values for inhibition of serum butyrylcholinesterase (BChE) were 3.5, 0.09 and 0.18 microM, respectively. Analysis of kinetic data indicated that the inhibition caused by malathion, carbofuran and paraquat was mixed for venom AChE. For BChE from human serum, the inhibition caused by malathion and paraquat was mixed and for carbofuran it was uncompetitive. The present results suggest a commercial paraquat preparation (a popular herbicide) inhibits cholinesterases with similar or higher potency than classical pesticide inhibitors. Furthermore, this inhibition was observed both in human serum and snake venom, a newly studied source of AChE.

  6. Clitocine targets Mcl-1 to induce drug-resistant human cancer cell apoptosis in vitro and tumor growth inhibition in vivo.

    PubMed

    Sun, Jian-Guo; Li, Hua; Li, Xia; Zeng, Xueli; Wu, Ping; Fung, Kwok-Pui; Liu, Fei-Yan

    2014-05-01

    Drug resistance is a major reason for therapy failure in cancer. Clitocine is a natural amino nucleoside isolated from mushroom and has been shown to inhibit cancer cell proliferation in vitro. In this study, we observed that clitocine can effectively induce drug-resistant human cancer cell apoptosis in vitro and inhibit tumor xenograft growth in vivo. Clitocine treatment inhibited drug-resistant human cancer cell growth in vitro in a dose- and time-dependent manner. Biochemical analysis revealed that clitocine-induced tumor growth inhibition is associated with activation of caspases 3, 8 and 9, PARP cleavage, cytochrome c release and Bax, Bak activation, suggesting that clitocine inhibits drug-resistant cancer cell growth through induction of apoptosis. Analysis of apoptosis regulatory genes indicated that Mcl-1 level was dramatically decreased after clitocine treatment. Over-expression of Mcl-1 reversed the activation of Bax and attenuated clitocine-induced apoptosis, suggesting that clitocine-induced apoptosis was at least partially by inducing Mcl-1 degradation to release Bax and Bak. Consistent with induction of apoptosis in vitro, clitocine significantly suppressed the drug-resistant hepatocellular carcinoma xenograft growth in vivo by inducing apoptosis as well as inhibiting cell proliferation. Taken together, our data demonstrated that clitocine is a potent Mcl-1 inhibitor that can effectively induce apoptosis to suppress drug-resistant human cancer cell growth both in vitro and in vivo, and thus holds great promise for further development as potentially a novel therapeutic agent to overcome drug resistance in cancer therapy.

  7. Integrated in silico-in vitro strategy for addressing cytochrome P450 3A4 time-dependent inhibition.

    PubMed

    Zientek, Michael; Stoner, Chad; Ayscue, Robyn; Klug-McLeod, Jacquelyn; Jiang, Ying; West, Michael; Collins, Claire; Ekins, Sean

    2010-03-15

    Throughout the past decade, the expectations from the regulatory agencies for safety, drug-drug interactions (DDIs), pharmacokinetic, and disposition characterization of new chemical entities (NCEs) by pharmaceutical companies seeking registration have increased. DDIs are frequently assessed using in silico, in vitro, and in vivo methodologies. However, a key gap in this screening paradigm is a full structural understanding of time-dependent inhibition (TDI) on the cytochrome P450 systems, particularly P450 3A4. To address this, a number of high-throughput in vitro assays have been developed. This work describes an automated assay for TDI using two concentrations at two time points (2 + 2 assay). Data generated with this assay for over 2000 compounds from multiple therapeutic programs were used to generate in silico Bayesian classification models of P450 3A4-mediated TDI. These in silico models were validated using several external test sets and multiple random group testing (receiver operator curve value >0.847). We identified a number of substructures that were likely to elicit TDI, the majority containing indazole rings. These in vitro and in silico approaches have been implemented as a part of the Pfizer screening paradigm. The Bayesian models are available on the intranet to guide synthetic strategy, predict whether a NCE is likely to cause a TDI via P450 3A4, filter for in vitro testing, and identify substructures important for TDI as well as those that do not cause TDI. This represents an integrated in silico-in vitro strategy for addressing P450 3A4 TDI and improving the efficiency of screening.

  8. Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

    SciTech Connect

    Burroughs, S.F.; Johnson, G.J. )

    1990-04-01

    beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of (14C)-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; but no irreversible inhibition of alpha 2 adrenergic receptors, measured with (3H)-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist (3H)-U46619 and antagonist (3H)-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ((Ca2+)i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in (Ca2+)i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane.

  9. Curcumin/turmeric solubilized in sodium hydroxide inhibits HNE protein modification--an in vitro study.

    PubMed

    Kurien, Biji T; Scofield, R Hal

    2007-03-21

    Free radical mediated lipid peroxidation has been implicated in multiple diseases. A major oxidation by-product of this deleterious process is 4-hydroxy-2-nonenal (HNE). HNE is cytotoxic, mutagenic and genotoxic and is involved in disease pathogenesis. Curcumin, a non-steroidal anti-inflammatory agent (occurring as the yellow pigment found in the rhizomes of the perennial herb Curcuma longa known as turmeric), has emerged as the newest "nutraceutical" agent that has been shown to be efficacious against colon cancer and other disorders, including correcting cystic fibrosis defects. Since curcumin has been reported to have anti-oxidant properties we hypothesized that it will inhibit HNE-modification of a protein substrate. Using an ELISA that employed HNE-modification of solid phase antigen following immobilization, we found that the curcumin solubilized in dilute alkali (5mM sodium hydroxide, pH 11) inhibited HNE-protein modification by 65%. Turmeric also inhibited HNE-protein modification similarly (65%) but at a much lower alkali level (130muM sodium hydroxide, pH 7.6). Alkali by itself (5mM sodium hydroxide, pH 11) was found to enhance HNE modification by as much as 267%. Curcumin/turmeric has to inhibit this alkali enhanced HNE-modification prior to inhibiting the normal HNE protein modification induced by HNE. Thus, inhibition of HNE-modification could be a mechanism by which curcumin exerts its antioxidant effects. The pH at which the inhibition of HNE modification of substrate was observed was close to the physiological pH, making this formulation of curcumin potentially useful practically.

  10. Yeast α-glucosidase inhibition by isoflavones from plants of Leguminosae as an in vitro alternative to acarbose.

    PubMed

    Choi, Chun Whan; Choi, Yeon Hee; Cha, Mi-Ran; Yoo, Dae Seok; Kim, Young Sup; Yon, Gyu Hwan; Hong, Kyung Sik; Kim, Young Ho; Ryu, Shi Yong

    2010-09-22

    In the course of searching for new classes of α-glucosidase inhibitors originated from natural resources, 11 kinds of isoflavones, i.e., medicarpin (1), formononetin (2), mucronulatol (3), (3R)-calussequinone (5), (3R)-5'-methoxyvestitol (6), tectorigenin (7), biochanin A (8), tuberosin (9), calycosin (10), daidzein (11), and genistein (12), as well as a flavone, liquritigenin (4), were isolated as active principles responsible for the yeast α-glucosidase inhibitory activity from two leguminous plant extracts, i.e., the heartwood extract of Dalbergia odorifera and the roots extract of Pueraria thunbergiana. Each components (1-12) demonstrated a significantly potent inhibition on yeast α-glucosidase in a dose dependent manner when the p-nitrophenyl-α-D-glucopyranoside was used as a substrate in vitro. The concentration required for 50% enzyme inhibition (IC50) were calculated as 2.93 mM (1), 0.51 mM (2), 3.52 mM (7) 0.35 mM (8), 3.52 mM (9), 0.85 mM (11), and 0.15 mM (12) when that of reference drug acarbose was evaluated as 9.11 mM, in vitro. However, isoflavone glycosides, i.e., puerarin (13), daidzin (14), formononetin-7-O-β-glucopyranoside (15), and genistin (16), exhibited a relatively poor inhibitory activity on yeast α-glucosidase as compared with the corresponding isoflavone (2, 11, 12), respectively.

  11. Broad HIV-1 inhibition in vitro by vaccine-elicited CD8+ T cells in African adults

    PubMed Central

    Mutua, Gaudensia; Farah, Bashir; Langat, Robert; Indangasi, Jackton; Ogola, Simon; Onsembe, Brian; Kopycinski, Jakub T; Hayes, Peter; Borthwick, Nicola J; Ashraf, Ambreen; Dally, Len; Barin, Burc; Tillander, Annika; Gilmour, Jill; De Bont, Jan; Crook, Alison; Hannaman, Drew; Cox, Josephine H; Anzala, Omu; Fast, Patricia E; Reilly, Marie; Chinyenze, Kundai; Jaoko, Walter; Hanke, Tomáš; HIV-CORE 004 study group, the

    2016-01-01

    We are developing a pan-clade HIV-1 T-cell vaccine HIVconsv, which could complement Env vaccines for prophylaxis and be a key to HIV cure. Our strategy focuses vaccine-elicited effector T-cells on functionally and structurally conserved regions (not full-length proteins and not only epitopes) of the HIV-1 proteome, which are common to most global variants and which, if mutated, cause a replicative fitness loss. Our first clinical trial in low risk HIV-1-negative adults in Oxford demonstrated the principle that naturally mostly subdominant epitopes, when taken out of the context of full-length proteins/virus and delivered by potent regimens involving combinations of simian adenovirus and poxvirus modified vaccinia virus Ankara, can induce robust CD8+ T cells of broad specificities and functions capable of inhibiting in vitro HIV-1 replication. Here and for the first time, we tested this strategy in low risk HIV-1-negative adults in Africa. We showed that the vaccines were well tolerated and induced high frequencies of broadly HIVconsv-specific plurifunctional T cells, which inhibited in vitro viruses from four major clades A, B, C, and D. Because sub-Saharan Africa is globally the region most affected by HIV-1/AIDS, trial HIV-CORE 004 represents an important stage in the path toward efficacy evaluation of this highly rational and promising vaccine strategy. PMID:27617268

  12. Protein related to DAN and cerberus (PRDC) inhibits osteoblastic differentiation and its suppression promotes osteogenesis in vitro.

    PubMed

    Ideno, Hisashi; Takanabe, Rieko; Shimada, Akemi; Imaizumi, Kazuhiko; Araki, Ryoko; Abe, Masumi; Nifuji, Akira

    2009-02-01

    Protein related to DAN and cerberus (PRDC) is a secreted protein characterized by a cysteine knot structure, which binds bone morphogenetic proteins (BMPs) and thereby inhibits their binding to BMP receptors. As an extracellular BMP antagonist, PRDC may play critical roles in osteogenesis; however, its expression and function in osteoblastic differentiation have not been determined. Here, we investigated whether PRDC is expressed in osteoblasts and whether it regulates osteogenesis in vitro. PRDC mRNA was found to be expressed in the pre-osteoblasts of embryonic day 18.5 (E18.5) mouse calvariae. PRDC mRNA expression was elevated by treatment with BMP-2 in osteoblastic cells isolated from E18.5 calvariae (pOB cells). Forced expression of PRDC using adenovirus did not affect cell numbers, whereas it suppressed exogenous BMP activity and endogenous levels of phosphorylated Smad1/5/8 protein. Furthermore, PRDC inhibited the expression of bone marker genes and bone-like mineralized matrix deposition in pOB cells. In contrast, the reduction of PRDC expression by siRNA elevated alkaline phosphatase activity, increased endogenous levels of phosphorylated Smad1/5/8 protein, and promoted bone-like mineralized matrix deposition in pOB cells. These results suggest that PRDC expression in osteoblasts suppresses differentiation and that reduction of PRDC expression promotes osteogenesis in vitro. PRDC is accordingly identified as a potential novel therapeutic target for the regulation of bone formation.

  13. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane.

    PubMed

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W

    2002-05-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay.

  14. Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment.

    PubMed

    Matusica, Dusan; Alfonsi, Fabienne; Turner, Bradley J; Butler, Tim J; Shepheard, Stephanie R; Rogers, Mary-Louise; Skeldal, Sune; Underwood, Clare K; Mangelsdorf, Marie; Coulson, Elizabeth J

    2016-02-01

    The p75 neurotrophin receptor (p75(NTR); also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75(NTR)-derived trophic cell-permeable peptide, c29, to inhibit p75(NTR)-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1(G93A) mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75(NTR) cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75(NTR) in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75(NTR) cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1(G93A) mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo. © 2016. Published by The Company of Biologists Ltd.

  15. Inhibition of EGFR-AKT Axis Results in the Suppression of Ovarian Tumors In Vitro and in Preclinical Mouse Model

    PubMed Central

    Gupta, Parul; Srivastava, Sanjay K.

    2012-01-01

    Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 µmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer. PMID:22952709

  16. An unusual polyanion from Physarum polycephalum that inhibits homologous DNA polymerase. alpha. in vitro

    SciTech Connect

    Fischer, H.; Erdmann, S.; Holler, E. )

    1989-06-13

    From extracts of microplasmodia of Physarum polycephalum and their culture medium, an unusual substance was isolated which inhibited homologous DNA polymerase {alpha} of this slime mold but not {beta}-like DNA polymerase and not heterologous DNA polymerases. Analysis, especially NMR spectroscopy, revealed the major component to be an anionic polyester of L-malic acid and the inhibition to be due to poly(L-malate) in binding reversibly to DNA polymerase {alpha}. The mode of inhibition is competitive with substrate DNA and follows an inhibition constant K{sub i} = 10 ng/mL. Inhibition is reversed in the presence of spermine, spermidine, poly(ethylene imine), and calf thymus histone H1. According to its ester nature, the inhibitor is slightly labile at neutral and instable at acid and alkaline conditions. Its largest size corresponds to a molecular mass of 40-50 kDa, but the bulk of the material after purification has lower molecular masses. The inhibitory activity depends on the polymer size and has a minimal size requirement.

  17. Inhibition of human breast and colorectal cancer cells by Viburnum foetens L. extracts in vitro

    PubMed Central

    Waheed, Abdul; Bibi, Yamin; Nisa, Sobia; Chaudhary, Fayyaz M; Sahreen, Sumaira; Zia, Muhammad

    2013-01-01

    Objective To investigate efficacy of Viburnum foetens (V. foetens) extracts against different cancer lines. Methods The crude extract and fractions of V. foetens are evaluated against MDA MB-468 and Caco-2 cancer cell lines by using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide) assays. These extracts are also tested against breast carcinoma and human colon adenocarcinoma through NRU (neutral red uptake) assay. Results The crude extract inhibited the cancerous cell growth in a dose dependent manner. From the MTT assay it is obvious that the ethylacetate fraction significantly inhibited the growth of Caco-2 (93.44%) cell. Similarly, the methanol and ethylacetate fractions shows 99% and 96% inhibition of MCF-7 and Caco-2 cell lines by NRU assay. Furthermore, the ethylacetate fraction also exhibited momentous inhibition of MDA MB-468 cells in both assays. Other fractions i.e. chloroform, hexane also inhibited cancer cell proliferation at a significant level. Natural products exhibited significant activity against multiple cancerous cells. Conclusions In this framework, we can speculate that the present study will be helpful in the identification and isolation of novel anticancer drug compounds from the crude extract (i.e., methanol and ethyl acetate fractions) of V. foetens.

  18. Heparin fragments inhibit human vascular smooth muscle cell proliferation in vitro

    SciTech Connect

    Selden, S.C.; Johnson, W.V.; Maciag, T.

    1986-03-01

    The authors have examined the effect of heparin on human abdominal aortic smooth muscle cell growth. Cell proliferation was inhibited by more than 90% at a concentration of 20 ..mu..g/ml in a 12 day growth assay using heparin from Sigma, Upjohn or Calbiochem. Additionally, 200 ..mu..g/ml Upjohn heparin inhibits /sup 3/H-thymidine incorporation by 50% in short term assays using serum or purified platelet-derived growth factor (25-100ng/ml) to initiate the cell cycle. Homogeneous size classes of heparin fragments were prepared by nitrous acid cleavage and BioGel P-10 filtration chromatography. Deca-, octa-, hexa-, tetra-, and di-saccharides inhibited proliferation by 90% at concentrations of 280, 320, 260, 180 and 100 ..mu..g/ml, respectively, in a 12 day growth assay. These data confirm the work of Castellot et.al. and extend the range of inhibitory fragments down to the tetra- and di-saccharide size. These data suggest, therefore, that di-saccharide subunit of heparin is sufficient to inhibit vascular smooth muscle cell proliferation. The authors are now examining the role of the anhydromannose moiety on the reducing end of the nitrous acid generated fragments as a possible mediator of heparin-induced inhibition of vascular smooth muscle cell proliferation.

  19. Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro.

    PubMed

    Töyräs, Anu; Ollikainen, Jouko; Taskinen, Markku; Mönkkönen, Jukka

    2003-07-01

    Bisphosphonates are antiresorptive drugs used for the treatment of metabolic bone diseases. They can be divided into two different pharmacological classes: nitrogen-containing and non-nitrogen-containing bisphosphonates. Non-nitrogen-containing bisphosphonates, like clodronate, are metabolised to a toxic ATP-analogue preventing osteoclast mediated bone resorption. Nitrogen-containing bisphosphonates, including alendronate, prevent osteoclast function by inhibiting the mevalonate pathway. Clodronate is known to have anti-inflammatory properties while alendronate induces cytokine secretion from lipopolysaccharide- (LPS) induced macrophages. This study investigates whether the cytotoxicity and cytokine production induced by alendronate and LPS could be counteracted by clodronate or products of mevalonate pathway: oxidized low density lipoprotein (ox-LDL), farnesol and geranylgeraniol. Treatment with alendronate increased LPS-induced secretion of IL-1beta, IL-6 and TNF-alpha from RAW 264 macrophages 2.4-, 1.4- and 1.8-fold, respectively. This treatment was cytotoxic for macrophages as indicated by lowered cell viability. Clodronate and ox-LDL both counteracted the cytokine secretion and cytotoxicity of alendronate. Farnesol and geranylgeraniol did neither reverse the cytokine secretion nor reduce the cytotoxicity of alendronate. Clodronate and ox-LDL were able to counteract the effects of alendronate on macrophages in vitro, probably by their known ability to inhibit DNA binding activity of transcription factors, nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1). These findings suggest that inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronate-induced apoptosis and prevention of osteoclast function.

  20. Interleukin 2 inhibits in vitro growth of human T cell lines carrying retrovirus.

    PubMed

    Sugamura, K; Nakai, S; Fujii, M; Hinuma, Y

    1985-05-01

    Four human T cell lines, TL-Mor, TL-Su, TL-TerI, and TL-OmI, carrying human T cell leukemia virus (HTLV), were established previously. TL-Mor, TL-Su, and TL-TerI were derived from interleukin 2 (IL-2)-dependent parental cell lines cloned from peripheral blood leukocytes (PBL) of three healthy HTLV carriers, while TL-OmI was directly established from PBL of a patient with adult T cell leukemia. These four TL cell lines grow autonomously without IL-2. When they were cultured in the presence of IL-2, their growth was inhibited after a few days. This growth inhibition depended on the dose of IL-2, and the effective dose significantly promoted growth of their parental IL-2-dependent cell lines. The growth inhibition is demonstrated to be due to specific binding of IL-2 to receptors on the TL cells.

  1. Seapolynol Extracted from Ecklonia cava Inhibits Adipocyte Differentiation in Vitro and Decreases Fat Accumulation in Vivo.

    PubMed

    Jeon, Hui-Jeon; Choi, Hyeon-Son; Lee, Yeon-Joo; Hwang, Ji-Hyun; Lee, Ok-Hwan; Seo, Min-Jung; Kim, Kui-Jin; Lee, Boo-Yong

    2015-12-04

    Seapolynol (SN) is a polyphenol mixture derived from Ecklonia cava. We evaluated the effects of SN on lipid accumulation in adipocytes, zebrafish, and mice. SN effectively inhibited lipid accumulation in three experimental models by suppressing adipogenic factors. Triglyceride synthetic enzymes such as diacylglycerol acyltransferase 1 (DGAT1) and GPAT3 were also downregulated by SN. This SN-induced inhibition of adipogenic factors was shown to be due to the regulatory effect of SN on early adipogenic factors; SN downregulated the expression of Krueppel-like factor 4 (KLF4), KLF5, CCAAT-enhancer-binding protein β (C/EBPβ), C/EBPδ, and Protein C-ets-2 (ETS2), while KLF2, an anti-early adipogenic factor, was upregulated by SN. SN-mediated inhibition in early adipogenesis was closely correlated with the inhibition of mitotic clonal expansion via cell cycle arrest. SN inhibited cell cycle progression by suppressing cell cycle regulators, such as cyclin A, cyclinD, and pRb but increased p27, a cell cycle inhibitor. In a mouse study, SN effectively reduced body weight and plasma lipid increases induced by a high-fat diet; triglycerides, total cholesterol, and low-density lipoprotein (LDL) levels were markedly reduced by SN. Moreover, SN remarkably improved high-fat-diet-induced hepatic lipid accumulation. Furthermore, SN activated AMP-activated protein kinase-α (AMPKα), an energy sensor, to suppress acetyl-coA carboxylase (ACC), inhibiting lipid synthesis. Our study suggests that SN may be an edible agent that can play a positive role in prevention of metabolic disorders.

  2. Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets.

    PubMed Central

    Lambillotte, C; Gilon, P; Henquin, J C

    1997-01-01

    The direct effects of glucocorticoids on pancreatic beta cell function were studied with normal mouse islets. Dexamethasone inhibited insulin secretion from cultured islets in a concentration-dependent manner: maximum of approximately 75% at 250 nM and IC50 at approximately 20 nM dexamethasone. This inhibition was of slow onset (0, 20, and 40% after 1, 2, and 3 h) and only slowly reversible. It was prevented by a blocker of nuclear glucocorticoid receptors, by pertussis toxin, by a phorbol ester, and by dibutyryl cAMP, but was unaffected by an increase in the fuel content of the culture medium. Dexamethasone treatment did not affect islet cAMP levels but slightly reduced inositol phosphate formation. After 18 h of culture with or without 1 microM dexamethasone, the islets were perifused and stimulated by a rise in the glucose concentration from 3 to 15 mM. Both phases of insulin secretion were similarly decreased in dexamethasone-treated islets as compared with control islets. This inhibition could not be ascribed to a lowering of insulin stores (higher in dexamethasone-treated islets), to an alteration of glucose metabolism (glucose oxidation and NAD(P)H changes were unaffected), or to a lesser rise of cytoplasmic Ca2+ in beta cells (only the frequency of the oscillations was modified). Dexamethasone also inhibited insulin secretion induced by arginine, tolbutamide, or high K+. In this case also the inhibition was observed despite a normal rise of cytoplasmic Ca2+. In conclusion, dexamethasone inhibits insulin secretion through a genomic action in beta cells that leads to a decrease in the efficacy of cytoplasmic Ca2+ on the exocytotic process. PMID:9022074

  3. Ginkgolic acid inhibits HIV protease activity and HIV infection in vitro

    PubMed Central

    Lü, Jian-Ming; Yan, Shaoyu; Jamaluddin, Saha; Weakley, Sarah M.; Liang, Zhengdong; Siwak, Edward B.; Yao, Qizhi; Chen, Changyi

    2012-01-01

    Summary Background Several HIV protease mutations, which are resistant to clinical HIV protease inhibitors (PIs), have been identified. There is a great need for second-generation PIs with different chemical structures and/or with an alternative mode of inhibition. Ginkgolic acid is a natural herbal substance and a major component of the lipid fraction in the nutshells of the Ginkgo biloba tree. The objective of this study was to determine whether ginkgolic acid could inhibit HIV protease activity in a cell free system and HIV infection in human cells. Material/Methods Purified ginkgolic acid and recombinant HIV-1 HXB2 KIIA protease were used for the HIV protease activity assay. Human peripheral blood mononuclear cells (PBMCs) were used for HIV infection (HIV-1SF162 virus), determined by a p24gag ELISA. Cytotoxicity was also determined. Results Ginkgolic acid (31.2 μg/ml) inhibited HIV protease activity by 60%, compared with the negative control, and the effect was concentration-dependent. In addition, ginkgolic acid treatment (50 and 100 μg/ml) effectively inhibited the HIV infection at day 7 in a concentration-dependent manner. Ginkgolic acid at a concentration of up to 150 μg/ml demonstrated very limited cytotoxicity. Conclusions Ginkgolic acid effectively inhibits HIV protease activity in a cell free system and HIV infection in PBMCs without significant cytotoxicity. Ginkgolic acid may inhibit HIV protease through different mechanisms than current FDA-approved HIV PI drugs. These properties of ginkgolic acid make it a promising therapy for HIV infection, especially as the clinical problem of viral resistance to HIV PIs continues to grow. PMID:22847190

  4. Rb deficiency accelerates progression of carcinoma of the urinary bladder in vivo and in vitro through inhibiting autophagy and apoptosis.

    PubMed

    Wang, Cheng-Yuan; Xu, Zhi-Bin; Wang, Jiang-Ping; Jiao, Yong; Zhang, Bo

    2017-02-22

    Urinary bladder cancer is known as a common cancer diagnosed across the world and results in significant mortality and morbidity rates among patients. The retinoblastoma (Rb) protein, as a main tumor suppressor, controls cellular responses to potentially oncogenic stimulation. Rb phosphorylation could disrupt E2F complex formation, resulting in diverse transcription factor dysfunction. In our study, we investigated how Rb is involved in controlling urinary bladder cancer progression. The results indicate that Rb expression is reduced in mice with urinary bladder tumor, and its suppression leads to urinary bladder cancer progression in vivo and in vitro. Rb mutation directly results in tumor size with lower survival rate in vivo. Rb knockdown in vitro promoted bladder tumor cell proliferation, migration and invasion. Interestingly, Rb knockout and knockdown result in autophagy and apoptosis inhibition via suppressing p53 and caspase-3 signaling pathways, enhancing bladder cancer development in vitro and in vivo. These findings reveal that Rb deficiency accelerated urinary bladder cancer progression, exposing an important role of Rb in suppressing urinary bladder cancer for treatment in the future.

  5. Inhibition of the in vitro growth of Babesia bigemina, Babesia caballi and Theileria equi parasites by trifluralin analogues.

    PubMed

    G Silva, Marta; Knowles, Donald P; Antunes, Sandra; Domingos, Ana; Esteves, Maria A; Suarez, Carlos E

    2017-06-01

    Bovine and equine babesiosis caused by Babesia bovis, Babesia bigemina and Babesia caballi, along with equine theileriosis caused by Theileria equi are global tick-borne hemoprotozoan diseases characterized by fever, anemia, weight losses and abortions. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the hosts for life. Antiprotozoal drugs are important for managing infection and disease. Previous research demonstrated that trifluralin analogues, designated (TFLAs) 1-15, which specifically bind to regions of alpha-tubulin protein in plants and protozoan parasites, have the ability to inhibit the in vitro growth of B. bovis. The inhibitory activity of TFLAs 1-15 minus TFLA 5 was tested in vitro against cultured B. bigemina, B. caballi and T. equi. The four TFLAs with greatest inhibitory activity were then analyzed for hemolytic activity and toxicity against erythrocytes. All TFLAs tested in the study showed inhibitory effects against the three parasite species. TFLA 2, TFLA 11, TFLA 13 and TFLA 14 were the most effective inhibitors for the three species tested, with estimated IC50 between 5.1 and 10.1μM at 72h. The drug's solvent (DMSO/ethanol) did not statistically affect the growth of the parasites nor cause hemolysis. Also, TFLA 2, 13 and 14 did not cause statistically significant hemolytic activity on bovine and equine erythrocytes at 15μM, and TFLA 2, 11 and 13 had no detectable toxic effects on bovine and equine erythrocytes at 15μM, suggesting that these drugs do not compromise erythrocyte viability. The demonstrated ability of the trifluralin analogues to inhibit in vitro growth of Babesia spp. and Theileria equi, and their lack of toxic effects on erythrocytes supports further in vivo testing and eventually their development as novel alternatives for the treatment of babesiosis and theileriosis. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  6. High-power helium-neon laser irradiation inhibits the growth of traumatic scars in vitro and in vivo.

    PubMed

    Shu, Bin; Ni, Guo-Xin; Zhang, Lian-Yang; Li, Xiang-Ping; Jiang, Wan-Ling; Zhang, Li-Qun

    2013-05-01

    This study explored the inhibitory effect of the high-power helium-neon (He-Ne) laser on the growth of scars post trauma. For the in vitro study, human wound fibroblasts were exposed to the high-power He-Ne laser for 30 min, once per day with different power densities (10, 50, 100, and 150 mW/cm(2)). After 3 days of repeated irradiation with the He-Ne laser, fibroblast proliferation and collagen synthesis were evaluated. For in vivo evaluation, a wounded animal model of hypertrophic scar formation was established. At postoperative day 21, the high-power He-Ne laser irradiation (output power 120 mW, 6 mm in diameter, 30 min each session, every other day) was performed on 20 scars. At postoperative day 35, the hydroxyproline content, apoptosis rate, PCNA protein expression and FADD mRNA level were assessed. The in vitro study showed that the irradiation group that received the power densities of 100 and 150 mW/cm(2) showed decreases in the cell proliferation index, increases in the percentage of cells in the G0/G1 phase, and decreases in collagen synthesis and type I procollagen gene expression. In the in vivo animal studies, regions exposed to He-Ne irradiation showed a significant decrease in scar thickness as well as decreases in hydroxyproline levels and PCNA protein expression. Results from the in vitro and in vivo studies suggest that repeated irradiation with a He-Ne laser at certain power densities inhibits fibroblast proliferation and collagen synthesis, thereby inhibits the growth of hypertrophic scars.

  7. The involvement of nitric oxide in ultraviolet-B-inhibited pollen germination and tube growth of Paulownia tomentosa in vitro.

    PubMed

    He, Jun-Min; Bai, Xiao-Ling; Wang, Rui-Bin; Cao, Bing; She, Xiao-Ping

    2007-10-01

    The role of nitric oxide (NO) in the ultraviolet-B radiation (UV-B)-induced reduction of in vitro pollen germination and tube growth of Paulownia tomentosa Steud. was studied. Results showed that exposure of the pollen to 0.4 and 0.8 W m(-2) UV-B radiation for 2 h resulted in not only the reduction of pollen germination and tube growth but also the enhancement of NO synthase (NOS, EC 1.14.13.39) activity and NO production in pollen grain and tube. Also, exogenous NO donors sodium nitroprusside and S-nitrosoglutathione inhibited both pollen germination and tube growth in a dose-dependence manner. NOS inhibitor N(G)-nitro-l-Arg-methyl eater (l-NAME) and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) not only largely prevented the NO generation but also partly reversed the UV-B-inhibited pollen germination and tube growth. These results indicate that UV-B radiation inhibits pollen germination and tube growth partly via promoting NO production in pollen grain and tube by a NOS-like enzyme. Additionally, a guanylyl cyclase inhibitor 6-anilino-5,8-quinolinequinone (LY-83583) prevented both the UV-B- and NO donors-inhibited pollen germination and tube growth, suggesting that the NO function is mediated by cyclic guanosine 5'-monophosphate. However, the effects of c-PTIO, l-NAME and LY-83583 on the UV-B-inhibited pollen germination and tube growth were only partial, suggesting that there are NO-independent pathways in UV-B signal networks.

  8. Acidic mucopolysaccharide from Holothuria leucospilota has antitumor effect by inhibiting angiogenesis and tumor cell invasion in vivo and in vitro.

    PubMed

    Zhang, Weiwei; Lu, Yin; Xu, Bo; Wu, Jiaming; Zhang, Lijuan; Gao, Ming; Zheng, Shizhong; Wang, Aiyun; Zhang, Changbin; Chen, Lei; Lei, Na

    2009-08-01

    Acidic mucopolysaccharide from Holothuria Leucospilota (HS) may affect some steps in metastasis cascade. In vitro, HS inhibited the growth of B16F10 cells and proliferation of VEGF-induced HUVEC dose-dependently compared to the control, VEGF-induced capillary-like tube networks and the numbers of migratory and invasive cells were significantly inhibited by HS in a dose-dependent manner under the cytotoxic doses. Additionally, VEGF-induced vessel sprouting of rat aortic ring was also inhibited by HS. It also has been demonstrated that the invasive ability of B16F10 melanoma cells through the Matrigel-embedded Boyden chamber was suppressed by 0.5 muM HS. The protein level secreted by B16F10 cells of MMP-2,-9 and VEGF were decreased by HS treatment. In vivo, a tumor growth inhibition study was carried out using mice bearing B16F10 cells model of metastasis, no matter experimental or spontaneous, showed that HS at 5.2, 11.6 and 26 mg/kg (weight of mice) could markedly decreased the metastatic tumors in mouse lung in a dose-dependent manner. In CAM assay and Matrigel plug assay in vivo, HS (50 microg/egg and 100 microg/egg) inhibited new blood vessel formation on the growing chick chorioallantoic membrane, and HS (5.2 and 26 mg/kg body weight) reduced the vessel density in Matrigel plugs implanted in mice. Taken together, these results demonstrate that HS has antimetastasic properties possibly via its antiangiogenesis induced by downregulation of VEGF and suppression of invasive ability of cancer cells mediated by downregulation of MMP-2, -9 and their activities.

  9. Synthesis of 2-acylated and sulfonated 4-hydroxycoumarins: In vitro urease inhibition and molecular docking studies.

    PubMed

    Rashid, Umer; Rahim, Fazal; Taha, Muhammad; Arshad, Muhammad; Ullah, Hayat; Mahmood, Tariq; Ali, Muhammad

    2016-06-01

    Sixteen 4-hydroxycoumarin derivatives were synthesized, characterized through EI-MS and (1)H NMR and screened for urease inhibitory potential. Three compounds exhibited better urease inhibition than the standard inhibitor thiourea (IC50=21±0.11μM) while other four compounds exhibited good to moderate inhibition with IC50 values between 29.45±1.1μM and 69.53±0.9μM. Structure activity relationship was established on the basis of molecular docking studies, which helped to predict the binding interactions of the most active compounds.

  10. In vitro growth inhibition of human cancer cells by novel honokiol analogs

    PubMed Central

    Lin, Jyh Ming; Prakasha Gowda, A. S.; Sharma, Arun K.; Amin, Shantu

    2012-01-01

    Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluate possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK11 and cyclin B1protien levels in A549 cells. PMID:22533983

  11. sTLR4/MD-2 complex inhibits colorectal cancer in vitro and in vivo by targeting LPS

    PubMed Central

    Meng, Jie; Wei, Liuhua; Wu, Chunlin; Zhang, Qiaoyun; Wei, Dong; Chen, Xiang; Wu, Hao; Chen, Xiaoli; Dai, Shengming

    2016-01-01

    Colorectal cancer (CRC) is aggressive and associated with TLR4-MD-2 signaling. Toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) were highly expressed in human CRC. The soluble form of extracellular TLR4 domain (sTLR4) and MD-2 may have important roles in binding lipopolysaccharide (LPS). In this study, sTLR4 and MD-2 protein and prepared sTLR4/MD-2 complex were synthesized successfully to restrain LPS-TLR4/MD-2 activation by competing with cellular membrane TLR4 for binding LPS. The sTLR4/MD-2 complex can significantly attenuate LPS induced pro-inflammatory and migration cytokine production in vitro and in vivo, and inhibit the effect of LPS on the cell cycle, migration and invasion of human CRC cells in vitro. Administration of sTLR4/MD-2 complex protected mice from tumor both in xenograft and implantation metastasis model. The sTLR4/MD-2 complex treated mice had smaller tumor, less body weight loss and lower expression of inflammatory cytokines. Here, the azoxymethane/dextran sulfate sodium salt (AOM/DSS) murine model was used as an experimental platform to simulate the physiological and pathological processes of cancers associated with chronic intestinal inflammation. AOM/DSS-induced tumors were inhibited in mice treated by sTLR4/MD-2 complex. It is demonstrated in our study that sTLR4/MD-2 complex could inhibit CRC by competing with binding LPS, raising the complex's possibility of a new prevention agent against CRC. PMID:27409669

  12. Maresin 1 Inhibits Epithelial-to-Mesenchymal Transition in Vitro and Attenuates Bleomycin Induced Lung Fibrosis in Vivo.

    PubMed

    Wang, Yaxin; Li, Ruidong; Chen, Lin; Tan, Wen; Sun, Zhipeng; Xia, Haifa; Li, Bo; Yu, Yuan; Gong, Jie; Tang, Min; Ji, Yudong; Yuan, Shiying; Shanglong Yao; Shang, You

    2015-11-01

    Lung fibrosis is an aggressive disease with uncontrolled fibrotic response and no effective therapeutic treatment. Epithelial-to-mesenchymal transition (EMT) has been proved to be an important pathological feature in lung fibrosis. In this study, we investigated whether MaR1, a kind of proresolving lipid mediators, could inhibit TGF-β1-induced EMT in vitro and lung fibrosis in vivo. In vitro study, mouse type II alveolar epithelial cells were treated with different does of MaR1 for 30 min and were exposed to TGF-β1 for 48 h. In vivo study, C57BL/6 mice were administered bleomycin intratracheally. After 14 days, MaR1 was injected intraperitoneally daily for 7 days. In day 28, mice were sacrificed. The results demonstrate that treatment of mouse type II alveolar epithelial cells with MaR1 (10 nM) significantly prevents TGF-β1-induced fibronectin and α-SMA expression and restores E-Cadherin level. The down-regulation of profibrotic molecules of MaR1 is associated with suppression of Smad2/3 and Akt phosphorylation. In vivo, MaR1 treatment significantly prolongs survival rate and attenuates destruction of lung architecture, as well as collagen deposition after bleomycin inhalation. TGF-β1 concentration in bronchoalveolar lavage and fibrotic markers (fibronectin and α-SMA) in lung tissues are inhibited by MaR1 administration. These data indicate that MaR1 inhibits TGF-β1-induced EMT and attenuates bleomycin-induced pulmonary fibrosis. MaR1 may be a promising strategy for alleviation of lung fibrosis.

  13. Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine

    PubMed Central

    Telinius, Niklas; Mohanakumar, Sheyanth; Majgaard, Jens; Kim, Sukhan; Pilegaard, Hans; Pahle, Einar; Nielsen, Jørn; de Leval, Marc; Aalkjaer, Christian; Hjortdal, Vibeke; Boedtkjer, Donna Briggs

    2014-01-01

    Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4 ± 4.9% of control in the presence of 20 nmol l−1 nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo. PMID:25172950

  14. Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine.

    PubMed

    Telinius, Niklas; Mohanakumar, Sheyanth; Majgaard, Jens; Kim, Sukhan; Pilegaard, Hans; Pahle, Einar; Nielsen, Jørn; de Leval, Marc; Aalkjaer, Christian; Hjortdal, Vibeke; Boedtkjer, Donna Briggs

    2014-11-01

    Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4 ± 4.9% of control in the presence of 20 nmol l(-1) nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

  15. Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4.

    PubMed Central

    Drivdahl, R H; Kutter, E M

    1990-01-01

    The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. We examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D+ or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D+ or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D+ strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with [gamma-32P]ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly. It thus appears likely that gpalc inhibits transcript elongation on cytosine-containing DNA by interacting with actively transcribing core polymerase as a complex with the enzyme and cytosine-rich stretches of the template. Images PMID:2185231

  16. Methylselenol, a selenium metabolite, inhibits colon cancer cell growth in vitro and in vivo

    USDA-ARS?s Scientific Manuscript database

    Methylselenol is hypothesized to be a critical selenium (Se) metabolite for anticancer activity. Submicromolar methylselenol exposure inhibited cell growth and led to an increase in the G1 and G2 fractions with a concomitant drop in the S-phase, and an induction of apoptosis in cancerous colon HCT11...

  17. Myrsinoic acid B inhibits the production of hydrogen sulfide by periodontal pathogens in vitro.

    PubMed

    Ito, Satomi; Shimura, Susumu; Tanaka, Tomoko; Yaegaki, Ken

    2010-06-01

    Recently, we reported that myrsinoic acid B purified from Myrsine seguinii inhibited methyl mercaptan (CH(3)SH) production by Fusobacterium nucleatum JCM8532. Since hydrogen sulfide (H(2)S) is the main component of physiological halitosis, while CH(3)SH is involved in pathological oral halitosis, the objective of this study is to determine whether myrsinoic acid B inhibits H(2)S production by oral microorganisms. F. nucleatum, Porphyromonas gingivalis and Treponema denticola were incubated with myrsinoic acid B and a substrate such as l-cysteine or l-methionine. H(2)S or CH(3)SH concentration in the headspace air, was determined using a gas chromatograph. The concentration of myrsinoic acid B inhibiting 50% (IC(50)) of H(2)S production by F. nucleatum was 0.142 µg ml(-1), and the IC(50) of P. gingivalis and T. denticola were 2.71 µg ml(-1) and 28.9 µg ml(-1), respectively. The presence of pyruvate, a by-product of H(2)S production, was determined. The IC(50) values of myrsinoic acid B for pyruvate production were 22.9 µg ml(-1) for F. nucleatum, 87.7 µg ml(-1) for P. gingivalis and 165 µg ml(-1) for T. denticola. We concluded that myrsinoic acid B inhibited the production of both H(2)S and pyruvate by periodontal pathogens.

  18. Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models

    PubMed Central

    Zhou, Hong; Gao, Jinsheng; Zhou, Li; Li, Xin; Li, Weidong; Li, Xuejun; Xia, Yin

    2012-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease characterized by massive enlargement of fluid-filled cysts in the kidney. However, there is no effective therapy yet for this disease. To examine whether ginkgolide B, a natural compound, inhibits cyst development, a Madin-Darby canine kidney (MDCK) cyst model, an embryonic kidney cyst model, and a PKD mouse model were used. Interestingly, ginkgolide B significantly inhibited MDCK cyst formation dose dependently, with up to 69% reduction by 2 μM ginkgolide B. Ginkgolide B also significantly inhibited cyst enlargement in the MDCK cyst model, embryonic kidney cyst model, and PKD mouse model. To determine the underlying mechanisms, the effect of ginkgolide B on MDCK cell viability, proliferation, apoptosis, chloride transporter CFTR activity, and intracellular signaling pathways were also studied. Ginkgolide B did not affect cell viability, proliferation, and expression and activity of the chloride transporter CFTR that mediates cyst fluid secretion. Ginkgolide B induced cyst cell differentiation and altered the Ras/MAPK signaling pathway. Taken together, our results demonstrate that ginkgolide B inhibits renal cyst formation and enlargement, suggesting that ginkgolide B might be developed into a novel candidate drug for ADPKD. PMID:22338085

  19. Cyclooxgenase-2 Inhibiting Perfluoropoly (Ethylene Glycol) Ether Theranostic Nanoemulsions—In Vitro Study

    PubMed Central

    Patel, Sravan Kumar; Zhang, Yang; Pollock, John A.; Janjic, Jelena M.

    2013-01-01

    Cylcooxgenase-2 (COX-2) expressing macrophages, constituting a major portion of tumor mass, are involved in several pro-tumorigenic mechanisms. In addition, macrophages are actively recruited by the tumor and represent a viable target for anticancer therapy. COX-2 specific inhibitor, celecoxib, apart from its anticancer properties was shown to switch macrophage phenotype from tumor promoting to tumor suppressing. Celecoxib has low aqueous solubility, which may limit its tumor inhibiting effect. As opposed to oral administration, we propose that maximum anticancer effect may be achieved by nanoemulsion mediated intravenous delivery. Here we report multifunctional celecoxib nanoemulsions that can be imaged by both near-infrared fluorescence (NIRF) and 19F magnetic resonance. Celecoxib loaded nanoemulsions showed a dose dependent uptake in mouse macrophages as measured by 19F NMR and NIRF signal intensities of labeled cells. Dramatic inhibition of intracellular COX-2 enzyme was observed in activated macrophages upon nanoemulsion uptake. COX-2 enzyme inhibition was statistically equivalent between free drug and drug loaded nanoemulsion. However, nanoemulsion mediated drug delivery may be advantageous, helping to avoid systemic exposure to celecoxib and related side effects. Dual molecular imaging signatures of the presented nanoemulsions allow for future in vivo monitoring of the labeled macrophages and may help in examining the role of macrophage COX-2 inhibition in inflammation-cancer interactions. These features strongly support the future use of the presented nanoemulsions as anti-COX-2 theranostic nanomedicine with possible anticancer applications. PMID:23409048

  20. Grape-seed procyanidins inhibit the in vitro growth and invasion of pancreatic carcinoma cells.

    PubMed

    Chung, Yuan-Chiang; Huang, Chiu-Chen; Chen, Chin-Hui; Chiang, Hua-Che; Chen, Kuen-Bao; Chen, Yu-Jen; Liu, Chia-Ling; Chuang, Lu-Te; Liu, Ming; Hsu, Chih-Ping

    2012-04-01

    Grape-seed procyanidins (GSPs) can inhibit cell proliferation and invasiveness in various human cancers. However, the effect of GSP on pancreatic carcinoma cells has not been investigated. Pancreatic carcinoma cell lines MIA PaCa-2 and BxPC-3 treated with GSP were assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for increased apoptosis by annexin V labeling, for their adhesion and invasion potential by evaluating their ability to penetrate through a matrix gel-coated Boyden chamber, and for changes in the levels of proteins involved in cellular events by immunoblotting. Grape-seed procyanidin inhibited MIA PaCa-2 and BxPC-3 proliferation in a dose-dependent manner and induced G1-phase arrest of the cell cycle in BxPC-3 or mitochondria-mediated apoptosis in MIA PaCa-2. Grape-seed procyanidin also inhibited the adhesion and invasion potential of both cell lines in a dose-dependent manner, which are associated with the suppression of metalloproteases matrix metalloproteinase 9 or 2 (MMP-9 or -2) expression. Grape-seed procyanidin inhibited the proliferation of pancreatic carcinoma cells by cell cycle blockage or apoptotic induction. The invasiveness was also suppressed by GSP through down-regulation of MMP-2 or MMP-9 in pancreatic carcinoma cells. Grape-seed procyanidin is a potential chemotherapeutic or preventive agent for pancreatic carcinoma.

  1. Proanthocyanidins inhibit mitogenic and survival-signaling in vitro and tumor growth in vivo.

    PubMed

    Meeran, Syed Musthapa; Katiyar, Santosh Kumar

    2008-01-01

    We have previously shown that treatment of human epidermoid carcinoma A431 cells with grape seed proanthocyanidins (GSPs) induces apoptosis of A431 cells. Here, we report that treatment of A431 cells with GSPs inhibits constitutive as well as EGF-induced higher levels of phosphorylated proteins of MAPK family in a dose-dependent manner. This effect is associated with the reactivation of MAP kinase phosphatases. Western blot analysis reveals that GSPs decrease: (i) the levels of phosphatidylinositol 3-kinase (PI3K) and the phosphorylation of Akt at ser473, and (ii) the constitutive activation of NF-kappaB/p65. As NF-kappaB-targeted genes play crucial roles in tumor cell proliferation and differentiation, we assessed the effect of GSPs on proteins encoded by these genes. Treatment with GSPs results in inhibition of the expression of COX-2, iNOS, PCNA, cyclin D1 and MMP-9 in A431 cells compared with non-GSPs-treated controls. Treatment of athymic nude mice with GSPs by oral gavage (50 or 100 mg/kg body weight/mouse) reduces the growth of A431-xenografts in mice, which is associated with the inhibition of tumor cell proliferation in xenografts as indicated by the inhibition of mRNA expression of PCNA and cyclin D1, and of NF-kappaB activity. Together, the data suggest that GSPs might be effective in the treatment of skin cancers.

  2. Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro.

    PubMed

    da Silva, Cleide G; Bueno, Ana Rúbia F; Schuck, Patrícia F; Leipnitz, Guilhian; Ribeiro, César A J; Rosa, Rafael B; Dutra Filho, Carlos S; Wyse, Angela T S; Wannmacher, Clóvis M D; Wajner, Moacir

    2004-01-01

    D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.

  3. Dopamine inhibits ATP-induced responses in the cat petrosal ganglion in vitro.

    PubMed

    Alcayaga, Julio; Retamal, Mauricio; Cerpa, Verónica; Arroyo, Jorge; Zapata, Patricio

    2003-03-21

    The petrosal ganglion (PG) provides sensory innervation to the carotid sinus and carotid body through the carotid (sinus) nerve (CN). Application of either acetylcholine (ACh) or adenosine 5'-triphosphate (ATP) to the PG superfused in vitro activates CN fibers. Dopamine (DA) modulates the effects of ACh. We have previously shown that DA when applied to the PG modulates the effects of ACh on carotid sinus nerve fibers. We currently report the effects of DA on the ATP-induced responses in the isolated PG in vitro. While DA had no effect on the basal activity recorded from the CN, it reduced ATP-induced responses in a dose-dependent manner, when preceding ATP applications by 30 s. Our results suggest that DA-a transmitter present in a group of PG neurons and in carotid body cells-may act as an inhibitory modulator of ATP-evoked responses in PG neurons.

  4. Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro-in vivo correlations.

    PubMed

    Hu, S; Mathijssen, R H J; de Bruijn, P; Baker, S D; Sparreboom, A

    2014-02-18

    Several tyrosine kinase inhibitors (TKIs) can decrease docetaxel clearance in patients by an unknown mechanism. We hypothesised that these interactions are mediated by the hepatic uptake transporter OATP1B1. The influence of 16 approved TKIs on transport was studied in vitro using HEK293 cells expressing OATP1B1 or its mouse equivalent Oatp1b2. Pharmacokinetic studies were performed with Oatp1b2-knockout and OATP1B1-transgenic mice. All docetaxel-interacting TKIs, including sorafenib, were identified as potent inhibitors of OATP1B1 in vitro. Although Oatp1b2 deficiency in vivo was associated with increased docetaxel exposure, single- or multiple-dose sorafenib did not influence docetaxel pharmacokinetics. These findings highlight the importance of identifying proper preclinical models for verifying and predicting TKI-chemotherapy interactions involving transporters.

  5. High Iron-Sequestrating Bifidobacteria Inhibit Enteropathogen Growth and Adhesion to Intestinal Epithelial Cells In vitro

    PubMed Central

    Vazquez-Gutierrez, Pamela; de Wouters, Tomas; Werder, Julia; Chassard, Christophe; Lacroix, Christophe

    2016-01-01

    The gut microbiota plays an important role in host health, in particular by its barrier effect and competition with exogenous pathogenic bacteria. In the present study, the competition of Bifidobacterium pseudolongum PV8-2 (Bp PV8-2) and Bifidobacterium kashiwanohense PV20-2 (Bk PV20-2), isolated from anemic infant gut microbiota and selected for their high iron sequestration properties, was investigated against Salmonella Typhimurium (S. Typhi) and Escherichia coli O157:H45 (EHEC) by using co-culture tests and assays with intestinal cell lines. Single and co-cultures were carried out anaerobically in chemically semi-defined low iron (1.5 μM Fe) medium (CSDLIM) without and with added ferrous iron (30 μM Fe). Surface properties of the tested strains were measured by bacterial adhesion to solvent xylene, chloroform, ethyl acetate, and to extracellular matrix molecules, mucus II, collagen I, fibrinogen, fibronectin. HT29-MTX mucus-secreting intestinal cell cultures were used to study bifidobacteria competition, inhibition and displacement of the enteropathogens. During co-cultures in CSDLIM we observed strain-dependent inhibition of bifidobacterial strains on enteropathogens, independent of pH, organic acid production and supplemented iron. Bp PV8-2 significantly (P < 0.05) inhibited S. Typhi N15 and EHEC after 24 h compared to single culture growth. In contrast Bk PV20-2 showed less inhibition on S. Typhi N15 than Bp PV8-2, and no inhibition on EHEC. Affinity for intestinal cell surface glycoproteins was strain-specific, with high affinity of Bp PV8-2 for mucin and Bk PV20-2 for fibronectin. Bk PV20-2 showed high adhesion potential (15.6 ± 6.0%) to HT29-MTX cell layer compared to Bp PV8-2 (1.4 ± 0.4%). In competition, inhibition and displacement tests, Bp PV8-2 significantly (P < 0.05) reduced S. Typhi N15 and EHEC adhesion, while Bk PV20-2 was only active on S. Typhi N15 adhesion. To conclude, bifidobacterial strains selected for their high iron binding

  6. RHAMNAZIN INHIBITS PROLIFERATION AND INDUCES APOPTOSIS OF HUMAN JURKAT LEUKEMIA CELLS IN VITRO.

    PubMed

    Philchenkov, A A; Zavelevych, M P

    2015-01-01

    Antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a DNA topoisomerase II inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

  7. Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

    PubMed

    Healy, Laura D; Puy, Cristina; Fernández, José A; Mitrugno, Annachiara; Keshari, Ravi S; Taku, Nyiawung A; Chu, Tiffany T; Xu, Xiao; Gruber, András; Lupu, Florea; Griffin, John H; McCarty, Owen J T

    2017-04-13

    Activated protein C (APC) is a multi-functional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a non-human primate model of E. coli-induced sepsis, pre-treatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

  8. Comparison of continuous versus pulsed photodynamic antimicrobial therapy for inhibition of fungal keratitis isolates in vitro (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nolan, Nicholas; Durkee, Heather A.; Aguilar, Mariela C.; Arboleda, Alejandro; Relhan, Nidhi; Martinez, Anna; Rowaan, Cornelis; Gonzalez, Alex; Alawa, Karam A.; Amescua, Guillermo; Flynn, Harry W.; Miller, Darlene; Parel, Jean-Marie A.

    2017-02-01

    Fungal keratitis can lead to pain and impaired vision. Current treatment options include antifungal agents and therapeutic penetrating keratoplasty. An emerging option for the management of keratitis is photodynamic antimicrobial therapy (PDAT) which uses a photosensitizer rose bengal activated with green light. Utilizing a pulsed irradiation, rather than the standard continuous irradiation may have a similar antimicrobial effect with less total energy. This study is to compare pulsed and continuous rose bengal mediated PDAT for inhibition of six fungal isolates on agar plates: Fusarium solani, Fusarium keratoplasticum, Aspergillus fumigatus, Candida albicans, Paecilomyces variotti, and Pseudoallescheria boydii. Isolates were mixed with 0.1% rose bengal and exposed to three irradiation conditions: (1) 30-minute continuous (10.8J/cm2), (2) 15-minute continuous (5.4J/cm2), (3) 30-minute pulsed (5.4J/cm2). Plates were photographed at 72 hours and analyzed with custom software. At 72 hours, 30-minute continuous rose bengal mediated PDAT inhibited all six fungal species. Fungal inhibition was analogous between 30-minute continuous and 30-minute pulsed test groups, with the exception of A. fumigatus. The 15-minute continuous irradiation was less effective when compared to both 30-minute continuous and 30-minute pulsed groups. These in vitro results demonstrate the potential strength of pulsed rose bengal mediated PDAT as an adjunct treatment modality for fungal keratitis.

  9. Synthesis, Characterization, and in Vitro Antitumor Activity of Ruthenium(II) Polypyridyl Complexes Tethering EGFR-Inhibiting 4-Anilinoquinazolines.

    PubMed

    Du, Jun; Kang, Yan; Zhao, Yao; Zheng, Wei; Zhang, Yang; Lin, Yu; Wang, Zhaoying; Wang, Yuanyuan; Luo, Qun; Wu, Kui; Wang, Fuyi

    2016-05-02

    Ruthenium-based anticancer complexes are promising antitumor agents for their low system toxicity and versatile chemical structures. Epidermal growth factor receptor (EGFR) has been found to be overexpressed in a broad range of tumor cells and is regarded as a drug target in developing novel antitumor drugs. In this work, five ruthenium(II) polypyridyl complexes containing EGFR-inhibiting 4-anilinoquinazoline pharmacophores were synthesized and characterized. These complexes showed both high EGFR-inhibiting activity and strong DNA minor groove-binding activity. In vitro antiproliferation screening demonstrated that the prepared ruthenium complexes are highly cytotoxic against a series of cancer cell lines, in particular non-small-cell lung A549 and human epidermoid carcinoma A431. Fluorescence-activated cell sorting analysis and fluorescence microscopy revealed that the most active complex, K4, induced much more late-stage cell apoptosis and necrosis than gefitinib, the first EGFR-targeting antitumor drug in clinical use. These results indicate that the ruthenium(II) polypyridyl complexes bearing EGFR-inhibiting 4-anilinoquinazolines possess highly active dual-targeting anticancer activity and are promising in developing new anticancer agents.

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