Manuka honey inhibits adhesion and invasion of medically important wound bacteria in vitro.

PubMed

Maddocks, Sarah Elizabeth; Jenkins, Rowena Eleri; Rowlands, Richard Samuel; Purdy, Kevin John; Cooper, Rose Agnes

2013-12-01

To characterize the effect of manuka honey on medically important wound bacteria in vitro, focusing on its antiadhesive properties. Crystal violet biofilm assays, fluorescent microscopy, protein adhesion assay and gentamicin protection assay were used to determine the impact of manuka honey on biofilm formation, human protein binding and adherence to/invasion into human keratinocytes. Manuka honey effectively disrupted and caused extensive cell death in biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes. Sublethal doses of manuka honey inhibited bacterial adhesion to the fibronectin, fibrinogen and collagen. Manuka honey impaired adhesion of laboratory and clinical isolates of S. aureus, P. aeruginosa and S. pyogenes to human keratinocytes in vitro, and inhibited invasion by S. pyogenes and homogeneous vancomycin intermediate S. aureus. Manuka honey can directly affect bacterial cells embedded in a biofilm and exhibits antiadhesive properties against three common wound pathogens.

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.

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.

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.

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.

In Vitro Inhibition of Human Papillomavirus Following Use of a Carrageenan-Containing Vaginal Gel

PubMed Central

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.

2016-01-01

Objective 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. Methods Sexually active US women between 19–35 years 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 8–12 hours after intercourse versus those who applied gel before and after intercourse returned 1, 4 or 8–12 hours 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. Results 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 8–12 hours, respectively. Higher carrageenan concentrations were associated with higher levels of PsV16 inhibition (rho=0.69). Conclusions 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. PMID:27625046

Inhibition of in vitro Histophilus somni biofilm production by recombinant Hsp60 antibodies.

PubMed

Zarankiewicz, T; Madej, J; Galli, J; Bajzert, J; Stefaniak, T

2012-01-01

Histophilus somni is an opportunistic pathogen causing respiratory, genitourinary and generalized infections in cattle. An important virulence factor is its ability to produce a biofilm. The aim of this work was to confirm that H. somni Hsp60 (Gro-EL) is a constituent of the biofilm produced by this bacterium in vitro and to check whether or not the presence of a specific antibody within the culture medium can inhibit biofilm production. Biofilm production by H. somni cultured in vitro was confirmed by crystalline violet staining. The presence of Hsp60 in the biofilm was confirmed by using specific antibodies produced in a mouse and goat hyperimmunized with H. somni recombinant Hsp60 (rHsp60). Large complexes of biofilm stained with Hsp60 antibodies were microscopically detected. This indicates that the Hsp60 protein is a common constituent of the biofilm produced by H. somni in vitro. In a second experiment, mouse serum containing anti-H. somni rHsp60 antibodies was added to an H. somni culture. It was found that the presence of anti-rHsp60 antibodies in the culture medium inhibited biofilm production in vitro. Only small biofilm particles were seen in the presence of the specific antibody, whereas in control cultures (without specific antiserum) large biofilm complexes were produced. The results indicate that antibodies specific to Hsp60 may be useful for preventing H. somni biofilm formation in vitro. If this also occurs in vivo, it may be helpful for eradicating H. somni infection in cattle through the elimination of carriers. Further in vivo studies are needed to confirm this idea.

Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burroughs, S.F.; Johnson, G.J.

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; butmore » 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.« less

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

Derricin and Derricidin Inhibit Wnt/β-Catenin Signaling and Suppress Colon Cancer Cell Growth In Vitro

PubMed Central

Fonseca, Barbara F.; Predes, Danilo; Cerqueira, Debora M.; Reis, Alice H.; Amado, Nathalia G.; Cayres, Marina C. L.; Kuster, Ricardo M.; Oliveira, Felipe L.; Mendes, Fabio A.; Abreu, Jose G.

2015-01-01

Overactivation of the Wnt/β-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/β-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/β-catenin pathway. Both chalcones are able to affect the cell distribution of β-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/β-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/β-catenin pathway and colon cancer cell growth in vitro. PMID:25775405

PARTIAL INHIBITION OF IN VITRO POLLEN GERMINATION BY SIMULATED SOLAR ULTRAVIOLET-B RADIATION

EPA Science Inventory

Pollen from four temperate-latitude taxa were treated with UV radiation in a portion of the UV-B (280-320 nm) waveband during in vitro germination. Inhibition of germination was noted in this pollen compared to samples treated identically except for the exclusion of the UV-B port...

An in vitro approach to potential methadone metabolic-inhibition interactions.

PubMed

Bomsien, Stephanie; Skopp, Gisela

2007-09-01

The aim of this study was to assess the drug interaction potential of psychotropic medication on methadone N-demethylation using cDNA-expressed cytochrome P450 CYP enzymes. Methadone was incubated with various drugs (n = 10) and cDNA-expressed CYP3A4, CYP2D6, CYP2B6, CYP2C19 and CYP1A2 enzymes to screen for their inhibition potency. The nature of enzyme selective activity for inhibition was further investigated for potent inhibitors. To test for a mechanism-based component in inhibition, all substances were tested with preincubation and without. 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) concentration was determined by liquid chromatography/tandem mass spectrometry following liquid/liquid extraction. Formation of EDDP was catalysed by CYP3A4, CYP2D6 and CYP2C19. The N-demethylation of methadone was preferentially inhibited by amitriptyline, buprenorphine, methylenedioxymethamphetamine (MDMA) and zolpidem. Both amitriptyline and buprenorphine were strong, reversible inhibitors of CYP3A4. Similarly, amitriptyline and MDMA were identified as inhibitors of CYP2D6. Zolpidem revealed a mechanism-based inhibition of CYP3A4. Amitriptyline, MDMA and zolpidem are likely to slow down conversion of methadone and to increase its area under the curve (AUC). A consideration of the in vitro evidence of drug-methadone interactions should help to improve patient care during methadone maintenance treatment.

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

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.

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...

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.

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.

Inhibition of p53 Mutant Peptide Aggregation In Vitro by Cationic Osmolyte Acetylcholine Chloride.

PubMed

Chen, Zhaolin; Kanapathipillai, Mathumai

2017-01-01

Mutations of tumor suppressor protein p53 are present in almost about 50% of all cancers. It has been reported that the p53 mutations cause aggregation and subsequent loss of p53 function, leading to cancer progression. Here in this study we focus on the inhibitory effects of cationic osmolyte molecules acetylcholine chloride, and choline on an aggregation prone 10 amino acid p53 mutant peptide WRPILTIITL, and the corresponding wildtype peptide RRPILTIITL in vitro. The characterization tools used for this study include Thioflavin- T (ThT) induced fluorescence, transmission electron microscopy (TEM), congo red binding, turbidity, dynamic light scattering (DLS), and cell viability assays. The results show that acetylcholine chloride in micromolar concentrations significantly inhibit p53 mutant peptide aggregation in vitro, and could be promising candidate for p53 mutant/ misfolded protein aggregation inhibition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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.

Clinically Relevant Concentrations of Ketamine Inhibit Osteoclast Formation In Vitro in Mouse Bone Marrow Cultures.

PubMed

Du, Erxia; McAllister, Patrick; Venna, Venugopal Reddy; Xiao, Liping

2017-04-01

Ketamine has been used safely in clinics for decades for analgesia and anesthesia. It is increasingly popular in clinical practice due to its new uses and importance for emergency procedures. It is known that ketamine is sequestered in the bone marrow and the major receptors for ketamine, noncompetitive N-methyl-d-aspartate receptors (NMDARs), are expressed in osteoclasts (OCs) and osteoblasts. However, the impact of ketamine on OCs or osteoblasts is unknown. In this study, we investigated the effects of ketamine on osteoclastogenesis and regulation of NMDARs expression in vitro. Bone marrows (BMs) or bone marrow macrophages (BMMs) were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) with or without ketamine for up to 6 days. OC formation peaked at day 5. On day 5 of culture, ketamine inhibited OC formation from both BM and BMM cultures at clinically relevant concentrations (3-200 µM). Ketamine inhibited RANKL-induced expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) in BMM cultures. Inhibition of ketamine on RANKL-induced osteoclastogenesis is associated with down-regulation of NMDARs. In addition, ketamine significantly inhibited the M-CSF induced migration of BMMs, inhibited cell fusion and significantly increased mature OC apoptosis. We conclude that clinically relevant concentrations of ketamine inhibit OC formation in both BM and BMM cultures in vitro through inhibiting migration and fusion process and enhancing mature OC apoptosis. It is likely that ketamine regulates osteoclastogenesis, at least in part, via its effects on NMDAR expression. J. Cell. Biochem. 118: 914-923, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Redox and non-redox mechanism of in vitro cyclooxygenase inhibition by natural quinones.

PubMed

Landa, Premysl; Kutil, Zsofia; Temml, Veronika; Vuorinen, Anna; Malik, Jan; Dvorakova, Marcela; Marsik, Petr; Kokoska, Ladislav; Pribylova, Marie; Schuster, Daniela; Vanek, Tomas

2012-03-01

In this study, ten anthra-, nine naphtho-, and five benzoquinone compounds of natural origin and five synthetic naphthoquinones were assessed, using an enzymatic in vitro assay, for their potential to inhibit cyclooxygenase-1 and -2 (COX-1 and COX-2), the key enzymes of the arachidonic acid cascade. IC₅₀ values comparable with COX reference inhibitor indomethacin were recorded for several quinones (primin, alkannin, diospyrin, juglone, 7-methyljuglone, and shikonin). For some of the compounds, we suggest the redox potential of quinones as the mechanism responsible for in vitro COX inhibition because of the quantitative correlation with their pro-oxidant effect. Structure-relationship activity studies revealed that the substitutions at positions 2 and 5 play the key roles in the COX inhibitory and pro-oxidant actions of naphthoquinones. In contrast, the redox mechanism alone could not explain the activity of primin, embelin, alkannin, and diospyrin. For these four quinones, molecular modeling suggested similar binding modes as for conventional nonsteroidal anti-inflammatory drugs (NSAIDs). © Georg Thieme Verlag KG Stuttgart · New York.

Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27.

PubMed

Sang, Dong-Ping; Li, Ru-Jun; Lan, Qing

2014-06-01

Quercetin is an effective Hsp27 inhibitor and has been reported to facilitate tumor cell apoptosis. The aim of this study was to investigate whether quercetin could sensitize human glioblastoma cells to temozolomide (TMZ) in vitro. Both U251 and U87 human glioblastoma cells were treated with quercetin and/or TMZ for 48 h. Cell viability was detected using the MTT assay. Cell apoptosis was analyzed with caspase-3 activity kits and flow cytometry. Hsp27 expression and phosphorylation were examined using Western blot analysis. RNA interference using Hsp27 siRNA oligos was performed to knock down the gene expression of Hsp27. TMZ (200 or 400 μmol/L) alone effectively inhibited the viability of U251 and U87 cells. When combined with quercetin (30 μmol/L), TMZ (100 μmol/L) significantly inhibited the cell viability, and the inhibition of TMZ (200 and 400 μmol/L) was enhanced. TMZ or quercetin anole did not affect caspase-3 activity and cell apoptosis, while TMZ combined with quercetin significantly increased caspase-3 activity and induced cell apoptosis. TMZ anole significantly increased Hsp27 phosphorylation in U251 and U87 cells, while quercetin or Hsp27 siRNA oligos combined with TMZ attenuated TMZ-induced Hsp27 phosphorylation and significantly inhibited Hsp27 expression. Combined treatment with TMZ and quercetin efficiently suppressed human glioblastoma cell survival in vitro.

Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli - an in vitro study

PubMed Central

2010-01-01

Background Probiotic bacteria are suggested to play a role in the maintenance of oral health. Such health promoting bacteria are added to different commercial probiotic products. The aim of the study was to investigate the ability of a selection of lactobacilli strains, used in commercially available probiotic products, to inhibit growth of oral mutans streptococci and C. albicans in vitro. Methods Eight probiotic lactobacilli strains were tested for growth inhibition on three reference strains and two clinical isolates of mutans streptococci as well as two reference strains and three clinical isolates of Candida albicans with an agar overlay method. Results At concentrations ranging from 109 to 105 CFU/ml, all lactobacilli strains inhibited the growth of the mutans streptococci completely with the exception of L. acidophilus La5 that executed only a slight inhibition of some strains at concentrations corresponding to 107 and 105 CFU/ml. At the lowest cell concentration (103 CFU/ml), only L. plantarum 299v and L. plantarum 931 displayed a total growth inhibition while a slight inhibition was seen for all five mutans streptococci strains by L. rhamnosus LB21, L. paracasei F19, L. reuteri PTA 5289 and L. reuteri ATCC 55730. All the tested lactobacilli strains reduced candida growth but the effect was generally weaker than for mutans streptococci. The two L. plantarum strains and L. reuteri ATCC 55730 displayed the strongest inhibition on Candida albicans. No significant differences were observed between the reference strains and the clinical isolates. Conclusion The selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of oral mutans streptococci and Candida albicans in vitro. PMID:20598145

Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli--an in vitro study.

PubMed

Hasslöf, Pamela; Hedberg, Maria; Twetman, Svante; Stecksén-Blicks, Christina

2010-07-02

Probiotic bacteria are suggested to play a role in the maintenance of oral health. Such health promoting bacteria are added to different commercial probiotic products. The aim of the study was to investigate the ability of a selection of lactobacilli strains, used in commercially available probiotic products, to inhibit growth of oral mutans streptococci and C. albicans in vitro. Eight probiotic lactobacilli strains were tested for growth inhibition on three reference strains and two clinical isolates of mutans streptococci as well as two reference strains and three clinical isolates of Candida albicans with an agar overlay method. At concentrations ranging from 109 to 105 CFU/ml, all lactobacilli strains inhibited the growth of the mutans streptococci completely with the exception of L. acidophilus La5 that executed only a slight inhibition of some strains at concentrations corresponding to 107 and 105 CFU/ml. At the lowest cell concentration (103 CFU/ml), only L. plantarum 299v and L. plantarum 931 displayed a total growth inhibition while a slight inhibition was seen for all five mutans streptococci strains by L. rhamnosus LB21, L. paracasei F19, L. reuteri PTA 5289 and L. reuteri ATCC 55730. All the tested lactobacilli strains reduced candida growth but the effect was generally weaker than for mutans streptococci. The two L. plantarum strains and L. reuteri ATCC 55730 displayed the strongest inhibition on Candida albicans. No significant differences were observed between the reference strains and the clinical isolates. The selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of oral mutans streptococci and Candida albicans in vitro.

Complete Genome Sequences of Three Bacillus amyloliquefaciens Strains That Inhibit the Growth of Listeria monocytogenes In Vitro.

PubMed

Tran, Thao D; Huynh, Steven; Parker, Craig T; Hnasko, Robert; Gorski, Lisa; McGarvey, Jeffery A

2018-06-21

Here, we report the complete genome sequences of three Bacillus amyloliquefaciens strains isolated from alfalfa, almond drupes, and grapes that inhibited the growth of Listeria monocytogenes strain 2011L-2857 in vitro We also report multiple gene clusters encoding secondary metabolites that may be responsible for the growth inhibition of L. monocytogenes . Copyright © 2018 Tran et al.

Nitric oxide induced by Indian ginseng root extract inhibits Infectious Bursal Disease virus in chicken embryo fibroblasts in vitro.

PubMed

Ganguly, Bhaskar; Umapathi, Vijaypillai; Rastogi, Sunil Kumar

2018-01-01

Infectious Bursal Disease is a severe viral disease of chicken responsible for serious economic losses to poultry farmers. The causative agent, Infectious Bursal Disease virus, is inhibited by nitric oxide. Root extract of the Indian ginseng, Withania somnifera , inhibits Infectious Bursal Disease virus in vitro. Also, Withania somnifera root extract is known to induce nitric oxide production in vitro. Therefore, the present study was undertaken to determine if the inhibitory activity of Withania somnifera against Infectious Bursal Disease virus was based on the production of nitric oxide. We show that besides other mechanisms, the inhibition of Infectious Bursal Disease virus by Withania somnifera involves the production of nitric oxide. Our results also highlight the paradoxical role of nitric oxide in the pathogenesis of Infectious Bursal Disease.

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

2017-07-03

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.

TGFβ Pathway Inhibition Redifferentiates Human Pancreatic Islet β Cells Expanded In Vitro

PubMed Central

Toren-Haritan, Ginat; Efrat, Shimon

2015-01-01

In-vitro expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of β-cell-derived (BCD) cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT). Nevertheless, BCD cells maintain open chromatin structure at β-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor β (TGFβ) pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion in vitro involves upregulation of the TGFβ pathway. Blocking TGFβ pathway activation using short hairpin RNA (shRNA) against TGFβ Receptor 1 (TGFBR1, ALK5) transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with ALK5 shRNA results in their redifferentiation, as judged by expression of β-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. ALK5 inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following ex-vivo expansion. PMID:26418361

Saw palmetto extracts potently and noncompetitively inhibit human alpha1-adrenoceptors in vitro.

PubMed

Goepel, M; Hecker, U; Krege, S; Rübben, H; Michel, M C

1999-02-15

We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have alpha1-adrenoceptor antagonistic properties in vitro. Preparations of beta-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic alpha1-adrenoceptors and [3H]prazosin binding to cloned human alpha1A- and alpha1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. Up to the highest concentration which could be tested, preparations of beta-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human alpha1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined alpha1-adrenoceptor antagonists. Saw palmetto extracts have alpha1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined alpha1-adrenoceptor antagonists, alpha1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction.

Interleukin-1 and tumor necrosis factor alpha inhibit repair of the porcine meniscus in vitro

PubMed Central

Hennerbichler, Alfred; Moutos, Franklin T.; Hennerbichler, Diana; Weinberg, J. Brice; Guilak, Farshid

2011-01-01

OBJECTIVE Injury or removal of the knee meniscus leads to progressive joint degeneration, and current surgical therapies for meniscal tears seek to maximally preserve meniscal structure and function. However, the factors that influence intrinsic repair of the meniscus are not well understood. The goal of this study was to investigate the capacity of meniscus tissue to repair a simulated defect in vitro and to examine the effect of pro-inflammatory cytokines on this process. METHODS Cylindrical explants were harvested from the outer one-third of medial porcine menisci. To simulate a full-thickness defect, a central core was removed and reinserted immediately into the defect. Explants were cultured for 2, 4, or 6 weeks in serum-containing media in the presence or absence of interleukin-1 (IL-1) or tumor necrosis factor alpha (TNF-alpha), and meniscal repair was investigated using mechanical testing and fluorescence confocal microscopy. RESULTS Meniscal lesions in untreated samples showed a significant capacity for intrinsic repair in vitro, with increasing cell accumulation and repair strength over time in culture. In the presence of IL-1 or TNF-alpha, no repair was observed despite the presence of abundant viable cells. CONCLUSIONS This study demonstrates that the meniscus exhibits an intrinsic repair response in vitro. However, the presence of pro-inflammatory cytokines completely inhibited repair. These findings suggest that increased levels of pro-inflammatory cytokines post-injury or under arthritic conditions may inhibit meniscal repair. Therefore, inhibition of these cytokines may provide a means of accelerating repair of damaged or injured menisci in vivo. PMID:17448702

Inhibition of venom serine proteinase and metalloproteinase activities by Renealmia alpinia (Zingiberaceae) extracts: comparison of wild and in vitro propagated plants.

PubMed

Patiño, Arley Camilo; Benjumea, Dora María; Pereañez, Jaime Andrés

2013-09-16

The plant Renealmia alpinia has been used in folk medicine to treat snakebites in the northwest region of Colombia. In addition, it has been shown to neutralize edema-forming, hemorrhagic, lethal, and defibrin(ogen)ating activities of Bothrops asper venom. In this work, extracts of Renealmia alpinia obtained by micropropagation (in vitro) and from specimens collected in the wild were tested and compared in their capacity to inhibit enzymatic and toxic activities of a snake venom metalloproteinase isolated from Bothrops atrox (Batx-I) venom and a serine proteinase (Cdc SII) from Crotalus durissus cumanensis venom. We have investigated the inhibition capacity of Renealmia alpinia extracts on enzymatic and toxic actions of isolated toxins, a metalloproteinase and a serine proteinase. The protocols investigated included inhibition of proteolytic activity on azocasein, inhibition of proteolytic activity on fibrinogen, inhibition of pro-coagulant activity, inhibition of hemorrhagic activity and inhibition of edema-forming activity. Colorimetric assays detected the presence of terpenoids, flavonoids, tannins and coumarins in Renealmia alpinia extracts. Renealmia alpinia extracts inhibited the enzymatic, hemorrhagic and fibrinogenolytic activities of Batx-I. Extracts also inhibited coagulant, defibrin(ogen)ating and edema-forming activities of Cdc SII. Results highlight that Renealmia alpinia in vitro extract displayed comparable inhibitory capacity on venom proteinases that Renealmia alpinia wild extract. No alteration was observed in the electrophoretic pattern of venom proteinases after incubation with Renealmia alpinia extracts, thus excluding proteolytic degradation or protein denaturation/precipitation as a mechanism of inhibition. Our results showed that Renealmia alpinia wild and in vitro extracts contain compounds that neutralize metallo- and serine proteinases present in snake venoms. The mechanism of inhibition is not related to proteolytic degradation of the

A preliminary investigation of the enzymatic inhibition of 5alpha-reduction and growth of prostatic carcinoma cell line LNCap-FGC by natural astaxanthin and Saw Palmetto lipid extract in vitro.

PubMed

Anderson, Mark L

2005-01-01

Inhibition of 5alpha-reductase has been reported to decrease the symptoms of benign prostate hyperplasia (BPH) and possibly inhibit or help treat prostate cancer. Saw Palmetto berry lipid extract (SPLE) is reported to inhibit 5alpha-reductase and decrease the clinical symptoms of BPH. Epidemiologic studies report that carotenoids such as lycopene may inhibit prostate cancer. In this investigation the effect of the carotenoid astaxanthin, and SPLE were examined for their effect on 5alpha-reductase inhibition as well as the growth of prostatic carcinoma cells in vitro. These studies support patent #6,277,417 B1. The results show astaxanthin demonstrated 98% inhibition of 5alpha-reductase at 300 microg/mL in vitro. Alphastat, the combination of astaxanthin and SPLE, showed a 20% greater inhibition of 5alpha-reductase than SPLE alone n vitro. A nine day treatment of prostatic carcinoma cells with astaxanthin in vitro produced a 24% decrease in growth at 0.1 mcg/mL and a 38% decrease at 0.01 mcg/mL. SPLE showed a 34% decrease at 0.1 mcg/mL. Low levels of carotenoid astaxanthin inhibit 5alpha-reductase and decrease the growth of human prostatic cancer cells in vitro. Astaxanthin added to SPLE shows greater inhibition of 5alpha-reductase than SPLE alone in vitro.

Inhibitory effect of ebselen on cerebral acetylcholinesterase activity in vitro: kinetics and reversibility of inhibition.

PubMed

Martini, Franciele; Bruning, César Augusto; Soares, Suelen Mendonca; Nogueira, Cristina Wayne; Zeni, Gilson

2015-01-01

Ebselen is a synthetic organoselenium compound that has been considered a potential pharmacological agent with low toxicity, showing antioxidant, anti-inflammatory and neuroprotective effects. It is bioavailable, blood-brain barrier permeant and safe based on cellular toxicity and Phase I-III clinical trials. There is evidence that ebselen inhibits acetylcholinesterase (AChE) activity, an enzyme that plays a key role in the cholinergic system by hydrolyzing acetylcholine (ACh), in vitro and ex vivo. This system has a well-known relationship with cognitive process, and AChE inhibitors, such as donepezil and galantamine, have been used to treat cognitive deficits, mainly in the Alzheimer's Disease (AD). However, these drugs have poor bioavailability and a number of side effects, including gastrointestinal upsets and hepatotoxicity. In this way, this study aimed to evaluate the effect of ebselen on cerebral AChE activity in vitro and to determine the kinetic profile and the reversibility of inhibition by dialysis. Ebselen inhibited the cerebral AChE activity with an IC50 of 29 µM, similar to IC50 found with pure AChE from electric eel, demonstrating a mixed and reversible inhibition of AChE, since it increased Km and decreased Vmax. The AChE activity was recovered within 60 min of dialysis. Therefore, the use of ebselen as a therapeutic agent for treatment of AD should be considered, although memory behavior tasks are needed to support such hypothesis.

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. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

PubMed Central

Woodman, P G; Adamczewski, J P; Hunt, T; Warren, G

1993-01-01

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment. Images PMID:8334308

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

PubMed

Woodman, P G; Adamczewski, J P; Hunt, T; Warren, G

1993-05-01

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

Iron inhibits hydroxyapatite crystal growth in vitro.

PubMed

Guggenbuhl, Pascal; Filmon, Robert; Mabilleau, Guillaume; Baslé, Michel F; Chappard, Daniel

2008-07-01

Hemochromatosis is a known cause of osteoporosis in which the pathophysiology of bone loss is largely unknown and the role of iron remains questionable. We have investigated the effects of iron on the growth of hydroxyapatite crystals in vitro on carboxymethylated poly(2-hydroxyethyl methacrylate) pellets. This noncellular and enzyme-independent model mimics the calcification of woven bone (composed of calcospherites made of hydroxyapatite crystals). Polymer pellets were incubated with body fluid containing iron at increasing concentrations (20, 40, 60 micromol/L). Hydroxyapatite growth was studied by chemical analysis, scanning electron microscopy, and Raman microscopy. When incubated in body fluid containing iron, significant differences were observed with control pellets. Iron was detected at a concentration of 5.41- to 7.16-fold that of controls. In pellets incubated with iron, there was a approximately 3- to 4-fold decrease of Ca and P and a approximately 1.3- to 1.4-fold increase in the Ca/P ratio. There was no significant difference among the iron groups of pellets, but a trend to a decrease of Ca with the increase of iron concentration was noted. Calcospherite diameters were significantly lower on pellets incubated with iron. Raman microspectroscopy showed a decrease in crystallinity (measured by the full width of the half height of the 960 Deltacm(-1) band) with a significant increase in carbonate substitution (measured by the intensity ratio of 1071 to 960 Deltacm(-1) band). Energy dispersive x-ray analysis identified iron in the calcospherites. In vitro, iron is capable to inhibit bone crystal growth with significant changes in crystallinity and carbonate substitution.

Ebselen inhibits the activity of acetylcholinesterase globular isoform G4 in vitro and attenuates scopolamine-induced amnesia in mice.

PubMed

Martini, Franciele; Pesarico, Ana P; Brüning, César A; Zeni, Gilson; Nogueira, Cristina W

2018-02-05

There is a well-known relationship between the cholinergic system and learning, memory, and other common cognitive processes. The process for researching and developing new drugs has lead researchers to repurpose older ones. This study investigated the effects of ebselen on the activity of acethylcholinesterase (AChE) isoforms in vitro and in an amnesia model induced by scopolamine in Swiss mice. In vitro, ebselen at concentrations equal or higher than 10 μM inhibited the activity of cortical and hippocampal G4/AChE, but not G1/AChE isoform. Treatment of mice with ebselen (50 mg/kg, i.p.) was effective against impairment of spatial recognition memory in both Y-maze and novel object recognition tests induced by scopolamine (1 mg/kg, i.p.). Ebselen (50 mg/kg) inhibited hippocampal AChE activity in mice. The present study demonstrates that ebselen inhibited the G4/AChE isoform in vitro and elicited an anti-amnesic effect in a mouse model induced by scopolamine. These findings reveal ebselen as a potential compound in terms of opening up valid therapeutic avenues for the treatment of memory impairment diseases. © 2018 Wiley Periodicals, Inc.

Arctigenin inhibits prostate tumor cell growth in vitro and in vivo

PubMed Central

Wang, Piwen; Solorzano, Walter; Diaz, Tanya; Magyar, Clara E.; Henning, Susanne M.; Vadgama, Jaydutt V.

2017-01-01

The low bioavailability of most phytochemicals limits their translation to humans. We investigated whether arctigenin, a novel anti-inflammatory lignan from the seeds of Arctium lappa, has favorable bioavailability/potency against prostate cancer. The anticarcinogenic activity of arctigenin was investigated both in vitro using the androgen-sensitive LNCaP and LAPC-4 human prostate cancer cells and pre-malignant WPE1-NA22 cells, and in vivo using xenograft mouse models. Arctigenin at lower doses (< 2μM) significantly inhibited the proliferation of LNCaP and LAPC-4 cells by 30-50% at 48h compared to control, and inhibited WPE1-NA22 cells by 75%, while did not affect normal prostate epithelial cells. Male severe combined immunodeficiency (SCID) mice were implanted subcutaneously with LAPC-4 cells for in vivo studies. In one experiment, the intervention started one week after tumor implantation. Mice received arctigenin at 50mg/kg (LD) or 100mg/kg (HD) b.w. daily or vehicle control by oral gavage. After 6 weeks, tumor growth was inhibited by 50% (LD) and 70% (HD) compared to control. A stronger tumor inhibitory effect was observed in a second experiment where arctigenin intervention started two weeks prior to tumor implantation. Arc was detectable in blood and tumors in Arc groups, with a mean value up to 2.0 μM in blood, and 8.3 nmol/g tissue in tumors. Tumor levels of proliferation marker Ki67, total and nuclear androgen receptor, and growth factors including VEGF, EGF, and FGF-β were significantly decreased by Arc, along with an increase in apoptosis marker of Bax/Bcl-2 ratio. Genes responsive to arctigenin were identified including TIMP3 and ZNF185, and microRNAs including miR-126-5p, and miR-21-5p. This study provides the first in vivo evidence of the strong anticancer activity of arctigenin in prostate cancer. The effective dose of arctigenin in vitro is physiologically achievable in vivo, which provides a high promise in its translation to human application

Arctigenin inhibits prostate tumor cell growth in vitro and in vivo.

PubMed

Wang, Piwen; Solorzano, Walter; Diaz, Tanya; Magyar, Clara E; Henning, Susanne M; Vadgama, Jaydutt V

2017-06-01

The low bioavailability of most phytochemicals limits their translation to humans. We investigated whether arctigenin, a novel anti-inflammatory lignan from the seeds of Arctium lappa , has favorable bioavailability/potency against prostate cancer. The anticarcinogenic activity of arctigenin was investigated both in vitro using the androgen-sensitive LNCaP and LAPC-4 human prostate cancer cells and pre-malignant WPE1-NA22 cells, and in vivo using xenograft mouse models. Arctigenin at lower doses (< 2μM) significantly inhibited the proliferation of LNCaP and LAPC-4 cells by 30-50% at 48h compared to control, and inhibited WPE1-NA22 cells by 75%, while did not affect normal prostate epithelial cells. Male severe combined immunodeficiency (SCID) mice were implanted subcutaneously with LAPC-4 cells for in vivo studies. In one experiment, the intervention started one week after tumor implantation. Mice received arctigenin at 50mg/kg (LD) or 100mg/kg (HD) b.w. daily or vehicle control by oral gavage. After 6 weeks, tumor growth was inhibited by 50% (LD) and 70% (HD) compared to control. A stronger tumor inhibitory effect was observed in a second experiment where arctigenin intervention started two weeks prior to tumor implantation. Arc was detectable in blood and tumors in Arc groups, with a mean value up to 2.0 μM in blood, and 8.3 nmol/g tissue in tumors. Tumor levels of proliferation marker Ki67, total and nuclear androgen receptor, and growth factors including VEGF, EGF, and FGF-β were significantly decreased by Arc, along with an increase in apoptosis marker of Bax/Bcl-2 ratio. Genes responsive to arctigenin were identified including TIMP3 and ZNF185, and microRNAs including miR-126-5p, and miR-21-5p. This study provides the first in vivo evidence of the strong anticancer activity of arctigenin in prostate cancer. The effective dose of arctigenin in vitro is physiologically achievable in vivo , which provides a high promise in its translation to human application.

A novel antiangiogenic peptide derived from hepatocyte growth factor inhibits neovascularization in vitro and in vivo

PubMed Central

Xu, Yi; Zhao, Hui; Zheng, Ying; Gu, Qing; Ma, Jianxing

2010-01-01

Purpose To study the antiangiogenic activity of two small peptides (H-RN and H-FT) derived from the hepatocyte growth factor kringle 1 domain (HGF K1) using in vitro and in vivo assays. Methods RF/6A rhesus macaque choroid-retina endothelial cells were used for in vitro studies. The inhibiting effect of two peptides on a vascular endothelial growth factor (VEGF)-stimulated cell proliferation, cell migration, and endothelial cell tube formation were investigated. For in vivo assays, the antiangiogenic activity of H-RN and H-FT in the chick chorioallantoic membrane model (CAM) and a mice oxygen-induced retinopathy model (OIR) were studied. A recombinant mouse VEGF-neutralizing antibody, bevacizumab, and a scrambled peptide were used as two control groups in separate studies. Results H-RN effectively inhibited VEGF-stimulated RF/6A cell proliferation, migration, and tube formation on Matrigel™, while H-FT did not. H-RN was also able to inhibit angiogenesis when applied to the CAM, and had antineovascularization activity in the retinal neovascularization of a mouse OIR model when administrated as an intravitreous injection. The antiangiogenic activity of H-RN was not as strong as that of VEGF antibodies. The H-FT and scrambled peptide had no such activity. Conclusions H-RN, a new peptide derived from the HGF K1 domain, was shown to have antiangiogenic activity in vitro and in vivo. It may lead to new potential drug discoveries and the development of new treatments for pathological retinal angiogenesis. PMID:21031024

Cytochrome P450 2C9-natural antiarthritic interactions: Evaluation of inhibition magnitude and prediction from in vitro data.

PubMed

Tan, Boon Hooi; Ahemad, Nafees; Pan, Yan; Palanisamy, Uma Devi; Othman, Iekhsan; Yiap, Beow Chin; Ong, Chin Eng

2018-04-01

Many dietary supplements are promoted to patients with osteoarthritis (OA) including the three naturally derived compounds, glucosamine, chondroitin and diacerein. Despite their wide spread use, research on interaction of these antiarthritic compounds with human hepatic cytochrome P450 (CYP) enzymes is limited. This study aimed to examine the modulatory effects of these compounds on CYP2C9, a major CYP isoform, using in vitro biochemical assay and in silico models. Utilizing valsartan hydroxylase assay as probe, all forms of glucosamine and chondroitin exhibited IC 50 values beyond 1000 μM, indicating very weak potential in inhibiting CYP2C9. In silico docking postulated no interaction with CYP2C9 for chondroitin and weak bonding for glucosamine. On the other hand, diacerein exhibited mixed-type inhibition with IC 50 value of 32.23 μM and K i value of 30.80 μM, indicating moderately weak inhibition. Diacerein's main metabolite, rhein, demonstrated the same mode of inhibition as diacerein but stronger potency, with IC 50 of 6.08 μM and K i of 1.16 μM. The docking of both compounds acquired lower CDOCKER interaction energy values, with interactions dominated by hydrogen and hydrophobic bondings. The ranking with respect to inhibition potency for the investigated compounds was generally the same in both in vitro enzyme assay and in silico modeling with order of potency being diacerein/rhein > various glucosamine/chondroitin forms. In vitro-in vivo extrapolation of inhibition kinetics (using 1 + [I]/K i ratio) demonstrated negligible potential of diacerein to cause interaction in vivo, whereas rhein was predicted to cause in vivo interaction, suggesting potential interaction risk with the CYP2C9 drug substrates. Copyright © 2018 John Wiley & Sons, Ltd.

In vitro inhibition of oral Candida albicans by chicken egg yolk antibody (IgY).

PubMed

Wang, X Z; Fan, B; Liu, L G; Hu, X Y; Li, R Y; Wei, Y; Wan, Z; Deng, X L

2008-06-01

This study was conducted to measure Candida albicans-specific chicken egg yolk antibody (IgY) inhibition of fluconazole-sensitive and resistant strains of C. albicans in order to assess potential use in the prevention and treatment of oral candidiasis. In this study, laying hens were immunized, and IgY was extracted by water dilution. The Minimal Inhibitory Concentrations (MICs) of IgY for inhibiting C. albicans growth were determined using the broth microdilution method from the CLSI M27-A2 protocol. Fluconazole (FLC) was used as the control. The results were analyzed with the chi(2) test. The anti-Candida titer of anti-C. albicans IgY was 1:12,000. The concentration of the IgY extract that effectively inhibited the growth of C. albicans was between 1.25 g/l and 5.0 g/l, and the efficacy rate was 82.98% during the observed 24-48 h time period. No correlation was recorded between the drug resistance of FLC and growth inhibition by IgY. It was concluded that anti-C. albicans IgY inhibited the growth of C. albicans in vitro and there was no correlation between the drug resistance of FLC and the growth inhibition by IgY (P > 0.99).

Effective inhibition of nasopharyngeal carcinoma in vitro and in vivo by targeting glycolysis with oxamate.

PubMed

Li, Xiaobing; Lu, Wenhua; Hu, Yumin; Wen, Shijun; Qian, Chaonan; Wu, Wenjing; Huang, Peng

2013-11-01

Elevated aerobic glycolysis in cancer cells (Warburg effect) has been observed in many tumor types including nasopharyngeal carcinoma (NPC), which can often be detected clinically using FDG-PET. However, the role of glycolysis in supporting the growth of NPC cells and its therapeutic implications still remain to be investigated. In the present study, we showed that the LDH inhibitor oxamate significantly suppressed NPC cell proliferation in vitro and tumor growth in vivo, yet exhibited minimum toxicity to normal nasopharyngeal epithelial cells in vitro and was well tolerated in mice. Moreover, oxamate exhibited cytotoxic effect in NPC cells under hypoxia. Mechanistic study showed that oxamate significantly inhibited LDH activity, leading to a substantial decrease in glucose uptake and lactate production. Combination of oxamate with a mitochondrial respiratory complex I inhibitor resulted in a significant depletion of cellular ATP and a synergistic killing of cancer cells. Our results suggest that inhibition of glycolysis by oxamate is an effective therapeutic strategy for treatment of NPC and that combination of this compound with mitochondrial-targeted agents may improve the therapeutic activity.

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...

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

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

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.

In Vitro Modulation of Renin-Angiotensin System Enzymes by Amaranth (Amaranthus hypochondriacus) Protein-Derived Peptides: Alternative Mechanisms Different from ACE Inhibition.

PubMed

Quiroga, Alejandra V; Aphalo, Paula; Nardo, Agustina E; Añón, María C

2017-08-30

Among the factors affecting the development of cardiovascular diseases, hypertension is one of the most important. Research done on amaranth proteins has demonstrated their hypotensive capacity in vivo and in vitro; nevertheless, the mechanism underlying this effect remains unclear. The aim of this study was to analyze in vitro the inhibition of peptides derived from an amaranth hydrolysate (AHH) on other RAS enzymes other than ACE. The chymase and renin activities were studied. AHH was not able to inhibit chymase activity, although a dose-response effect was found on renin activity (IC 50 0.6 mg/mL). To provide an approach to the renin inhibition mechanism, we analyzed AHH renin inhibition kinetics and performed a structural characterization of the peptides involved in the effect in terms of molecular size and hydrophobicity. Results suggest that amaranth peptides exhibit renin competitive inhibition behavior. Renin inhibition potency was directly related to peptide hydrophobicity. RP-HPLC separation of AHH and subsequent analysis of the peptide sequences showed 6 peptides belonging to 11S globulin (that can be grouped into 3 families) that would be responsible for renin inhibition. These results demonstrate that Amaranthus hypochondriacus seeds are an adequate source of peptides with renin inhibitory properties that could be used in functional food formulations.

Radiosensitization of Pancreatic Cancer Cells In Vitro and In Vivo through Poly (ADP-ribose) Polymerase Inhibition with ABT-888.

PubMed

Tuli, Richard; Surmak, Andrew J; Reyes, Juvenal; Armour, Michael; Hacker-Prietz, Amy; Wong, John; DeWeese, Theodore L; Herman, Joseph M

2014-05-13

To determine whether poly (ADP-ribose) polymerase-1/2 (PARP-1/2) inhibition enhances radiation-induced cytotoxicity of pancreatic adenocarcinoma in vitro and in vivo, and the mechanism by which this occurs. Pancreatic carcinoma cells were treated with ABT-888, radiation, or both. In vitro cell viability, apoptosis, and PARP activity were measured. Orthotopic xenografts were generated in athymic mice and treated with ABT-888 (25mg/kg), radiation (5Gy), both, or no treatment. Mice were monitored with bioluminescence imaging. In vitro, treatment with ABT-888 and radiation led to higher rates of cell death after 8days (P < .01). Co-treatment with 5Gy and 1, 10 or 100μmol/l of ABT-888 led to dose enhancement factors of 1.29, 1.41 and 2.36, respectively. Caspase activity was not significantly increased when treated with ABT-888 (10 μmol/l) alone (1.28-fold, P = .08), but became significant when radiation was added (2.03-fold, P < .01). PARP activity increased post-radiation and was abrogated following co-treatment with ABT-888. In vivo, treatment with ABT-888, radiation or both led to tumor growth inhibition (TGI) of 8, 30 and 39days, and survival at 60days of 0%, 0% and 40%, respectively. ABT-888 with radiation significantly enhanced tumor response in vitro and in vivo. ABT-888 inhibited PAR protein polymerization resulting in dose-dependent feedback up-regulation of PARP and p-ATM suggesting increased DNA damage. This translated into enhancement in TGI and survival with radiation in vivo. In vitro PAR levels correlated with levels of tumor apoptosis suggesting potential as a predictive biomarker. These data are being used to support a Phase I study in locally advanced pancreatic cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibition of lymphocyte proliferation and antibody production in vitro by silica, talc, bentonite or Corynebacterium parvum: involvement of peroxidative processes.

PubMed

Hoffeld, J T

1983-05-01

This study was undertaken to determine whether and by what means particles which induce granulomata in vivo can affect murine spleen lymphoproliferative and antibody responses in vitro. Particles of silica, talc, Bentonite or C. parvum cells inhibited lipopolysaccharide- or concanavalin A-stimulated proliferation and sheep red blood cell-induced antibody response in vitro. The inhibition required at least 48 hours exposure of the cells to the particles. The late onset of inhibition and its reproducibility at different cell or mitogen concentrations implicated particle-induced injury to both phagocytes and lymphocytes. Either alpha-tocopherol or 2-mercaptoethanol prevented the particle-induced inhibition of spleen cell responses. alpha-Tocopherol and 2-mercaptoethanol have in common the capacity to protect cells against membrane lipid peroxidation. The inhibitory peroxidative process(es) implicated by these studies are most likely attributable to: (a) stimulation of oxidative metabolism of phagocytic cells by particles; and (b) iron-catalyzed peroxidation directly by the particles. These data may be relevant in understanding the pathogenesis of and devising therapeutic approaches toward various granulomatous conditions.

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat.

PubMed

Algeelani, Sara; Alam, Novera; Hossain, Md Amin; Mikus, Gerd; Greenblatt, David J

2018-08-01

1. Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer's data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat. 2. Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors. 3. The 50% inhibitory concentrations (IC 50 ) of cobicistat and ritonavir were at least 50 µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC 50 0.7 mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC 50 17 µM). 4. Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.

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...

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.

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.

Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression.

PubMed

Mack, D R; Michail, S; Wei, S; McDougall, L; Hollingsworth, M A

1999-04-01

Probiotic agents, live microorganisms with beneficial effects for the host, may offer an alternative to conventional antimicrobials in the treatment and prevention of enteric infections. The probiotic agents Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing pathogenic Escherichia coli to HT-29 intestinal epithelial cells but did not inhibit adherence to nonintestinal HEp-2 cells. HT-29 cells were grown under conditions that induced high levels of either MUC2 or MUC3 mRNA, but HEp-2 cells expressed only minimal levels of MUC2 and no MUC3 mRNA. Media enriched for MUC2 and MUC3 mucin were added exogenously to binding assays and were shown to be capable of inhibiting enteropathogen adherence to HEp-2 cells. Incubation of L. plantarum 299v with HT-29 cells increased MUC2 and MUC3 mRNA expression levels. From these in vitro studies, we propose the hypothesis that the ability of probiotic agents to inhibit adherence of attaching and effacing organisms to intestinal epithelial cells is mediated through their ability to increase expression of MUC2 and MUC3 intestinal mucins.

Casticin, a flavonoid isolated from Vitex rotundifolia, inhibits prolactin release in vivo and in vitro

PubMed Central

Ye, Qi; Zhang, Qiao-yan; Zheng, Cheng-jian; Wang, Yang; Qin, Lu-ping

2010-01-01

Aim: To investigate the anti-hyperprolactinemia activity of casticin, a flavonoid isolated from Vitex rotundifolia, and elucidate its molecular mechanism. Methods: Hyperprolactinemia (MIHP) was induced by administration of metoclopramide dihydrochloride (50 mg/kg, tid, ip, for 10 d) in SD rats and the primary pituitary cells were prepared from the pituitary glands of the SD rats. Prolactin concentrations were measured using a radioimmunoassay. Cell viability was measured using an MTT assay. The mRNA expression of estrogen receptor alpha and beta in rat pituitary cells was measured using semi-quantitative RT-PCR analysis. Results: The level of serum prolactin in the MIHP model group was 2.1 fold higher than that in the untreated control group (P<0.01). Casticin (10, 20, and 40 mg/kg, ip, for 7 d) reduced serum prolactin levels by 33.9%, 54.3%, and 64.7%, respectively (P<0.01). The positive control drug bromocriptine 1 mg/kg decreased the serum prolactin concentration in MIHP rats by 44.9%. 17β-Estradiol (E2) significantly increased the proliferation of pituitary cells and casticin (1 and 10 μmol/L) markedly inhibited E2-induced pituitary cell proliferation by 27.7% and 42.1%, respectively. Stimulation of pituitary cells with E2 increased prolactin secretion into the cell culture supernatants, and casticin (0.1, 1, and 10 μmol/L) significantly inhibited the prolactin release stimulated by E2 in a concentration-dependent manner. Casticin (1 and 10 μmol/L) significantly inhibited ERα mRNA expression in pituitary cells stimulated with E2 (P<0.01) but increased ERβ mRNA expression at a concentration of 10 μmol/L (P<0.01). However, casticin had no effects on proliferation and prolectin release of the unstimulated primary pituitary cells in vitro. Conclusion: Casticin inhibited the release of prolactin from pituitary cells of SD rats stimulated with E2 in vivo and in vitro. These effects might be related with inhibiting the ERα mRNA expression and increasing the

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

2017-02-14

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.

miR-137 inhibits renal cell carcinoma growth in vitro and in vivo

PubMed Central

ZHANG, HONGXIA; LI, HONGJUN

2016-01-01

MicroRNA (miR)-137 has been reported to be underexpressed and involved in various cell processes and to have antitumor effects in a range of tumors, but so far not in renal cell carcinoma (RCC). The aim of the present study was to investigate the clinical significance and role of miR-137 in RCC. The expression levels of miR-137 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in 50 cases of paired RCC tissues and adjacent normal tissues, and in RCC cell lines. The role of miR-137 in the growth and survival of RCC cells was assessed with several in vitro approaches and in nude mice models. The results of the RT-qPCR showed that the miR-137 expression was downregulated in the RCC tissues and cell lines. The in vitro assay showed that ectopic expression of miR-137 robustly impaired RCC cell proliferation, migratory and invasive properties, and increased the induction of cell apoptosis properties. The in vivo assay demonstrated that enforced miR-137 suppressed tumor growth in xenograft nude mice models. In addition, miR-137 was indicated to inhibit the activation of phosphoinositide 3 kinase/protein kinase B signal pathway, which contributes to the inhibition of RCC growth. These findings indicate that miR-137 functions as tumor suppressor in RCC, suggesting that miR-137 may be a potential therapeutic target for RCC. PMID:27347205

Coffee inhibition of CYP3A4 in vitro was not translated to a grapefruit-like pharmacokinetic interaction clinically.

PubMed

Dresser, George K; Urquhart, Brad L; Proniuk, Julianne; Tieu, Alvin; Freeman, David J; Arnold, John Malcolm; Bailey, David G

2017-10-01

Grapefruit can augment oral medication bioavailability through irreversible (mechanism-based) inhibition of intestinal CYP3A4. Supplementary data from our recent coffee-drug interaction clinical study showed some subjects had higher area under the plasma drug concentration - time curve (AUC) and plasma peak drug concentration (Cmax) of the CYP3A4 probe felodipine compared to aqueous control. It was hypothesized that coffee might interact like grapefruit in responsive individuals. Beans from six geographical locations were consistently brewed into coffee that was separated chromatographically to a methanolic fraction for in vitro inhibition testing of CYP3A4 metabolism of felodipine at 1% coffee strength. The effect of simultaneous incubation and 10-min preincubation with coffee fractions determined whether coffee had direct and mechanism-based inhibitory activity. A subsequent five-way randomized balanced controlled crossover clinical study evaluated the clinical pharmacokinetic interaction with single-dose felodipine. Grapefruit juice, water, or three of the in vitro tested coffees were ingested at 300 mL alone 1 h before and then with felodipine. In vitro, all six coffees decreased felodipine metabolism for both simultaneous and preincubation exposure compared to corresponding control. Five coffees demonstrated mechanism-based inhibition. Grapefruit increased felodipine AUC 0-8 (25 vs. 13 ng.h/mL, P < 0.001) and Cmax (5.8 vs. 2.7 ng/mL, P < 0.001) and decreased dehydrofelodipine/felodipine AUC 0-8 ratio (0.84 vs. 1.29, P < 0.001), while the three coffees caused no change in these parameters compared to water. Despite high in vitro potency of CYP3A4 inhibition, the coffees did not cause a clinical pharmacokinetic interaction possibly from insufficient amount of inhibitor(s) in coffee reaching intestinal CYP3A4 during the absorption phase of felodipine. The results of this study highlight the need for follow-up clinical testing when in vitro results

Inhibition of platelet aggregation and in vitro free radical scavenging activity of dried fruiting bodies of Pleurotus eous.

PubMed

Suseem, S R; Saral, Mary

2015-07-01

To evaluate the ethyl acetate, methanol and aqueous extracts of dried fruiting bodies of Pleurotus eous for its anti-platelet activity on human volunteer's blood. And also to analyze the free radical scavenging property of the extracts of P.eous by using various in vitro models. Anti-platelet activity of dried fruiting bodies of P.eous was evaluated by in vitro model using blood platelets. Inhibition of platelet aggregation was monitored after pre-incubation of platelets with the crude extracts of mushroom P.eous. Antioxidant activities of extracts of P.eous were evaluated by different in vitro experiments, namely, 1, 1-diphenyl-2-picryl hydrazyl (DPPH), superoxide, hydroxyl radical and lipid peroxide radical models. Crude extracts of mushroom P.eous inhibited platelet aggregation dose-dependently which was induced by adenosine diphosphate (ADP). At a maximum concentration of 10 mg/mL, methanol extract effected 64.02% inhibition of lipid per-oxidation and 50.12% scavenging effect on superoxide anion radical. Aqueous extract of P.eous have shown 69.43% chelating ability on ferrous ions, 24.27% scavenging effect on hydroxyl radical and 49.57% scavenging effect on DPPH radical at 10 mg/mL. Increasing concentrations of the extract were found to cause progressively decreasing of the intensity of absorbance. Anti-platelet effects could be related in part to the polyphenolic compounds present in the extracts. Antioxidant activity results indicated the free radical scavenging property of the extracts of P.eous which might be due to the high content of phenolic compounds and flavonoids.

Inhibition of human cancer cell line growth and human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro.

PubMed

Chen, Huan-Huan; Zhou, Hui-Jun; Fang, Xin

2003-09-01

Artemisinin derivatives artesunate (ART) and dihydroartemisinin are remarkable anti-malarial drugs with low toxicity to humans. In the present investigation, we find they also inhibited tumor cell growth and suppressed angiogenesis in vitro. The anti-cancer activity was demonstrated by inhibition (IC(50)) of four human cancer cell lines: cervical cancer Hela, uterus chorion cancer JAR, embryo transversal cancer RD and ovarian cancer HO-8910 cell lines growth by the MTT assay. IC(50) values ranged from 15.4 to 49.7 microM or from 8.5 to 32.9 microM after treatment with ART or dihydroartemisinin for 48 h, indicating that dihydroartemisinin was more effective than ART in inhibiting cancer cell lines. The anti-angiogenic activities were tested on in vitro models of angiogenesis, namely, proliferation, migration and tube formation of human umbilical vein endothelial (HUVE) cells. We investigated the inhibitory effects of ART and dihydroartemisinin on HUVE cells proliferation by cell counting, migration into the scratch wounded area in HUVE cell monolayers and microvessel tube-like formation on collagen gel. The results showed ART and dihydroartemisinin significantly inhibited angiogenisis in a dose-dependent form in range of 12.5-50 microM and 2.5-50 microM, respectively. They indicated that dihydroartemisinin was more effective than ART in inhibiting angiogenesis either. These results and the known low toxicity are clues that ART and dihydroartemisinin may be promising novel candidates for cancer chemotherapy.

Bioinformatical and in vitro approaches to essential oil-induced matrix metalloproteinase inhibition.

PubMed

Zeidán-Chuliá, Fares; Rybarczyk-Filho, José L; Gursoy, Mervi; Könönen, Eija; Uitto, Veli-Jukka; Gursoy, Orhan V; Cakmakci, Lutfu; Moreira, José C F; Gursoy, Ulvi K

2012-06-01

Essential oils carry diverse antimicrobial and anti-enzymatic properties. Matrix metalloproteinase (MMP) inhibition characteristics of Salvia fruticosa Miller (Labiatae), Myrtus communis Linnaeus (Myrtaceae), Juniperus communis Linnaeus (Cupressaceae), and Lavandula stoechas Linnaeus (Labiatae) essential oils were evaluated. Chemical compositions of the essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS). Bioinformatical database analysis was performed by STRING 9.0 and STITCH 2.0 databases, and ViaComplex software. Antibacterial activity of essential oils against periodontopathogens was tested by the disc diffusion assay and the agar dilution method. Cellular proliferation and cytotoxicity were determined by commercial kits. MMP-2 and MMP-9 activities were measured by zymography. Bioinformatical database analyses, under a score of 0.4 (medium) and a prior correction of 0.0, gave rise to a model of protein (MMPs and tissue inhibitors of metalloproteinases) vs. chemical (essential oil components) interaction network; where MMPs and essential oil components interconnected through interaction with hydroxyl radicals, molecular oxygen, and hydrogen peroxide. Components from L. stoechas potentially displayed a higher grade of interaction with MMP-2 and -9. Although antibacterial and growth inhibitory effects of essential oils on the tested periodontopathogens were limited, all of them inhibited MMP-2 in vitro at concentrations of 1 and 5 µL/mL. Moreover, same concentrations of M. communis and L. stoechas also inhibited MMP-9. MMP-inhibiting concentrations of essential oils were not cytotoxic against keratinocytes. We propose essential oils of being useful therapeutic agents as MMP inhibitors through a mechanism possibly based on their antioxidant potential.

Hydroxyapatite nanoparticles inhibit the growth of human glioma cells in vitro and in vivo.

PubMed

Chu, Sheng-Hua; Feng, Dong-Fu; Ma, Yan-Bin; Li, Zhi-Qiang

2012-01-01

Hydroxyapatite nanoparticles (nano-HAPs) have been reported to exhibit antitumor effects on various human cancers, but the effects of nano-HAPs on human glioma cells remain unclear. The aim of this study was to explore the inhibitory effect of nano-HAPs on the growth of human glioma U251 and SHG44 cells in vitro and in vivo. Nano-HAPs could inhibit the growth of U251 and SHG44 cells in a dose- and time-dependent manner, according to methyl thiazoletetrazolium assay and flow cytometry. Treated with 120 mg/L and 240 mg/L nano-HAPs for 48 hours, typical apoptotic morphological changes were noted under Hoechst staining and transmission electron microscopy. The tumor growth of cells was inhibited after the injection in vivo, and the related side effects significantly decreased in the nano-HAP-and-drug combination group. Because of the function of nano-HAPs, the expression of c-Met, SATB1, Ki-67, and bcl-2 protein decreased, and the expression of SLC22A18 and caspase-3 protein decreased noticeably. The findings indicate that nano-HAPs have an evident inhibitory action and induce apoptosis of human glioma cells in vitro and in vivo. In a drug combination, they can significantly reduce the adverse reaction related to the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).

Hydroxyapatite nanoparticles inhibit the growth of human glioma cells in vitro and in vivo

PubMed Central

Chu, Sheng-Hua; Feng, Dong-Fu; Ma, Yan-Bin; Li, Zhi-Qiang

2012-01-01

Hydroxyapatite nanoparticles (nano-HAPs) have been reported to exhibit antitumor effects on various human cancers, but the effects of nano-HAPs on human glioma cells remain unclear. The aim of this study was to explore the inhibitory effect of nano-HAPs on the growth of human glioma U251 and SHG44 cells in vitro and in vivo. Nano-HAPs could inhibit the growth of U251 and SHG44 cells in a dose- and time-dependent manner, according to methyl thiazoletetrazolium assay and flow cytometry. Treated with 120 mg/L and 240 mg/L nano-HAPs for 48 hours, typical apoptotic morphological changes were noted under Hoechst staining and transmission electron microscopy. The tumor growth of cells was inhibited after the injection in vivo, and the related side effects significantly decreased in the nano-HAP-and-drug combination group. Because of the function of nano-HAPs, the expression of c-Met, SATB1, Ki-67, and bcl-2 protein decreased, and the expression of SLC22A18 and caspase-3 protein decreased noticeably. The findings indicate that nano-HAPs have an evident inhibitory action and induce apoptosis of human glioma cells in vitro and in vivo. In a drug combination, they can significantly reduce the adverse reaction related to the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). PMID:22888225

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

PubMed Central

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

2017-01-01

Background 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. Methodology/Principal findings 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. Conclusions/Significance 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. PMID:28614380

Prediction of in vivo drug-drug interactions based on mechanism-based inhibition from in vitro data: inhibition of 5-fluorouracil metabolism by (E)-5-(2-Bromovinyl)uracil.

PubMed

Kanamitsu, S I; Ito, K; Okuda, H; Ogura, K; Watabe, T; Muro, K; Sugiyama, Y

2000-04-01

The fatal drug-drug interaction between sorivudine, an antiviral drug, and 5-fluorouracil (5-FU) has been shown to be caused by a mechanism-based inhibition. In this interaction, sorivudine is converted by gut flora to (E)-5-(2-bromovinyl)uracil (BVU), which is metabolically activated by dihydropyrimidine dehydrogenase (DPD), and the activated BVU irreversibly binds to DPD itself, thereby inactivating it. In an attempt to predict this interaction in vivo from in vitro data, inhibition of 5-FU metabolism by BVU was investigated by using rat and human hepatic cytosol and human recombinant DPD. Whichever enzyme was used, increased inhibition was observed that depended on the preincubation time of BVU and enzyme in the presence of NADPH and BVU concentration. The kinetic parameters obtained for inactivation represented by k(inact) and K'(app) were 2.05 +/- 1.52 min(-1), 69.2 +/- 60.8 microM (rat hepatic cytosol), 2.39 +/- 0.13 min(-1), 48.6 +/- 11.8 microM (human hepatic cytosol), and 0.574 +/- 0.121 min(-1), 2.20 +/- 0.57 microM (human recombinant DPD). The drug-drug interaction in vivo was predicted quantitatively based on a physiologically based pharmacokinetic model, using pharmacokinetic parameters obtained from the literature and kinetic parameters for the enzyme inactivation obtained in the in vitro studies. In rats, DPD was predicted to be completely inactivated by administration of BVU and the area under the curve of 5-FU was predicted to increase 11-fold, which agreed well with the reported data. In humans, a 5-fold increase in the area under the curve of 5-FU was predicted after administration of sorivudine, 150 mg/day for 5 days. Mechanism-based inhibition of drug metabolism is supposed to be very dangerous. We propose that such in vitro studies should be carried out during the drug-developing phase so that in vivo drug-drug interactions can be predicted.

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

2017-04-15

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 IC 50 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 IC 50 values between 0.049 and 166μM, whereas four analogs inhibited also MAO-B with IC 50 values between 82 and 376μM. 7-Me-AMT provided the lowest IC 50 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. Copyright © 2017 Elsevier B.V. All rights reserved.

Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semler, B.L.; Hanecak, R.; Dorner, L.F.

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 ofmore » the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site.« less

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

PubMed Central

Kang, Minyong; Jeong, Chang Wook; Ku, Ja Hyeon; Kwak, Cheol; Kim, Hyeon Hoe

2014-01-01

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer. PMID:24815071

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...

In vitro inhibition of monkeypox virus production and spread by Interferon-β

PubMed Central

2012-01-01

Background The Orthopoxvirus genus contains numerous virus species that are capable of causing disease in humans, including variola virus (the etiological agent of smallpox), monkeypox virus, cowpox virus, and vaccinia virus (the prototypical member of the genus). Monkeypox is a zoonotic disease that is endemic in the Democratic Republic of the Congo and is characterized by systemic lesion development and prominent lymphadenopathy. Like variola virus, monkeypox virus is a high priority pathogen for therapeutic development due to its potential to cause serious disease with significant health impacts after zoonotic, accidental, or deliberate introduction into a naïve population. Results The purpose of this study was to investigate the prophylactic and therapeutic potential of interferon-β (IFN-β) for use against monkeypox virus. We found that treatment with human IFN-β results in a significant decrease in monkeypox virus production and spread in vitro. IFN-β substantially inhibited monkeypox virus when introduced 6-8 h post infection, revealing its potential for use as a therapeutic. IFN-β induced the expression of the antiviral protein MxA in infected cells, and constitutive expression of MxA was shown to inhibit monkeypox virus infection. Conclusions Our results demonstrate the successful inhibition of monkeypox virus using human IFN-β and suggest that IFN-β could potentially serve as a novel safe therapeutic for human monkeypox disease. PMID:22225589

Delta-9 tetrahydrocannabinol (THC) inhibits lytic replication of gamma oncogenic herpesviruses in vitro.

PubMed

Medveczky, Maria M; Sherwood, Tracy A; Klein, Thomas W; Friedman, Herman; Medveczky, Peter G

2004-09-15

The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication. Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method. Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68. THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development

Esculin and its oligomer fractions inhibit adhesion and migration of U87 glioblastoma cells and in vitro angiogenesis.

PubMed

Mokdad-Bzeouich, Imen; Kovacic, Hervé; Ghedira, Kamel; Chebil, Latifa; Ghoul, Mohamed; Chekir-Ghedira, Leila; Luis, José

2016-03-01

Cancer metastasis is the major cause of cancer-related death. Chemoprevention is defined as the use of natural or synthetic substances to prevent cancer formation or cancer progress. In the present study, we investigate the antitumor activity of esculin and its oligomer fractions in U87 glioblastoma cells. We showed that esculin and its oligomers reduced U87 cell growth in a dose dependent manner. They also inhibited cell adhesion to collagen IV and vitronectin by interfering with the function of their respective receptors α2β1 and αvβ5 integrins. Furthermore, the tested samples were able to reduce migration of U87 cells towards another extracellular matrix fibronectin. Moreover, esculin and its oligomer fractions inhibited in vitro angiogenesis of endothelial cells (HMEC-1). In summary, our data provide the first evidence that esculin and its oligomer fractions are able to reduce adhesion, migration of glioblastoma cells and in vitro angiogenesis. Esculin and its oligomers may thus exert multi-target functions against cancer cells.

Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion.

PubMed Central

Pellegatta, F.; Lu, Y.; Radaelli, A.; Zocchi, M. R.; Ferrero, E.; Chierchia, S.; Gaja, G.; Ferrero, M. E.

1996-01-01

1. Leukocyte-endothelial cell interactions play an important role during ischaemia-reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2. Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia-reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3. In basal conditions, defibrotide (1000 micrograms ml-1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% +/- 3.6 (P < 0.05), and after endothelial cell stimulation (TNF-alpha, 500 u ml-1) or after leukocyte stimulation (fMLP, 10(-7) M), it inhibited leukocyte adhesion by 26.5% +/- 3.4 and 32.4% +/- 1.8, respectively (P < 0.05). 4. In adhesion blockage experiments, the use of the monoclonal antibody anti-CD31 (5 micrograms ml-1) did not demonstrate a significant inhibitory effect whereas use of the monoclonal antibody anti-LFA-1 (5 micrograms ml-1) significantly interfered with the effect of defibrotide. 5. This result was confirmed in NIH/3T3-ICAM-1 transfected cells. 6. We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM-1/LFA-1 adhesion system is involved in the defibrotide mechanism of action. PMID:8762067

Aqueous extracts of Roselle (Hibiscus sabdariffa Linn.) varieties inhibit α-amylase and α-glucosidase activities in vitro.

PubMed

Ademiluyi, Adedayo O; Oboh, Ganiyu

2013-01-01

This study sought to investigate the inhibitory effect of aqueous extracts of two varieties (red and white) of Hibiscus sabdariffa (Roselle) calyces on carbohydrate hydrolyzing enzymes (α-amylase and α-glucosidase), with the aim of providing the possible mechanism for their antidiabetes properties. Aqueous extracts were prepared (1:100 w/v) and the supernatant used for the analysis. The extracts caused inhibition of α-amylase and α-glucosidase activities in vitro.The IC(50) revealed that the red variety (25.2 μg/mL) exhibited higher α-glucosidase inhibitory activity than the white variety (47.4 μg/mL), while the white variety (90.5 μg/mL) exhibited higher α-amylase inhibitory activity than the red variety (187.9 μg/mL). However, the α-glucosidase inhibitory activities of both calyces were higher than that of their α-amylase. In addition, the red variety possessed higher antioxidant capacity as exemplified by the (•)OH scavenging abilities, Fe(2+) chelating ability, and inhibition of Fe(2+)-induced pancreatic lipid peroxidation in vitro. The enzyme inhibitory activities and antioxidant properties of the roselle extracts agreed with their phenolic content. Hence, inhibition of α-amylase and α-glucosidase, coupled with strong antioxidant properties could be the possible underlying mechanism for the antidiabetes properties of H. sabdariffa calyces; however, the red variety appeared to be more potent.

Environmental monitoring using acetylcholinesterase inhibition in vitro. A case study in two Mexican lagoons.

PubMed

Rodríguez-Fuentes, G; Gold-Bouchot, G

2000-01-01

Cholinesterase inhibition is considered a specific biomarker of exposure and effect for organophosphorous pesticides. Its use for monitoring has been hindered, particularly in tropical countries where organophosphates are widely used for malaria and dengue control, because of the frequent lack of suitable controls. An in vitro technique is proposed as a biochemical method for monitoring pollutant mixtures in sediment toxicity tests. Brain homogenate from the fish Oreochromis niloticus is used as the enzyme source. Optimum incubation time, extraction solvent and effect of crude oil on acetylcholinesterase (AChE) are reported. The method described was used in sediments from two Mexican lagoons, located in an oil extraction area where pesticides are used in agriculture and vector control campaigns. AChE inhibitions from 3 to 21% were found in these lagoons, even in the presence of high concentrations of petroleum.

Bulgarian propolis induces analgesic and anti-inflammatory effects in mice and inhibits in vitro contraction of airway smooth muscle.

PubMed

Paulino, Niraldo; Dantas, Andreia Pires; Bankova, Vassya; Longhi, Daniela Taggliari; Scremin, Amarilis; de Castro, Solange Lisboa; Calixto, João Batista

2003-11-01

Propolis is a bee product, which has long been used in folk medicine for the management of different diseases. In this study we evaluated the analgesic and anti-inflammatory effects of a standard ethanolic extract of Bulgarian propolis (Et-Blg) in mice and its in vitro effect on airway smooth muscle. Et-Blg inhibited acetic acid-induced abdominal contortions with an ID(50) = 7.4 +/- 0.7 mg. kg(-1). In the formalin test, the extract caused a significant reduction in pain in mice treated with 100 mg. kg(-1) Et-Blg during the neurogenic phase and for the inflammatory phase with all doses of the extract, with an ID(50) = 2.5 +/- 0.4 mg. kg(-1). Et-Blg inhibited also the capsaicin-induced ear edema in mice; however, this extract was ineffective when assessed in the tail-flick and hot-plate thermal assays. The analgesic effect of Et-Blg was associated with the inhibition of inflammatory responses and not to a simple irritation of nervous terminals. In vitro, this extract inhibited the contraction of trachea smooth muscle induced by histamine (IC(50) = 50 +/- 5 microg. mL(-1)), capsaicin (IC(50) = 26.8 +/- 3 microg. mL(-1)), 80 mM KCl (IC(50) = 27.8 +/- 3 microg. mL(-1)), and carbachol (IC(50) = 54 +/- 2 microg. mL(-1)).

Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisone, G.; Wu, C.F.; Consolo, S.

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,more » 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.« less

In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.).

PubMed

Poovitha, Sundar; Parani, Madasamy

2016-07-18

α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo. The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose. Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.

The flavonoid tangeretin inhibits invasion of MO4 mouse cells into embryonic chick heart in vitro.

PubMed

Bracke, M E; Vyncke, B M; Van Larebeke, N A; Bruyneel, E A; De Bruyne, G K; De Pestel, G H; De Coster, W J; Espeel, M F; Mareel, M M

1989-01-01

Tangeretin, a flavonoid from citrus plants, was found to inhibit the invasion of MO4 cells (Kirsten murine sarcoma virus transformed fetal mouse cells) into embryonic chick heart fragments in vitro. The flavonoid appeared to be chemically stable in tissue culture medium, and the anti-invasive effect was reversible on omission of the molecule from the medium. Unlike (+)-catechin, another anti-invasive flavonoid, tangeretin bound poorly to extracellular matrix. It did not alter fucosylated surface glycopeptides of MO4 cells. Tangeretin seemed not to act as a microtubule inhibitor, as immunocytochemistry revealed no disturbance of the cytoplasmic microtubule complex. However, at anti-invasive concentrations of tangeretin, cell proliferation and thymidine incorporation appeared to be inhibited. When cultured on an artificial substrate, treated MO4 cells were less elongated, covered a larger surface area and exhibited a slower directional migration than untreated cells. From the decrease in ATP content in MO4 cells after tangeretin treatment, we deduce that this flavonoid inhibits a number of intracellular processes, which leads to an inhibition of cell motility and hence of invasion.

AGE-RELATED BRAIN CHOLINESTERASE INHIBITION KINETICS FOLLOWING IN VITRO INCUBATION WITH CHLORPYRIFOS-OXON AND DIAZINON-OXON

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 valuesmore » 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.« less

Tyrphostin AG17 inhibits adipocyte differentiation in vivo and in vitro.

PubMed

Camacho, Alberto; Segoviano-Ramírez, Juan Carlos; Sánchez-Garcia, Adriana; de Jesus Herrera-de la Rosa, Jose; García-Juarez, Jaime; Hernandez-Puente, Carlos Alberto; Calvo-Anguiano, Geovana; Maltos-Uro, Sergio Rodolfo; Olguin, Alejandra; Gojon-Romanillos, Gabriel; Gojon-Zorrilla, Gabriel; Ortiz-Lopez, Rocio

2018-05-29

Excessive subcutaneous adiposity in obesity is associated to positive white adipocyte tissue (WAT) differentiation (adipogenesis) and WAT expandability. Here, we hypothesized that supplementation with the insulin inhibitor and mitochondrial uncoupler, Tyrphostin (T-AG17), in vitro and in vivo inhibits adipogenesis and adipocyte hypertrophy. We used a 3T3-L1 proadipocyte cell line to identify the potential effect of T-AG17 on adipocyte differentiation and fat accumulation in vitro. We evaluated the safety of T-AG17 and its effects on physiological and molecular metabolic parameters including hormonal profile, glucose levels, adipogenesis and adipocyte hypertrophy in a diet-induced obesity model using C57BL/6 mice. We found that T-AG17 is effective in preventing adipogenesis and lipid synthesis in the 3T3-L1 cell line, as evidenced by a significant decrease in oil red staining (p < 0.05). In obese C57BL/6 mice, oral administration of T-AG17 (0.175 mg/kg for 2 weeks) lead to decreased fat accumulation and WAT hypertrophy. Further, T-AG17 induced adipocyte apoptosis by activating caspase-3. In the hepatocytes of obese mice, T-AG17 promoted an increase in the size of lipid inclusions, which was accompanied by glycogen accumulation. T-AG17 did not alter serum biochemistry, including glucose, insulin, leptin, free fatty acids, creatinine, and aspartate aminotransferase. T-AG17 promotes adipocyte apoptosis in vivo and is an effective modulator of adipocyte differentiation and WAT hypertrophy in vitro and in vivo. Therefore, T-AG17 may be useful as a pharmacological obesity treatment.

Chlorogenic acid inhibits hepatocellular carcinoma in vitro and in vivo.

PubMed

Yan, Yuan; Liu, Na; Hou, Ni; Dong, Lei; Li, Jie

2017-08-01

Curative treatment of patients with hepatocellular carcinoma (HCC) is poor. There is an urgent need to develop more effective strategies for the chemoprevention of HCC. Chlorogenic acid (CGA), a type of polyphenol present in the diet, especially from coffee, has many biological activities. Patients with viral hepatitis who drank coffee everyday experienced a reduction in the incidence of HCC. In the present study, we evaluated the effects of CGA on HCC. CGA inhibited the proliferation of HepG2 cells in vitro and the progression of HepG2 xenograft in vivo. CGA induced the inactivation of ERK1/2 and suppressed the expression of MMP-2 and MMP-9 in HepG2 xenograft tissue. These data demonstrate that CGA can prevent the progression of HCC through multiple pathways. CGA appears to be an effective chemopreventive agent for hepatocellular carcinoma. Copyright © 2017 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 ofmore » 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.« less

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.

FLAX OIL FROM TRANSGENIC LINUM USITATISSIMUM SELECTIVELY INHIBITS IN VITRO PROLIFERATION OF HUMAN CANCER CELL LINES.

PubMed

Gebarowski, Tomasz; Gebczak, Katarzyna; Wiatrak, Benita; Kulma, Anna; Pelc, Katarzyna; Czuj, Tadeusz; Szopa, Jan; Gasiorowski, Kazimierz

2017-03-01

Emulsions made of oils from transgenic flaxseeds significantly decreased in vitro proliferation of six tested human cancer cell lines in 48-h cultures, as assessed with the standard sulforhodamine assay. However, the emulsions also increased proliferation rate of normal human dermal fibroblasts and, to a lower extend, of keratinocytes. Both inhibition of in vitro proliferation of human cancer cell lines and stimulation of proliferation of normal dermal fibroblasts and keratinocytes were especially strong with the emulsion type B and with emulsion type M. Oils from seeds of transgenic flax type B and M should be considered as valuable adjunct to standard cytostatic therapy of human cancers and also could be applied to improve the treatment of skin lesions in wound healing.

In vitro and in situ inhibition of carotenoid biosynthesis in Capsicum annuum by bleaching herbicides.

PubMed

Simkin, A J; Breitenbach, J; Kuntz, M; Sandmann, G

2000-10-01

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).

Duloxetine Inhibits Effects of MDMA (“Ecstasy") In Vitro and in Humans in a Randomized Placebo-Controlled Laboratory Study

PubMed Central

Nicola, Valentina G.; Vischer, Nerina; Donzelli, Massimiliano; Krähenbühl, Stephan; Grouzmann, Eric; Huwyler, Jörg; Hoener, Marius C.; Liechti, Matthias E.

2012-01-01

This study assessed the effects of the serotonin (5-HT) and norepinephrine (NE) transporter inhibitor duloxetine on the effects of 3,4–methylenedioxy­methamphetamine (MDMA, ecstasy) in vitro and in 16 healthy subjects. The clinical study used a double-blind, randomized, placebo-controlled, four-session, crossover design. In vitro, duloxetine blocked the release of both 5-HT and NE by MDMA or by its metabolite 3,4-methylenedioxyamphetamine from transmitter-loaded human cells expressing the 5-HT or NE transporter. In humans, duloxetine inhibited the effects of MDMA including elevations in circulating NE, increases in blood pressure and heart rate, and the subjective drug effects. Duloxetine inhibited the pharmacodynamic response to MDMA despite an increase in duloxetine-associated elevations in plasma MDMA levels. The findings confirm the important role of MDMA-induced 5-HT and NE release in the psychotropic effects of MDMA. Duloxetine may be useful in the treatment of psychostimulant dependence. Trial Registration Clinicaltrials.gov NCT00990067 PMID:22574166

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®). Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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.

Gastrodin inhibits osteoclastogenesis via down-regulating the NFATc1 signaling pathway and stimulates osseointegration in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Feng; Shen, Yi; Liu, Bo

Bone is a rigid yet dynamic organ, and this dynamism is mediated by the delicate balance between osteoclastic bone resorption and osteoblastic bone formation. However, excessive activation of osteoclasts is responsible for many bone diseases such as osteoporosis, Paget disease, and tumor bone metastasis. Agents that could inhibit osteoclast formation or function are regarded as promising alternatives to treat osteoclast-related diseases. Recently, traditional Chinese medicine has attracted attention because of its multiple activities in bone metabolism. Among them, gastrodin has been reported as an anti-osteoporosis agent that reduces reactive oxygen species. However, the direct action of gastrodin on osteoclast differentiationmore » and bone resorption, and its underlying molecular mechanism, remain unknown. In this study, we investigated the effects of gastrodin on receptor activator NF-κB ligand (RANKL)-activated osteoclasts formation and bone resorption. Our results showed that gastrodin retarded RANKL-induced osteoclast differentiation efficiently by downregulating transcriptional and translational expression of nuclear factor of activated T cells cl (NFATc1), a major factor in RANKL-mediated osteoclastogenesis. Meanwhile, gastrodin prevented osteoclast maturation and migration by inhibiting the gene expression of dendrocyte expressed seven transmembrane protein (DC-STAMP), an osteoclastic-specific gene that controls cells fusion and movement. And gastrodin prevented RANKL-induced osteoclastic bone erosion in vitro. In addition, gastrodin also stimulated bone mesenchymal stem cell (BMSC) spreading and osseointegration in titanium plate. In summary, gastrodin could prevent osteoclasts formation and bone resorption via blockage of NFATc1 activity, and stimulate osseointegration in vitro. Gastrodin could be developed as a potent phytochemical candidate to treat osteolytic diseases. - Highlights: • Gastrodin suppresses osteoclasts formation and function in vitro

Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro.

PubMed

Fu, Dehao; He, Xianfeng; Yang, Shuhua; Xu, Weihua; Lin, Tao; Feng, Xiaobo

2011-06-30

To study the effects of zoledronic acid (ZA) on the vasculogenic mimicry of osteosarcoma cells in vitro. A Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy. The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting. ZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells. ZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

Oil Palm Phenolics Inhibit the In Vitro Aggregation of β-Amyloid Peptide into Oligomeric Complexes

PubMed Central

Koledova, Vera V.; Shin, Hyeari; Park, Jennifer H.; Tan, Yew Ai; Sambanthamurthi, Ravigadevi

2018-01-01

Alzheimer's disease is a severe neurodegenerative disease characterized by the aggregation of amyloid-β peptide (Aβ) into toxic oligomers which activate microglia and astrocytes causing acute neuroinflammation. Multiple studies show that the soluble oligomers of Aβ42 are neurotoxic and proinflammatory, whereas the monomers and insoluble fibrils are relatively nontoxic. We show that Aβ42 aggregation is inhibited in vitro by oil palm phenolics (OPP), an aqueous extract from the oil palm tree (Elaeis guineensis). The data shows that OPP inhibits stacking of β-pleated sheets, which is essential for oligomerization. We demonstrate the inhibition of Aβ42 aggregation by (1) mass spectrometry; (2) Congo Red dye binding; (3) 2D-IR spectroscopy; (4) dynamic light scattering; (5) transmission electron microscopy; and (6) transgenic yeast rescue assay. In the yeast rescue assay, OPP significantly reduces the cytotoxicity of aggregating neuropeptides in yeast genetically engineered to overexpress these peptides. The data shows that OPP inhibits (1) the aggregation of Aβ into oligomers; (2) stacking of β-pleated sheets; and (3) fibrillar growth and coalescence. These inhibitory effects prevent the formation of neurotoxic oligomers and hold potential as a means to reduce neuroinflammation and neuronal death and thereby may play some role in the prevention or treatment of Alzheimer's disease. PMID:29666700

Inhibition of in vitro and in vivo brown fat differentiation program by myostatin.

PubMed

Braga, Melissa; Pervin, Shehla; Norris, Keith; Bhasin, Shalender; Singh, Rajan

2013-06-01

Obesity arises mainly due to the imbalance between energy storage and its expenditure. Metabolically active brown adipose tissue (BAT) has recently been detected in humans and has been proposed as a new target for anti-obesity therapy because of its unique capacity to regulate energy expenditure. Myostatin (Mst), a negative regulator of muscle mass, has been identified as a potential target to regulate overall body composition. Although the beneficial effects of Mst inhibition on muscle mass are well known, its role in the regulation of lipid metabolism, and energy expenditure is not very clear. We tested the effects of Mst inhibition on the gene regulatory networks that control BAT differentiation using both in vivo and in vitro model systems. PRDM16 and UCP1, two key regulators of brown fat differentiation were significantly up regulated in levator-ani (LA) and gastrocnemius (Gastroc) muscles as well as in epididymal (Epi) and subcutaneous (SC) fat pads isolated from Mst knock out (Mst KO) male mice compared with wild type (WT) mice. Using mouse embryonic fibroblast (MEFs) primary cultures obtained from Mst KO group compared to the WT group undergoing adipogenic differentiation, we also demonstrate a significant increase in select genes and proteins that improve lipid metabolism and energy expenditure. Treatment of Mst KO MEFs with recombinant Mst protein significantly inhibited the gene expression levels of UCP1, PRDM16, PGC1-α/β as well as BMP7. Future studies to extend these findings and explore the therapeutic potential of Mst inhibition on metabolic disorders are warranted. Copyright © 2012 The Obesity Society.

Sofosbuvir inhibits hepatitis A virus replication in vitro assessed by a cell-based fluorescent reporter system.

PubMed

Jiang, Wang; Muhammad, Fawad; Ma, Pengjuan; Liu, Xiyu; Long, Gang

2018-06-01

Hepatitis A virus (HAV) infection remains a major cause of acute hepatitis worldwide and even leads to fulminant hepatitis. For screening antivirals against HAV in vitro, we develop a cell-based fluorescent reporter system named Huh-7.5.1-GA, in which HAV infection is visualized by green fluorescence protein (GFP) translocation from the cytosol into the nucleus. The reliability of Huh-7.5.1-GA for antiviral studies is validated by IFN-α, a known inhibitor of HAV replication, which impedes GFP translocation. Utilizing this in-vitro reporter system, we find that sofosbuvir, an FDA approved prodrug for the treatment of chronic hepatitis C, disturbs GFP translocation and inhibits HAV replication efficiently. In addition, we find that inhibition of HAV by sofosbuvir is hepatic-cell dependent, with IC50 (half-maximal inhibitory concentration) being 6.3 μM and 9.9 μM in Huh-7.5.1, quantified separately by RT-qPCR and image-based analysis. Therefore, our reporter system may serve as a high-throughput platform for screening potent antivirals against HAV. Sofosbuvir may be considered for treatment of hepatitis A, especially in re-infected patients who undergo liver transplantation due to HAV-induced liver failure. Copyright © 2018 Elsevier B.V. All rights reserved.

Importance of multi-P450 inhibition in drug-drug interactions: evaluation of incidence, inhibition magnitude and prediction from in vitro data

PubMed Central

Isoherranen, Nina; Lutz, Justin D; Chung, Sophie P; Hachad, Houda; Levy, Rene H; Ragueneau-Majlessi, Isabelle

2012-01-01

Drugs that are mainly cleared by a single enzyme are considered more sensitive to drug-drug interactions (DDIs) than drugs cleared by multiple pathways. However, whether this is true when a drug cleared by multiple pathways is co-administered with an inhibitor of multiple P450 enzymes (multi-P450 inhibition) is not known. Mathematically, simultaneous equipotent inhibition of two elimination pathways that each contributes half of the drug clearance is equal to equipotent inhibition of a single pathway that clears the drug. However, simultaneous strong or moderate inhibition of two pathways by a single inhibitor is perceived as an unlikely scenario. The aim of this study was (i) to identify P450 inhibitors currently in clinical use that can inhibit more than one clearance pathway of an object drug in vivo, and (ii) to evaluate the magnitude and predictability of DDIs caused by these multi-P450 inhibitors. Multi-P450 inhibitors were identified using the Metabolism and Transport Drug Interaction Database™. A total of 38 multi-P450 inhibitors, defined as inhibitors that increased the AUC or decreased the clearance of probes of two or more P450’s, were identified. Seventeen (45 %) multi-P450 inhibitors were strong inhibitors of at least one P450 and an additional 12 (32 %) were moderate inhibitors of one or more P450s. Only one inhibitor (fluvoxamine) was a strong inhibitor of more than one enzyme. Fifteen of the multi-P450 inhibitors also inhibit drug transporters in vivo, but such data are lacking on many of the inhibitors. Inhibition of multiple P450 enzymes by a single inhibitor resulted in significant (>2-fold) clinical DDIs with drugs that are cleared by multiple pathways such as imipramine and diazepam while strong P450 inhibitors resulted in only weak DDIs with these object drugs. The magnitude of the DDIs between multi-P450 inhibitors and diazepam, imipramine and omeprazole could be predicted using in vitro data with similar accuracy as probe substrate

Quantitative Analysis of Complex Drug-Drug Interactions Between Repaglinide and Cyclosporin A/Gemfibrozil Using Physiologically Based Pharmacokinetic Models With In Vitro Transporter/Enzyme Inhibition Data.

PubMed

Kim, Soo-Jin; Toshimoto, Kota; Yao, Yoshiaki; Yoshikado, Takashi; Sugiyama, Yuichi

2017-09-01

Quantitative analysis of transporter- and enzyme-mediated complex drug-drug interactions (DDIs) is challenging. Repaglinide (RPG) is transported into the liver by OATP1B1 and then is metabolized by CYP2C8 and CYP3A4. The purpose of this study was to describe the complex DDIs of RPG quantitatively based on unified physiologically based pharmacokinetic (PBPK) models using in vitro K i values for OATP1B1, CYP3A4, and CYP2C8. Cyclosporin A (CsA) or gemfibrozil (GEM) increased the blood concentrations of RPG. The time profiles of RPG and the inhibitors were analyzed by PBPK models, considering the inhibition of OATP1B1 and CYP3A4 by CsA or OATP1B1 inhibition by GEM and its glucuronide and the mechanism-based inhibition of CYP2C8 by GEM glucuronide. RPG-CsA interaction was closely predicted using a reported in vitro K i,OATP1B1 value in the presence of CsA preincubation. RPG-GEM interaction was underestimated compared with observed data, but the simulation was improved with the increase of f m,CYP2C8 . These results based on in vitro K i values for transport and metabolism suggest the possibility of a bottom-up approach with in vitro inhibition data for the prediction of complex DDIs using unified PBPK models and in vitro f m value of a substrate for multiple enzymes should be considered carefully for the prediction. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

In Vitro and in Vivo Mechanism of Bone Tumor Inhibition by Selenium-Doped Bone Mineral Nanoparticles.

PubMed

Wang, Yifan; Wang, Jianglin; Hao, Hang; Cai, Mingle; Wang, Shiyao; Ma, Jun; Li, Yan; Mao, Chuanbin; Zhang, Shengmin

2016-11-22

Biocompatible tissue-borne crystalline nanoparticles releasing anticancer therapeutic inorganic elements are intriguing therapeutics holding the promise for both tissue repair and cancer therapy. However, how the therapeutic inorganic elements released from the lattice of such nanoparticles induce tumor inhibition remains unclear. Here we use selenium-doped hydroxyapatite nanoparticles (Se-HANs), which could potentially fill the bone defect generated from bone tumor removal while killing residual tumor cells, as an example to study the mechanism by which selenium released from the lattice of Se-HANs induces apoptosis of bone cancer cells in vitro and inhibits the growth of bone tumors in vivo. We found that Se-HANs induced apoptosis of tumor cells by an inherent caspase-dependent apoptosis pathway synergistically orchestrated with the generation of reactive oxygen species. Such mechanism was further validated by in vivo animal evaluation in which Se-HANs tremendously induced tumor apoptosis to inhibit tumor growth while reducing systemic toxicity. Our work proposes a feasible paradigm toward the design of tissue-repairing inorganic nanoparticles that bear therapeutic ions in the lattice and can release them in vivo for inhibiting tumor formation.

Inhibition mechanism of lanthanum ion on the activity of horseradish peroxidase in vitro

NASA Astrophysics Data System (ADS)

Guo, Shaofen; Wang, Lihong; Lu, Aihua; Lu, Tianhong; Ding, Xiaolan; Huang, Xiaohua

2010-02-01

In order to understand the inhibition mechanism of lanthanum ion (La 3+) on the activity of horseradish peroxidase (HRP), the effects of La 3+ on the activity, electron transfer and conformation of HRP in vitro were investigated by using cyclic voltammetry (CV), atomic force microscopy (AFM), circular dichroism (CD), high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and inductively coupled plasma mass spectrometry (ICP-MS). It was found that La 3+ can combine with the amide groups of the polypeptide chain in HRP molecule, forming the complex of La 3+ and HRP (La-HRP). The formation of the La-HRP complex causes the destruction of the native structure of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure extent of active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the direct electrochemical and catalytic activities of HRP are decreased. It is a possible inhibition mechanism of La 3+ on the activity of peroxidase.

Traditional Uighur Medicine Karapxa decoction, inhibits liver xanthine oxidase and reduces serum uric acid concentrations in hyperuricemic mice and scavenges free radicals in vitro.

PubMed

Amat, Nurmuhammat; Umar, Anwar; Hoxur, Parida; Anaydulla, Mihrigul; Imam, Guzalnur; Aziz, Ranagul; Upur, Halmurat; Kijjoa, Anake; Moore, Nicholas

2015-04-25

Karapxa decoction (KD) is a Traditional Uighur Medicine used for hepatitis, cholecystitis, gastralgia, oedema, gout and arthralgia. Because of its purported effect in gout, its effects were tested in hyperuricemic mice models induced by yeast extract paste or potassium oxonate, as well as its capacity to scavenge free radicals in vitro. Hyperuricemia was induced in mice by yeast extract paste or potassium oxonate. KD was given orally for 14 days at 200, 400 and 800 mg/kg/day, with Allopurinol 10 mg/kg/day as positive control. Serum uric acid (UA), and liver xanthine oxidase activity (XO) were measured. Scavenging activity of KD on 1, 1-diphenyl-2-picrylhydrazyl radicals (DPP•), nitric oxide (•NO), superoxide (O2•-), efficiency against lipid peroxidation, and XO inhibition were determined in vitro. KD inhibited liver XO activity and reduced serum uric acid in hyperuricemic mice. KD also showed noticeable antioxidant activity, scavenging free radicals (DPP•, •NO and O2•-). It was effective against lipid peroxidation and inhibited XO in vitro. This study supports the traditional use of Karapxa decoction to treat hyperuricemia and gout.

In vitro assessment of competitive and time-dependent inhibition of the nevirapine metabolism by nortriptyline in rats.

PubMed

Usach, Iris; Ferrer, José-Maria; Peris, José-Esteban

2018-04-17

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) widely used as a component of High Active Antiretroviral Therapy (HAART) since it is inexpensive, readily absorbed after oral administration and non-teratogenic. In the present work, the mechanism of a previously described pharmacokinetic interaction between NVP and the antidepressant drug nortriptyline (NT) was studied using rat hepatic microsomes. The obtained results showed a competitive inhibition of the NVP metabolism by NT. The three main NVP metabolites (2-OH-NVP, 3-OH-NVP and 12-OH-NVP) where competitively inhibited with similar inhibitory constant values (K i  = 4.01, 3.97 and 4.40 μM, respectively). Time-dependent inhibition of the NVP metabolism was also detected, with a 2.5-fold reduction in the IC 50 values of NT for 2-, 3-, and 12-OH-NVP formation when NT was preincubated with the microsomal suspension in the presence of an NADPH-generating system. A concentration-dependent inhibition of the formation of NVP metabolites by the main NT metabolite (10-OH-NT) was also observed, however, the inhibitory potency of 10-OH-NT was much lower than that of the parent drug. The apparent hepatic intrinsic clearance of NVP determined in these in vitro experiments was used to predict the in vivo clearance of NVP using the "well-stirred" and the "parallel-tube" models, resulting in values close to those previously observed in vivo clearance. Finally, a good prediction of the increase in the plasma concentrations of NVP when co-administered with NT was obtained employing the inhibitory constant of NT determined in vitro and the estimated plasma concentration of NT entering the liver. Copyright © 2018. Published by Elsevier Inc.

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. (c) 2009 Wiley-Liss, Inc.

Extract of Artemisia lavandulaefolia Inhibits In Vitro Angiogenesis in Human Umbilical Vein Endothelial Cells

PubMed Central

Yi, Eui-Yeun; Han, Kyung-Suk; Kim, Yung-Jin

2014-01-01

Angiogenesis is important processes for tumor growth and metastasis. Anti-angiogenesis target therapy has recently been known to be new anti-cancer therapeutic strategies. Natural products such as traditional medicine comprise a major source of angiogenesis inhibitors. Artemisia lavandulaefolia has been known to use in the traditional medical practices. However, its molecular mechanism on the tumor protection and therapy was not clearly elucidated. In this study, we investigated the possibility that extract of A. lavandulaefolia inhibits in vitro angiogenesis. Therefore, we examined the effect of extract of A. lavandulaefolia on the vascular network formation of human umbilical vein endothelial cells (HUVECs). We found that the treatment of A. lavandulaefolia extract suppressed the tube formation of HUVECs without any influence on the viability of HUVECs. In addition, extract of A. lavandulaefolia inhibited the migration and invasion of HUVECs. These results suggest that extract of A. lavandulaefolia could be act for an angiogenic inhibitor. PMID:25574458

Inhibition of JAK3 and PKC via Immunosuppressive Drugs Tofacitinib and Sotrastaurin Inhibits Proliferation of Human B Lymphocytes In Vitro.

PubMed

Martina, M N; Ramirez Bajo, M J; Bañon-Maneus, E; Moya Rull, D; Hierro-Garcia, N; Revuelta, I; Campistol, J M; Rovira, J; Diekmann, F

2016-11-01

Antibody-mediated response in solid organ transplantation is critical for graft dysfunction and loss. The use of immunosuppressive agents partially inhibits the B-lymphocyte response leading to a risk of acute and chronic antibody-mediated rejection. This study evaluated the impact of JAK3 and PKC inhibitors tofacitinib (Tofa) and sotrastaurin (STN), respectively, on B-cell proliferation, apoptosis, and activation in vitro. Human B cells isolated from peripheral blood of healthy volunteers were cocultured with CD40 ligand-transfected fibroblasts as feeder cells in the presence of interleukin (IL) 2, IL-10, and IL-21. The cocultures were treated with immunosuppressants Tofa, STN, and rapamycin (as a control), to analyze the proliferation and apoptosis of B cells by means of Cyquant and flow cytometry, respectively. CD27 and IgG staining were applied to evaluate whether treatments modified the activation of B cells. Tofa and STN were able to inhibit B-cell proliferation to the same extent as rapamycin, without inducing cell apoptosis. After 6 days in coculture with feeder cells, all B cells showed CD27 memory B-cell phenotype. None of the immunosuppressive treatments modified the proportion between class-switched and non-class-switched memory B cells observed in nontreated cultures. The high predominance of CD27 + CD24 + phenotype was not modified by any immunosuppressive treatment. Our results show that Tofa and STN can suppress B-cell antibody responses to an extent similar to rapamycin, in vitro; therefore these compounds may be a useful therapy against antibody-mediated rejection in transplantation. Copyright © 2016. Published by Elsevier Inc.

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.

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.

Immunoregulatory cytokines in mouse placental extracts inhibit in vitro osteoclast differentiation of murine macrophages.

PubMed

Canellada, A; Custidiano, A; Abraham, F; Rey, E; Gentile, T

2013-03-01

Previous studies showed that placental extracts (PE) alleviates arthritic symptoms in animal models of arthritis. To evaluate whether murine PEs obtained at embryonic days 7.5 (PE7) and 17.5 (PE18) regulate RANKL-induced osteoclast differentiation, RAW 264.7 cells were cultured with RANKL and MCSF in presence or not of PEs. Tartrate-resistant acid phosphatase (TRAP) was stained and multinucleated TRAP positive cells were visualized under a light microscope. Cathepsin K and metalloprotease expression was assessed by RT-PCR and gelatin zymography respectively. NFATc1 expression was determined by immunoblot. To analyze NFAT-dependent transcription, macrophages were transfected with a luciferase reporter plasmid. Cytokines were determined in PEs by ELISA and immunoblot. Transforming growth factor (TGF)- beta and Interleukin (IL)-10 receptor were inhibited in cell cultures with specific antibodies. PE7 and PE18 inhibited RANKL-induced multinucleated TRAP positive cells, Cathepsin K expression and metalloprotease activity, as well as NFATc1 expression and activity, thereby inhibiting osteoclast differentiation of RAW cells. Inflammatory/Regulatory cytokine ratio was higher in PE7 than in PE18. Blocking TGF-beta abolished the effect of both, PE7 and PE18, on multinucleated TRAP positive cells and metalloprotease expression, whereas blocking IL-10 receptor reverted the effect of PE18 but not of PE7. Inhibition of osteoclast differentiation by PEs was not unexpected, since cytokines detected in extracts were previously found to regulate osteoclast differentiation. PEs inhibited osteoclast differentiation of macrophages in vitro. Downregulation of NFATc1 might be involved in this effect. Regulatory/Th2 cytokines play a role in the effect of PEs on osteoclast differentiation. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Comparative observation on inhibition of hemozoin formation and their in vitro and in vivo anti-schistosome activity displayed by 7 antimalarial drugs].

PubMed

Xue, Jian; Jiang, Bin; Liu, Cong-Shan; Sun, Jun; Xiao, Shu-Hua

2013-06-01

To observe and compare the inhibition of hemozoin formation and the in vitro as well as in vivo antischistosomal activity induced by seven antimalarial drugs. Inhibition of hemozoin formation displayed by chloroquine phosphate, quinine hydrochloride, quinidine, mefloquine hydrochloride, pyronaridine phosphate and lumefantrine at 25 micromol/L, and artemether at 100 micromol/L was performed by assay of inhibition of beta-hematin formation in 1 mol/L sodium acetate buffers containing hematin with various pH of 4.0, 4.2, 4.4, 4.6, 4.8, and 5.0. In in vitro antischistosomal study, the medium of RPMI 1640 supplemented by 10% calf serum was used to maintain the adult Schistosoma japonicum, and the 50% and 95% lethal concentrations (LC50 and LC95) to kill the adult worms of each drug were then determined. Meanwhile, the interaction of quinine, pyronaridine and chloroquine combined with hemin against adult schistosomes was also undertaken. As to in vivo test, the efficacy of seven antimalarial drugs administered orally or intraperitoneally to mice infected with adult schistosomes was observed. In the acidic acetate-hematin solution, 25 micromol/L pyronaridine showed significant inhibition of beta-hematin formation at pH 4.4-5.0 with inhibition rates of 81.3%-97.0%. At pH 4.6, the inhibition rates of beta-hematin formation in acetate-hematin solution induced by mefloquine, chloroquine or quinine at concentration of 25 beta mol/L were 79.7%, 72.8% or 65.8%, respectively, and the beta-hematin formation was continually inhibited by these 3 antimalarial drugs at pH 4.8 and 5.0 with inhibition rates of 83.1%-90.6%, 41.9%-49.0% or 53.2-62.0%. The inhibition rates of beta-hematin formation at pH 4.6 and 4.8-5.0 induced by lumefantrine 25 micromol/L were 74.3% and 40.4%-40.5%, respectively. While under the same concentration of quinidine, 53.4% and 50.9% inhibition rates of beta-hematin formation were observed at pH 4.8 and 5.0. As to artemether, higher concentration of 100

Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro

PubMed Central

Wang, Jing; Yang, Yangfan; Xu, Jiangang; Lin, Xianchai; Wu, Kaili

2013-01-01

Purpose To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. Methods Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFβS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFβS, FN, and Snail1 were analyzed using reverse transcriptase–polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. Results PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFβ1, TGFβ2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFβ1, and TGFβ2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. Conclusions PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity. PMID:24415895

Leptin inhibits testosterone secretion from adult rat testis in vitro.

PubMed

Tena-Sempere, M; Pinilla, L; González, L C; Diéguez, C; Casanueva, F F; Aguilar, E

1999-05-01

Leptin, the product of the ob gene, has emerged recently as a pivotal signal in the regulation of fertility. Although the actions of leptin in the control of reproductive function are thought to be exerted mainly at the hypothalamic level, the potential direct effects of leptin at the pituitary and gonadal level have been poorly characterised. In the present study, we first assessed the ability of leptin to regulate testicular testosterone secretion in vitro. Secondly, we aimed to evaluate whether leptin can modulate basal gonadotrophin and prolactin (PRL) release by incubated hemi-pituitaries from fasted male rats. To attain the first goal, testicular slices from prepubertal and adult rats were incubated with increasing concentrations (10(-9)-10(-7) M) of recombinant leptin. Assuming that in vitro testicular responsiveness to leptin may be dependent on the background leptin levels, testicular tissue from both food-deprived and normally-fed animals was used. Furthermore, leptin modulation of stimulated testosterone secretion was evaluated by incubation of testicular samples with different doses of leptin in the presence of 10 IU human chorionic gonadotrophin (hCG). In addition, analysis of leptin actions on pituitary function was carried out using hemi-pituitaries from fasted adult male rats incubated in the presence of increasing concentrations (10(-9)-10(-7) M) of recombinant leptin. Serum testosterone levels, and basal and hCG-stimulated testosterone secretion by incubated testicular tissue were significantly decreased by fasting in prepubertal and adult male rats. However, a significant reduction in circulating LH levels was only evident in adult fasted rats. Doses of 10(-9)-10(-7) M leptin had no effect on basal or hCG-stimulated testosterone secretion by testes from prepubertal rats, regardless of the nutritional state of the donor animal. In contrast, leptin significantly decreased basal and hCG-induced testosterone secretion by testes from fasted and fed

Redox-responsive microbeads containing thiolated pectin-doxorubicin conjugate inhibit tumor growth and metastasis: An in vitro and in vivo study.

PubMed

Cheewatanakornkool, Kamonrak; Niratisai, Sathit; Dass, Crispin R; Sriamornsak, Pornsak

2018-07-10

The objective of this study was to investigate the in vitro cytotoxicity and in vivo anticancer efficacy of redox-responsive microbeads containing thiolated pectin-doxorubicin (DOX) conjugate. Oral microbeads were coated with an enteric polymer to protect the drug from release in the upper gastrointestinal (GI) tract and allow redox-triggered drug release in the colon. Morphology, particle size, drug content, and in vitro drug release behavior of the microbeads were characterized; in vitro cytotoxicity was tested on mouse colon carcinoma, human colorectal adenocarcinoma, and human bone osteosarcoma cell lines. In vivo anticancer efficacy of coated microbeads was examined in BALB/c mice with murine colon carcinoma. These coated microbeads significantly inhibited the growth of all cell lines. The in vivo study confirmed delivery of DOX to the colorectal tumor site, redox-responsiveness, and anticancer efficacy of coated microbeads. Coated microbeads also effectively inhibited primary tumor growth and suppressed tumor metastases without gross toxicity to the non-target tissue. No noticeable damage was found in mouse GI tissues, indicating lack of DOX toxicity. These novel coated microbeads containing thiolated pectin-DOX conjugate may be a promising vehicle for targeted clinical delivery of DOX to the colorectal cancer site by oral administration. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

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.

In vitro inhibition of calcium oxalate crystallization and crystal adherence to renal tubular epithelial cells by Terminalia arjuna.

PubMed

Mittal, A; Tandon, S; Singla, S K; Tandon, C

2016-04-01

Urolithiasis is a multifactorial disease and remains a public health problem around the world. Of all types of renal stones, calcium oxalate (CaOx) is the most common composition formed in the urinary system of the patients with urolithiasis. The present study is aimed at evaluating the antiurolithiatic properties of the Tris-Cl extract (TE) of Terminalia arjuna (T. arjuna). The antilithiatic activity of TE of T. arjuna was investigated on nucleation, aggregation, and growth of the CaOx crystals, as well as its protective potency was tested on oxalate-induced cell injury of NRK-52E renal epithelial cells. Also, in vitro antioxidant activity of TE T. arjuna bark was also determined. The TE of T. arjuna exhibited a concentration-dependent inhibition of nucleation and growth of CaOx crystals. Inhibition of aggregation of CaOx crystals remains constant. When NRK-52E cells were injured by exposure to oxalate for 48 h, the TE prevented the cells from injury and CaOx crystal adherence resulting in increased cell viability in a dose-dependent manner. The TE also scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals with an IC50 at 51.72 µg/mL. The results indicated that T. arjuna is a potential candidate for phytotherapy against urolithiasis as it attains the ability to inhibit CaOx crystallization and scavenge DPPH free radicals in vitro along with a cytoprotective role.

Fucoxanthin, a Marine Carotenoid, Reverses Scopolamine-Induced Cognitive Impairments in Mice and Inhibits Acetylcholinesterase in Vitro

PubMed Central

Lin, Jiajia; Huang, Ling; Yu, Jie; Xiang, Siying; Wang, Jialing; Zhang, Jinrong; Yan, Xiaojun; Cui, Wei; He, Shan; Wang, Qinwen

2016-01-01

Fucoxanthin, a natural carotenoid abundant in edible brown seaweeds, has been shown to possess anti-cancer, anti-oxidant, anti-obesity and anti-diabetic effects. In this study, we report for the first time that fucoxanthin effectively protects against scopolamine-induced cognitive impairments in mice. In addition, fucoxanthin significantly reversed the scopolamine-induced increase of acetylcholinesterase (AChE) activity and decreased both choline acetyltransferase activity and brain-derived neurotrophic factor (BDNF) expression. Using an in vitro AChE activity assay, we discovered that fucoxanthin directly inhibits AChE with an IC50 value of 81.2 μM. Molecular docking analysis suggests that fucoxanthin likely interacts with the peripheral anionic site within AChE, which is in accordance with enzymatic activity results showing that fucoxanthin inhibits AChE in a non-competitive manner. Based on our current findings, we anticipate that fucoxanthin might exhibit great therapeutic efficacy for the treatment of Alzheimer’s disease by acting on multiple targets, including inhibiting AChE and increasing BDNF expression. PMID:27023569

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

Inhibition of pyrimidine biosynthesis de novo in Plasmodium falciparum by 2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone in vitro.

PubMed

Hammond, D J; Burchell, J R; Pudney, M

1985-01-01

The effects of the hydroxynaphthoquinone BW58C on some metabolite levels and the flux of H14CO3 through the de novo pyrimidine biosynthetic pathway of intact Plasmodium falciparum have been studied in vitro using HPLC techniques. 800 nM BW58C appeared to have no significant effect on the energy status of isolated P. falciparum, but at 0.1 nM it caused a dramatic decrease in the concentrations of pyrimidine nucleotides, specifically UTP, during 256 min of incubation. Although about one hour was required to achieve a significant decrease in pyrimidine nucleotide concentrations, a much more rapid inhibition of the flux of H14CO3 through the de novo pathway was found upon addition of 0.1 nM BW58C. This inhibition caused about a 10 fold increase in the radioactivity of carbamoyl-aspartate over a 64 min period, and an overall increase in the concentration of this metabolite of about 3 fold during 256 min of incubation. These effects of BW58C against P. falciparum in vitro are discussed in terms of inhibition of de novo pyrimidine biosynthesis at the site of dihydroorotate dehydrogenase.

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.

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.

Chronic nicotine inhibits the therapeutic effects of gemcitabine on pancreatic cancer in vitro and in mouse xenografts.

PubMed

Banerjee, Jheelam; Al-Wadei, Hussein A N; Schuller, Hildegard M

2013-03-01

Smoking is an established risk factor for pancreatic cancer and nicotine replacement therapy (NRT) often accompanies chemotherapy. The current study has tested the hypothesis that chronic exposure to low dose nicotine reduces the responsiveness of pancreatic cancer to the leading therapeutic for this cancer, gemcitabine. The effects of chronic nicotine (1 μm/L) on two pancreatic cancer cell lines in vitro and in a xenograft model were assessed by immunoassays, Western blots and cell proliferation assays. Exposure in vitro to nicotine for 7 days inhibited the gemcitabine-induced reduction in viable cells, gemcitabine-induced apoptosis as indicated by reduced expression of cleaved caspase-3 while inducing the phosphorylation of signalling proteins extracellular signal-regulated kinase (ERK), v-akt thymoma viral oncogene homolog (protein kinase B, AKT) and Src. Nicotine (1 μm/L) in the drinking water for 4 weeks significantly reduced the therapeutic response of mouse xenografts to gemcitabine while reducing the induction of cleaved caspase-3 and the inhibition of phosphorylated forms of multiple signalling proteins by gemcitabine in xenograft tissues. Our experimental data suggest that continued moderate smoking and NRT may negatively impact therapeutic outcomes of gemcitabine on pancreatic cancer and that clinical studies in cancer patients are now warranted. Copyright © 2012 Elsevier Ltd. All rights reserved.

Tox-Database.net: a curated resource for data describing chemical triggered in vitro cardiac ion channels inhibition

PubMed Central

2012-01-01

Background Drugs safety issues are now recognized as being factors generating the most reasons for drug withdrawals at various levels of development and at the post-approval stage. Among them cardiotoxicity remains the main reason, despite the substantial effort put into in vitro and in vivo testing, with the main focus put on hERG channel inhibition as the hypothesized surrogate of drug proarrhythmic potency. The large interest in the IKr current has resulted in the development of predictive tools and informative databases describing a drug's susceptibility to interactions with the hERG channel, although there are no similar, publicly available sets of data describing other ionic currents driven by the human cardiomyocyte ionic channels, which are recognized as an overlooked drug safety target. Discussion The aim of this database development and publication was to provide a scientifically useful, easily usable and clearly verifiable set of information describing not only IKr (hERG), but also other human cardiomyocyte specific ionic channels inhibition data (IKs, INa, ICa). Summary The broad range of data (chemical space and in vitro settings) and the easy to use user interface makes tox-database.net a useful tool for interested scientists. Database URL http://tox-database.net. PMID:22947121

Human adipose tissue-derived mesenchymal stem cells inhibit T-cell lymphoma growth in vitro and in vivo.

PubMed

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

2014-09-01

Human mesenchymal stem cells (hMSCs) are thought to be one of the most reliable stem cell sources for a variety of cell therapies. This study investigated the anti-tumor effect of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) on EL4 murine T-cell lymphoma in vitro and in vivo. The growth-inhibitory effect of hAT-MSCs on EL4 tumor cells was evaluated using a WST-1 cell proliferation assay. Cell-cycle arrest and apoptosis were investigated by flow cytometry and western blot. To evaluate an anti-tumor effect of hAT-MSCs on T-cell lymphoma in vivo, CM-DiI-labeled hAT-MSCs were circumtumorally injected in tumor-bearing nude mice, and tumor size was measured. hAT-MSCs inhibited T-cell lymphoma growth by altering cell-cycle progression and inducing apoptosis in vitro. hAT-MSCs inhibited tumor growth in tumor-bearing nude mice and prolonged survival time. Immunofluorescence analysis showed that hAT-MSCs migrated to tumor sites. hAT-MSCs suppress the growth of T-cell lymphoma, suggesting a therapeutic option for T-cell lymphoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The ibrutinib B-cell proliferation inhibition is potentiated in vitro by dexamethasone: Application to chronic lymphocytic leukemia.

PubMed

Manzoni, Delphine; Catallo, Régine; Chebel, Amel; Baseggio, Lucile; Michallet, Anne-Sophie; Roualdes, Olivier; Magaud, Jean-Pierre; Salles, Gilles; Ffrench, Martine

2016-08-01

New B-cell receptor-targeted therapies such as ibrutinib, a Bruton tyrosine kinase inhibitor, are now proposed for lymphoid pathologies. The putative benefits of its combination with glucocorticoids were evaluated here. We compared the effects of dexamethasone (DXM), ibrutinib and their in vitro combination on proliferation and metabolic stress markers in stimulated normal B-lymphocytes and in malignant lymphocytes from chronic lymphocytic leukemia (CLL) patients. In both cellular models, cell cycle progression was globally inhibited by DXM and/or ibrutinib. This inhibition was significantly amplified by DXM addition to ibrutinib and was related to a significant decrease in the expression of the cell cycle regulatory proteins CDK4 and cyclin E. Apoptosis increased especially with DXM/ibrutinib combination and was associated with a significant decrease in Mcl-1 expression. Treatment effects on metabolic stress were evaluated by DNA damage recognition after 53BP1 foci labeling. The percentage of cells with more than five 53BP1 foci decreased significantly with ibrutinib in normal and CLL lymphocytes. This decrease was strongly reinforced, in CLL, by DXM addition. Our data indicated that, in vitro, DXM potentiated antiproliferative effects of ibrutinib and decreased DNA damage in lymphoid B-cells. Thus their combination may be proposed for CLL treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of β,β-dimethylacrylshikonin on inhibition of human colorectal cancer cell growth in vitro and in vivo.

PubMed

Fan, Yingying; Jin, Shaoju; He, Jun; Shao, Zhenjun; Yan, Jiao; Feng, Ting; Li, Hong

2012-01-01

In traditional Chinese medicine, shikonin and its derivatives, has been used in East Asia for several years for the prevention and treatment of several diseases, including cancer. We previously identified that β,β-dimethylacrylshikonin (DA) could inhibit hepatocellular carcinoma growth. In the present study, we investigated the inhibitory effects of DA on human colorectal cancer (CRC) cell line HCT-116 in vitro and in vivo. A viability assay showed that DA could inhibit tumor cell growth in a time- and dose-dependent manner. Flow cytometry showed that DA blocks the cell cycle at G(0)/G(1) phase. Western blotting results demonstrated that the induction of apoptosis by DA correlated with the induction of pro-apoptotic proteins Bax, and Bid, and a decrease in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Furthermore, treatment of HCT-116 bearing nude mice with DA significantly retarded the growth of xenografts. Consistent with the results in vitro, the DA-mediated suppression of HCT-116 xenografts correlated with Bax and Bcl-2. Taken together, these results suggest that DA could be a novel and promising approach to the treatment of CRC.

Radiosensitization of human glioma cells by tamoxifen is associated with the inhibition of PKC-ι activity in vitro.

PubMed

Yang, Lei; Yuan, Xiaopeng; Wang, Jie; Gu, Cheng; Zhang, Haowen; Yu, Jiahua; Liu, Fenju

2015-07-01

The present study aimed to investigate the radiosensitizing effects of tamoxifen (TAM), a non-steroidal anti-estrogen drug, in human glioma A172 and U251 cells in vitro . A colony-forming assay revealed that TAM enhances radiosensitivity in A172 and U251 cells. Treatment with TAM also increased the percentage of apoptotic cells subsequent to ionizing radiation, and increased the expression of apoptotic markers, including cleaved caspase-3 and poly(ADP-ribose) polymerase. Ionizing radiation induced G2/M phase arrest, which was alleviated within 24 h when the radiation-induced DNA damage was repaired. However, flow cytometry analysis revealed that TAM treatment delayed the recovery of cell cycle progression. Additional examination demonstrated that TAM-mediated protein kinase C-ι (PKC-ι) inhibition may lead to the activation of pro-apoptotic B-cell lymphoma 2-associated death promoter, and the dephosphorylation of cyclin-dependent kinase 7, resulting in increased cell apoptosis and sustained G2/M phase arrest following exposure to radiation. The present data indicate that the radiosensitizing effects of TAM on glioma cells are partly due to the inhibition of PKC-ι activity in vitro .

Lactic acid bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo.

PubMed

Fuglsang, Anders; Rattray, Fergal P; Nilsson, Dan; Nyborg, Niels C B

2003-01-01

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 microg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 microg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II.

The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo.

PubMed

Okada, Masashi; Kuramoto, Kenta; Takeda, Hiroyuki; Watarai, Hikaru; Sakaki, Hirotsugu; Seino, Shizuka; Seino, Manabu; Suzuki, Shuhei; Kitanaka, Chifumi

2016-05-10

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted.

Berry components inhibit α-glucosidase in vitro: synergies between acarbose and polyphenols from black currant and rowanberry.

PubMed

Boath, Ashley S; Stewart, Derek; McDougall, Gordon J

2012-12-01

Polyphenol-rich extracts from certain berries inhibited α-glucosidase activity in vitro. The two most effective berry extracts, from black currant and rowanberry, inhibited α-glucosidase with IC(50) values respectively of 20 and 30μg GAE/ml and were as effective as the pharmaceutical inhibitor, acarbose. These berry extracts differed greatly in their polyphenol composition: black currant was dominated by anthocyanins (∼70% of total) whereas rowanberry was enriched in chlorogenic acids (65% total) and had low levels of anthocyanins. Both black currant and rowanberry extracts potentiated the inhibition caused by acarbose and could replace the inhibition lost by reducing the acarbose dose. However, no additive effects were noted when black currant and rowanberry extracts were added in combination. The mechanisms underlying the synergy between acarbose and the berry polyphenols and the lack of synergy between the berry components are discussed. These extracts exhibited the potential to replace acarbose (or reduce the dose required) in its current clinical use in improving post-prandial glycaemic control in type 2 diabetics. As a result, these polyphenols may offer a dietary means for type 2 diabetics to exercise glycaemic control. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro investigation of efficacy of new reactivators on OPC inhibited rat brain acetylcholinesterase.

PubMed

Atanasov, Vasil N; Petrova, Iskra; Dishovsky, Christophor

2013-03-25

Organophosphorus compounds (OPC) were developed as warfare nerve agents. They are also widely used as pesticides. The drug therapy of intoxication with OPC includes mainly combination of cholinesterase (ChE) reactivators and cholinolytics. There is no single ChE reactivator having an ability to reactivate sufficiently the inhibited enzyme due to the high variability of chemical structure of the inhibitors. The difficulties in reactivation of ChE activity and slight antidote effect regarding intoxication with some OPC are some of the reasons for continuous efforts to obtain new reactivators of ChE. The aim of the present study was to evaluate the efficacy of some ChE reactivators against OPC intoxication (tabun, paraoxon and dichlorvos) in in vitro experiments and to compare their activity to that known for some currently used oximes (obidoxime, HI-6, 2-PAM). Experiments were carried out using rat brain acetylcholinesterase (AChE). Reactivators showed different activity in the reactivation of rat brain AChE after dichlorvos, paraoxon and tabun inhibition. AChE was easier reactivated after paraoxon treatment. The best effect showed BT-07-4M, obidoxime, TMB-4 and BT-08 from the group of symmetric oximes, and Toxidin, BT-05 and BT-03 from asymmetric compounds. The reactivation of brain AChE inhibited with tabun demonstrated better activity of new compound BT-07-4M, TMB-4 and obidoxime from symmetric oximes, and BT-05 and BT-03 possessing asymmetric structure. All compounds showed low activity toward inhibition of AChE caused by dichlorvos. Comparison of two main structure types (symmetric/asymmetric) showed that the symmetric compounds reactivated better AChE, inhibited with this OPC, than asymmetric ones. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

AzaSite® inhibits Staphylococcus aureus and coagulase-negative Staphylococcus biofilm formation in vitro.

PubMed

Wu, Eric C; Kowalski, Regis P; Romanowski, Eric G; Mah, Francis S; Gordon, Y Jerold; Shanks, Robert M Q

2010-12-01

The aim of this study was to analyze the effect of azithromycin (AZM) 1% ophthalmic solution in DuraSite® (AzaSite®) on biofilm formation by Staphylococcus aureus and coagulase-negative staphylococci in vitro. Susceptible and resistant clinical strains (n = 8) of S. aureus and coagulase-negative staphylococci were challenged with serial dilutions of AzaSite® and its components: AZM, benzalkonium chloride (BAK), and the DuraSite drug delivery vehicle. After 20 h of incubation, bacterial growth was quantified using a spectrophotometer (A = 600 nm). Plates were stained with crystal violet and biofilm formation was quantified spectrophotometrically at A = 590 nm. AzaSite® and AZM inhibited bacterial growth (P < 0.05) and biofilm formation (P < 0.05) in AZM-susceptible strains at all studied dilutions. AZM-resistant strains treated with AzaSite® exhibited a significant reduction in biofilm formation (P < 0.05) at subinhibitory concentrations (1.25%-5%). AZM had no effect on bacterial growth in resistant strains but conferred a small reduction in biofilm formation at concentrations from 1.25 to 10 mg/mL in most strains. DuraSite® inhibited biofilm formation at concentrations between 10% and 2.5% in all studied strains (P < 0.05), without affecting bacterial growth. BAK inhibited bacterial growth and biofilm formation in all strains between concentrations of 0.042 and 0.375 mg/mL (P < 0.05). AzaSite®, AZM, or BAK prevented biofilm formation by inhibiting growth of AZM-susceptible strains. AzaSite®, AZM, and DuraSite® also reduced biofilm formation at subinhibitory concentrations for growth. Our data indicate that AZM has a moderate inhibitory effect on biofilm formation, whereas DuraSite® appears to play a greater role in the inhibition of staphylococcal biofilm formation by AzaSite®.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rowe, Alexander M.; Brundage, Kathleen M.; Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506

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 hypothesizedmore » 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.« less

Targeting the yin and the yang: combined inhibition of the tyrosine kinase c-Src and the tyrosine phosphatase SHP-2 disrupts pancreatic cancer signaling and biology in vitro and tumor formation in vivo.

PubMed

Gomes, Evan G; Connelly, Sarah F; Summy, Justin M

2013-07-01

Although c-Src (Src) has emerged as a potential pancreatic cancer target in preclinical studies, Src inhibitors have not demonstrated a significant therapeutic benefit in clinical trials. The objective of these studies was to examine the effects of combining Src inhibition with inhibition of the protein tyrosine phosphatase SHP-2 in pancreatic cancer cells in vitro and in vivo. SHP-2 and Src functions were inhibited by siRNA or small molecule inhibitors. The effects of dual Src/SHP-2 functional inhibition were evaluated by Western blot analysis of downstream signaling pathways; cell biology assays to examine caspase activity, viability, adhesion, migration, and invasion in vitro; and an orthotopic nude mouse model to observe pancreatic tumor formation in vivo. Dual targeting of Src and SHP-2 induces an additive or supra-additive loss of phosphorylation of Akt and ERK-1/2 and corresponding increases in expression of apoptotic markers, relative to targeting either protein individually. Combinatorial inhibition of Src and SHP-2 significantly reduces viability, adhesion, migration, and invasion of pancreatic cancer cells in vitro and tumor formation in vivo, relative to individual Src/SHP-2 inhibition. These data suggest that the antitumor effects of Src inhibition in pancreatic cancer may be enhanced through simultaneous inhibition of SHP-2.

Modified rice bran hemicellulose inhibits vascular endothelial growth factor-induced angiogenesis in vitro via VEGFR2 and its downstream signaling pathways

PubMed Central

ZHU, Xia; OKUBO, Aya; IGARI, Naoki; NINOMIYA, Kentaro; EGASHIRA, Yukari

2016-01-01

Angiogenesis is implicated in diverse pathological conditions such as cancer, rheumatoid arthritis, psoriasis, atherosclerosis, and retinal neovascularization. In the present study, we investigated the effects of modified rice bran hemicellulose (MRBH), a water-soluble hemicellulose preparation from rice bran treated with shiitake enzymes, on vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and its mechanism. We found that MRBH significantly inhibited VEGF-induced tube formation in human umbilical vein endothelial cells (HUVECs) co-cultured with human dermal fibroblasts. We also observed that MRBH dose-dependently suppressed the VEGF-induced proliferation and migration of HUVECs. Furthermore, examination of the anti-angiogenic mechanism indicated that MRBH reduced not only VEGF-induced activation of VEGF receptor 2 but also of the downstream signaling proteins Akt, extracellular signal-regulated protein kinase 1/2, and p38 mitogen-activated protein kinase. These findings suggest that MRBH has in vitro anti-angiogenic effects that are partially mediated through the inhibition of VEGF signaling. PMID:28439487

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

PubMed

Walker, Valerie; Mills, Graham A; Anderson, Mary E; Ingle, Brandall L; Jackson, John M; Moss, Charlotte L; Sharrod-Cole, Hayley; Skipp, Paul J

2017-02-01

1. Metabolic acidosis due to accumulation of l-5-oxoproline is a rare, poorly understood, disorder associated with acetaminophen treatment in malnourished patients with chronic morbidity. l-5-Oxoprolinuria signals abnormal functioning of the γ-glutamyl cycle, which recycles and synthesises glutathione. Inhibition of glutathione synthetase (GS) by N-acetyl-p-benzoquinone imine (NAPQI) could contribute to 5-oxoprolinuric acidosis in such patients. We investigated the interaction of NAPQI with GS in vitro. 2. Peptide mapping of co-incubated NAPQI and GS using mass spectrometry demonstrated binding of NAPQI with cysteine-422 of GS, which is known to be essential for GS activity. Computational docking shows that NAPQI is properly positioned for covalent bonding with cysteine-422 via Michael addition and hence supports adduct formation. 3. Co-incubation of 0.77 μM of GS with NAPQI (25-400 μM) decreased enzyme activity by 16-89%. Inhibition correlated strongly with the concentration of NAPQI and was irreversible. 4. NAPQI binds covalently to GS causing irreversible enzyme inhibition in vitro. This is an important novel biochemical observation. It is the first indication that NAPQI may inhibit glutathione synthesis, which is pivotal in NAPQI detoxification. Further studies are required to investigate its biological significance and its role in 5-oxoprolinuric acidosis.

Activation of PPARγ inhibits pro-inflammatory cytokines production by upregulation of miR-124 in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Dan; Shi, Liuyan; Xin, Wei

Peroxisome proliferator-activated receptor gamma (PPARγ) and miR-124 have been reported to play important roles in regulation of inflammation. However, the underlying anti-inflammatory mechanisms remain not well understood. In the present study, we demonstrated that the expression level of PPARγ is positively correlated with that of miR-124 in patients with sepsis. Activation of PPARγ upregulates miR-124 and in turn inhibits miR-124 target gene. PPARγ bound directly to PPRE in the miR-124 promoter region, and enhanced the promoter transcriptional activity. PPARγ-induced miR-124 is involved in the suppression of pro-inflammatory cytokine in vitro and in vivo. These results suggest that PPARγ-induced miR-124 inhibits the productionmore » of pro-inflammatory cytokines is a novel PPARγ anti-inflammatory mechanism and also indicate that miR-124 may be a potential therapeutic target for the treatment of inflammatory diseases. - Highlights: • The expression level of PPARγ is positively correlated with that of miR-124 in patients with sepsis. • PPARγ upregulates miR-124 and in turn inhibits miR-124 target gene. • PPARγ promotes miR-124 transcription through binding to miR-124 promoter region. • Inhibition of miR-124 attenuates the PPARγ-mediated suppression of proinflammatory cytokines in vitro. • PPARγ-induced miR-124 is involved in the suppression of pro-inflammatory cytokine in vivo.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 extentmore » 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.« less

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

PubMed

Saporito, M S; Warwick, R O

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 (all 1 microM) decreased K+ evoked 3H-5-HT release from superfused HYP slices by 25%. Bacitracin (BCN, 2 micrograms/ml), a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K+ evoked 3H-5-HT release. Phosphoramidon (PAN, 10 microM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K+ evoked 3H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 microM), enhanced both BN and NM-C inhibition of 3H-5-HT release. Bestatin (BST, 10 microM) had no effect on BN or NM-C inhibitory activity on 3H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3H-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 3H-5-HT uptake. These data suggest: a) that BN-like peptides may alter neurotransmission in the HYP by acting presynaptically on the 5-HT release mechanism; b) a similarity in the structural requirements for the BN induced inhibition of 5-HT release and BN evoked thermoregulatory disturbances; and c) that peptidases may selectively augment or reduce pharmacologic activity of BN-like peptides upon CNS administration.

In vitro ability of currently available oximes to reactivate organophosphate pesticide-inhibited human acetylcholinesterase and butyrylcholinesterase.

PubMed

Jun, Daniel; Musilova, Lucie; Musilek, Kamil; Kuca, Kamil

2011-01-01

We have in vitro tested the ability of common, commercially available, cholinesterase reactivators (pralidoxime, obidoxime, methoxime, trimedoxime and HI-6) to reactivate human acetylcholinesterase (AChE), inhibited by five structurally different organophosphate pesticides and inhibitors (paraoxon, dichlorvos, DFP, leptophos-oxon and methamidophos). We also tested reactivation of human butyrylcholinesterase (BChE) with the aim of finding a potent oxime, suitable to serve as a "pseudocatalytic" bioscavenger in combination with this enzyme. Such a combination could allow an increase of prophylactic and therapeutic efficacy of the administered enzyme. According to our results, the best broad-spectrum AChE reactivators were trimedoxime and obidoxime in the case of paraoxon, leptophos-oxon, and methamidophos-inhibited AChE. Methamidophos and leptophos-oxon were quite easily reactivatable by all tested reactivators. In the case of methamidophos-inhibited AChE, the lower oxime concentration (10(-5) M) had higher reactivation ability than the 10(-4) M concentration. Therefore, we evaluated the reactivation ability of obidoxime in a concentration range of 10(-3)-10(-7) M. The reactivation of methamidophos-inhibited AChE with different obidoxime concentrations resulted in a bell shaped curve with maximum reactivation at 10(-5) M. In the case of BChE, no reactivator exceeded 15% reactivation ability and therefore none of the oximes can be recommended as a candidate for "pseudocatalytic" bioscavengers with BChE.

Metabolic interactions between acetaminophen (paracetamol) and two flavonoids, luteolin and quercetin, through in-vitro inhibition studies.

PubMed

Cao, Lei; Kwara, Awewura; Greenblatt, David J

2017-12-01

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 (IC 50 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 IC 50 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.

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.

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

4-Chlorotetrazolo[1,5-a]quinoxaline inhibits activation of Syk kinase to suppress mast cells in vitro and mast cell-mediated passive cutaneous anaphylaxis in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Kui Lea; Ko, Na Young; Lee, Jun Ho

2011-12-15

4-Chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. We aimed to study the effects of 4-chlorotetrazolo[1,5-a]quinoxaline on activation of mast cells in vitro and in mice. 4-Chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited degranulation of mast cells in a dose-dependent manner, and also suppressed the expression and secretion of TNF-{alpha} and IL-4 in mast cells. Mechanistically, 4-chlorotetrazolo[1,5-a]quinoxaline inhibited activating phosphorylation of Syk and LAT, which are crucial for early Fc{epsilon}RI-mediated signaling events, as well as Akt and MAP kinases, which play essential roles in the production of various pro-inflammatory cytokines in mast cells. Notably, although 4-chlorotetrazolo[1,5-a]quinoxaline inhibited the activation of Fyn and Syk, minimal inhibition was observedmore » in mast cells in the case of Lyn. Furthermore, consistent with its in vitro activity, 4-chlorotetrazolo[1,5-a]quinoxaline significantly suppressed mast cell-mediated passive cutaneous anaphylaxis in mice. In summary, the results from this study demonstrate that 4-chlorotetrazolo[1,5-a]quinoxaline shows an inhibitory effect on mast cells in vitro and in vivo, and that this is mediated by inhibiting the activation of Syk in mast cells. Therefore, 4-chlorotetrazolo[1,5-a]quinoxaline could be useful in the treatment of mast cell-mediated allergic diseases. -- Highlights: Black-Right-Pointing-Pointer 4-chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. Black-Right-Pointing-Pointer The effect of 4-chlorotetrazolo[1,5-a]quinoxaline on mast cells was investigated. Black-Right-Pointing-Pointer 4-chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited Syk activation. Black-Right-Pointing-Pointer 4-chlorotetrazolo[1,5-a]quinoxaline could be useful for IgE-mediated allergy.« less

Luteolin-7-O-Glucoside Present in Lettuce Extracts Inhibits Hepatitis B Surface Antigen Production and Viral Replication by Human Hepatoma Cells in Vitro

PubMed Central

Cui, Xiao-Xian; Yang, Xiao; Wang, Hui-Jing; Rong, Xing-Yu; Jing, Sha; Xie, You-Hua; Huang, Dan-Feng; Zhao, Chao

2017-01-01

Hepatitis B virus (HBV) infection is endemic in Asia and chronic hepatitis B (CHB) is a major public health issue worldwide. Current treatment strategies for CHB are not satisfactory as they induce a low rate of hepatitis B surface antigen (HBsAg) loss. Extracts were prepared from lettuce hydroponically cultivated in solutions containing glycine or nitrate as nitrogen sources. The lettuce extracts exerted potent anti-HBV effects in HepG2 cell lines in vitro, including significant HBsAg inhibition, HBV replication and transcription inhibition, without exerting cytotoxic effects. When used in combination interferon-alpha 2b (IFNα-2b) or lamivudine (3TC), the lettuce extracts synergistically inhibited HBsAg expression and HBV replication. By using differential metabolomics analysis, Luteolin-7-O-glucoside was identified and confirmed as a functional component of the lettuce extracts and exhibited similar anti-HBV activity as the lettuce extracts in vitro. The inhibition rate on HBsAg was up to 77.4%. Moreover, both the lettuce extracts and luteolin-7-O-glucoside functioned as organic antioxidants and, significantly attenuated HBV-induced intracellular reactive oxygen species (ROS) accumulation. Luteolin-7-O-glucoside also normalized ROS-induced mitochondrial membrane potential damage, which suggests luteolin-7-O-glucoside inhibits HBsAg and HBV replication via a mechanism involving the mitochondria. Our findings suggest luteolin-7-O-glucoside may have potential value for clinical application in CHB and may enhance HBsAg and HBV clearance when used as a combination therapy. PMID:29270164

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.

Green tea extract inhibits proliferation of uterine leiomyoma cells in vitro and in nude mice

PubMed Central

ZHANG, Dong; AL-HENDY, Mohamed; RICHARD-DAVIS, Gloria; MONTGOMERY-RICE, Valerie; SHARAN, Chakradhari; RAJARATNAM, Veera; KHURANA, Anjali; AL-HENDY, Ayman

2010-01-01

Objective Investigate the effect of epigallocatechin gallate (EGCG), on rat leiomyoma (ELT3) cells in vitro and in nude mice model. Study Design ELT3 cells were treated with various concentrations of EGCG. Cell proliferation, PCNA and Cdk4 protein levels were evaluated. ELT3 cells were inoculated subcutaneously in female athymic nude mice. Animals were fed 1.25mg EGCG (in drinking water)/mouse/day. Tumors were collected and evaluated at 4 and 8 weeks post-treatment. Results Inhibitory effect of EGCG (200 μM) on ELT3 cells was observed after 24 h treatment (p<0.05). At ≥50μM, EGCG significantly decreased PCNA and Cdk4 protein levels (p<0.05). In vivo, EGCG treatment dramatically reduced the volume and weight of tumors at 4 and 8 weeks post-treatment (p<0.05). The PCNA and Cdk4 protein levels were significantly reduced in EGCG treated group (p<0.05). Conclusion EGCG effectively inhibits the proliferation and induce apoptosis in rat ELT3 uterine leiomyoma cells in vitro and in vivo. PMID:20074693

Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

PubMed Central

Wen, Jiexia; Pan, Sumin; Liang, Shuang; Zhong, Zhenyu; He, Ying; Lin, Hongyu; Li, Wenyan; Wang, Liyue; Li, Xiujin; Zhong, Fei

2013-01-01

Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo. PMID:24089666

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.

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

PubMed Central

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

2015-01-01

Introduction 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. Methods 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. Results 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. Conclusions PDI activity is inhibited by red wine and grape juice, identifying a potentially novel mechanism underlying the cardiovascular benefits attributed to wine consumption. PMID:26585763

Dendrobium officinale Orchid Extract Prevents Ovariectomy-Induced Osteoporosis in Vivo and Inhibits RANKL-Induced Osteoclast Differentiation in Vitro

PubMed Central

Wang, Qi; Zi, Cheng-Ting; Wang, Jing; Wang, Yu-Na; Huang, Ye-Wei; Fu, Xue-Qi; Wang, Xuan-Jun; Sheng, Jun

2018-01-01

Background: Dendrobium officinale, a traditional Chinese medical herb with high value that is widely used in Asia, possesses many positive effects on human health, including anti-chronic inflammation, anti-obesity, and immune modulation properties; however, whether D. officinale has inhibitory effects on postmenopausal osteoporosis remains unknown. Objective: We investigated the effects of D. officinale extract (DOE) on ovariectomy-induced bone loss in vivo and on osteoclastogenesis in vitro. Methods: In vivo, female rats were divided into a sham-operated (sham) group and five ovariectomized (OVX) subgroups: OVX with vehicle (OVX), OVX with Xian-Ling-Gu-Bao capsule (240 mg/kg body weight/day), and OVX with low-, medium-, and high-dose DOE (150, 300, and 600 mg/kg body weight/day, respectively). Animals in each group were administered their corresponding treatments for 13 weeks. Body weight, serum biochemical parameters, uterine and femoral physical parameters, bone mineral density (BMD), bone biomechanical properties, and bone microarchitecture were obtained. In vitro, the effects of DOE on osteoclastogenesis were examined using RAW264.7 cells. The effects of DOE on osteoclastogenesis and the expression of osteoclast-specific marker genes and proteins were determined. Results: DOE effectively ameliorated serum biochemical parameters, especially alleviated estradiol (E2) deficiency and maintained calcium and phosphorus homeostasis. DOE improved uterine and femoral physical parameters. In addition, DOE improved femoral BMD and biomechanical properties. DOE significantly ameliorated bone microarchitecture. Moreover, DOE inhibited osteoclastogenesis independent of its cytoxicity and suppressed the expression of osteoclast-specific marker genes and proteins. Conclusion: DOE can effectively prevent ovariectomy-induced bone loss in vivo and inhibit osteoclastogenesis in vitro. PMID:29379436

Dendrobium officinale Orchid Extract Prevents Ovariectomy-Induced Osteoporosis in Vivo and Inhibits RANKL-Induced Osteoclast Differentiation in Vitro.

PubMed

Wang, Qi; Zi, Cheng-Ting; Wang, Jing; Wang, Yu-Na; Huang, Ye-Wei; Fu, Xue-Qi; Wang, Xuan-Jun; Sheng, Jun

2017-01-01

Background: Dendrobium officinale , a traditional Chinese medical herb with high value that is widely used in Asia, possesses many positive effects on human health, including anti-chronic inflammation, anti-obesity, and immune modulation properties; however, whether D. officinale has inhibitory effects on postmenopausal osteoporosis remains unknown. Objective: We investigated the effects of D. officinale extract (DOE) on ovariectomy-induced bone loss in vivo and on osteoclastogenesis in vitro . Methods: In vivo , female rats were divided into a sham-operated (sham) group and five ovariectomized (OVX) subgroups: OVX with vehicle (OVX), OVX with Xian-Ling-Gu-Bao capsule (240 mg/kg body weight/day), and OVX with low-, medium-, and high-dose DOE (150, 300, and 600 mg/kg body weight/day, respectively). Animals in each group were administered their corresponding treatments for 13 weeks. Body weight, serum biochemical parameters, uterine and femoral physical parameters, bone mineral density (BMD), bone biomechanical properties, and bone microarchitecture were obtained. In vitro , the effects of DOE on osteoclastogenesis were examined using RAW264.7 cells. The effects of DOE on osteoclastogenesis and the expression of osteoclast-specific marker genes and proteins were determined. Results: DOE effectively ameliorated serum biochemical parameters, especially alleviated estradiol (E2) deficiency and maintained calcium and phosphorus homeostasis. DOE improved uterine and femoral physical parameters. In addition, DOE improved femoral BMD and biomechanical properties. DOE significantly ameliorated bone microarchitecture. Moreover, DOE inhibited osteoclastogenesis independent of its cytoxicity and suppressed the expression of osteoclast-specific marker genes and proteins. Conclusion: DOE can effectively prevent ovariectomy-induced bone loss in vivo and inhibit osteoclastogenesis in vitro .

Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics.

PubMed

Reidy, G F; Mehta, I; Murray, M

1989-05-01

The anti-parkinsonian agent orphenadrine has been shown to form an in vitro metabolic intermediate (MI) complex in hepatic microsomes isolated from phenobarbital (PB)-treated rats. The present study was undertaken to assess the cytochrome P-450 isozyme specificity of inhibition and MI complexation. Spectral studies with untreated and PB-induced rat hepatic microsomes confirmed earlier reports on the selectivity of P-450 complexation by orphenadrine; MI complex formation was only observed with PB-induced microsomes. Inhibition studies with the P-450 substrates androst-4-ene-3,17-dione (androstenedione) and 7-pentoxyresorufin revealed selective inhibition of P-450 PB-B/D-associated monooxygenase activity. Thus, in microsomes from untreated male rats, orphenadrine failed to significantly inhibit (less than 50% inhibition up to a concentration of 300 microM) any of the major pathways of P-450-associated androstenedione metabolism. Preincubation of these microsomal fractions with orphenadrine and NADPH was not associated with increased inhibition of androstenedione metabolism. However, in PB-induced microsomes, P-450 PB-B/D-specific androstenedione 16 beta-hydroxylase activity was significantly and selectively inhibited (IC50 = 90 microM). Preincubation of orphenadrine with NADPH-supplemented PB-induced microsomes for 2, 4, or 8 min before androstenedione addition resulted in increased inhibition toward 16 beta-hydroxylase activity, lowering the observed IC50 to 6.6, 0.47, and 0.06 microM), respectively. Preincubation did not affect the selectivity of inhibition. In the absence of preincubation, orphenadrine appeared to be a potent mixed (competitive/noncompetitive)-type inhibitor of P-450 PB-B/D-associated pentoxyresorufin O-depentylation (Ki = 3.8 microM). Preincubation of orphenadrine with NADPH-supplemented microsomal fractions for 4 min resulted in a 30-fold lowering of the apparent inhibitor constant (Ki = 0.13 microM) and a change in the apparent inhibition

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.

Inhibition of proanthocyanidin A2 on porcine reproductive and respiratory syndrome virus replication in vitro

PubMed Central

Chen, Yao; Duan, Mubing; Tian, Ge; Deng, Xianbo; Sun, Yankuo; Zhou, Tong; Zhang, Guihong; Chen, Weisan

2018-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely prevalent and endemic swine pathogen that causes significant economic losses for the global pig industry annually. Currently, the most prevalent strategy for PRRSV control remains the prevention of virus transmission, with highly effective therapeutic agents and vaccines still lacking. Proanthocyanidin A2 (PA2) belongs to the family of tea polyphenols, which have been reported to exhibit a range of biological activities including anti-oxidative, cardio-protective, anti-tumoural, anti-bacterial, anti-viral, and anti-inflammatory effects in vitro as well as in vivo. Here, we demonstrate that PA2 exhibits potent anti-viral activity against PRRSV infection in Marc-145 cells. Similar inhibitory effects were also found in porcine alveolar macrophages, the primary target cell type of PRRSV infection in pigs in vivo. For traditional type II PRRSV CH-1a strain and high pathogenic GD-XH strain and GD-HD strain, PA2 exhibited broad-spectrum and comparable inhibitory activities in vitro with EC50 ranging from 2.2 to 3.2 μg/ml. Treatment of PRRSV-infected Marc-145 cells with PA2 significantly inhibited viral RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. In addition, PA2 treatment reduced gene expressions of cytokines (TNF-α, IFN-α, IL-1β and IL-6) induced by PRRSV infection in PAMs. Mechanistically, PA2 inhibited PRRSV replication by targeting multiple pathways including blockade of viral entry and progeny virus release. Altogether, our findings suggest that PA2 has the potential to serve as a novel prophylactic and therapeutic strategies against PRRSV infection. PMID:29489892

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spaggiari, Dany, E-mail: dany.spaggiari@unige.ch

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6,more » 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC{sub 50} values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC{sub 50} values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC{sub 50} values

Cleavage of Poly(A)-Binding Protein by Enterovirus Proteases Concurrent with Inhibition of Translation In Vitro

PubMed Central

Joachims, Michelle; Van Breugel, Pieter C.; Lloyd, Richard E.

1999-01-01

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in

Bilirubin induces a calcium-dependent inhibition of multifunctional Ca2+/calmodulin-dependent kinase II activity in vitro.

PubMed

Churn, S B; DeLorenzo, R J; Shapiro, S M

1995-12-01

Excessive bilirubin levels in newborn infants result in long-term neurologic deficits that remain after bilirubin levels return to normal. Much of the observed neurologic deficits can be attributed to bilirubin-induced, delayed neuronal cell death. Inhibition of calcium/calmodulin-dependent kinase II (CaM kinase II) activity that precedes cell death is observed in conditions such as seizure activity, stroke, and glutamate excitotoxicity. Because neonatal bilirubin exposure results in neuronal loss in developing brain systems, we tested whether bilirubin exposure would induce an immediate inhibition of CaM activity, in vitro. P-81 filtration assay of basal and calcium-stimulated kinase activity was performed under standard kinase assay conditions. Bilirubin and/or albumin was added to the reaction vessels to determine the effect of these agents on kinase activity. Bilirubin exposure resulted in a concentration-dependent inhibition of CaM kinase II activity (IC50 = 16.78 microM). At concentrations above 50 microM, bilirubin exposure resulted in a 71 +/- 8% (mean +/- SD) inhibition of kinase activity (p < 0.001, t test, n = 10). Bilirubin exposure did not result in kinase inhibition if excessive bilirubin was removed by albumin binding before stimulation of kinase activity (106.9 +/- 9.6% control activity, n = 5). However, removal of bilirubin by binding with albumin after calcium addition did not restore kinase activity. (36.1 +/- 3.8% control activity, n = 5). Thus, once inhibition was observed, the activity could not be restored by addition of albumin. The data suggest that bilirubin exposure resulted in a calcium-dependent inhibition of CaM kinase II activity that, once induced, was not reversible by removing bilirubin by the addition of albumin. Because inhibition of CaM kinase II activity has been correlated with delayed neuronal cell death in many neuropathologic conditions, bilirubin-induced inhibition of this enzyme may be a cellular mechanism by which

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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-daltonmore » 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.« less

Withaferin A Inhibits the Proteasome Activity in Mesothelioma In Vitro and In Vivo

PubMed Central

Cheryan, Vino T.; Wu, Wenjuan; Cui, Cindy Qiuzhi; Polin, Lisa A.; Pass, Harvey I.; Dou, Q. Ping; Rishi, Arun K.; Wali, Anil

2012-01-01

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent. PMID:22912669

LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

PubMed Central

Bennett, Terry L.; Kraft, Shannon M.; Reaves, Barbara J.; Mima, Joji; O’Brien, Kevin M.; Starai, Vincent J.

2013-01-01

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis. PMID:23437241

In Vitro Ability of Currently Available Oximes to Reactivate Organophosphate Pesticide-Inhibited Human Acetylcholinesterase and Butyrylcholinesterase

PubMed Central

Jun, Daniel; Musilova, Lucie; Musilek, Kamil; Kuca, Kamil

2011-01-01

We have in vitro tested the ability of common, commercially available, cholinesterase reactivators (pralidoxime, obidoxime, methoxime, trimedoxime and HI-6) to reactivate human acetylcholinesterase (AChE), inhibited by five structurally different organophosphate pesticides and inhibitors (paraoxon, dichlorvos, DFP, leptophos-oxon and methamidophos). We also tested reactivation of human butyrylcholinesterase (BChE) with the aim of finding a potent oxime, suitable to serve as a “pseudocatalytic” bioscavenger in combination with this enzyme. Such a combination could allow an increase of prophylactic and therapeutic efficacy of the administered enzyme. According to our results, the best broad-spectrum AChE reactivators were trimedoxime and obidoxime in the case of paraoxon, leptophos-oxon, and methamidophos-inhibited AChE. Methamidophos and leptophos-oxon were quite easily reactivatable by all tested reactivators. In the case of methamidophos-inhibited AChE, the lower oxime concentration (10−5 M) had higher reactivation ability than the 10−4 M concentration. Therefore, we evaluated the reactivation ability of obidoxime in a concentration range of 10−3–10−7 M. The reactivation of methamidophos-inhibited AChE with different obidoxime concentrations resulted in a bell shaped curve with maximum reactivation at 10−5 M. In the case of BChE, no reactivator exceeded 15% reactivation ability and therefore none of the oximes can be recommended as a candidate for “pseudocatalytic” bioscavengers with BChE. PMID:21673941

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

Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herkert, N.M.; Schulz, S.; Wille, T.

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 ormore » 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.« less

Croton megalobotrys Müll Arg. and Vitex doniana (Sweet): Traditional medicinal plants in a three-step treatment regimen that inhibit in vitro replication of HIV-1.

PubMed

Tietjen, Ian; Gatonye, Teresia; Ngwenya, Barbara N; Namushe, Amos; Simonambanga, Sundana; Muzila, Mbaki; Mwimanzi, Philip; Xiao, Jianbo; Fedida, David; Brumme, Zabrina L; Brockman, Mark A; Andrae-Marobela, Kerstin

2016-09-15

Human Immunodeficiency Virus (HIV) strains resistant to licensed anti-retroviral drugs (ARVs) continue to emerge. On the African continent, uneven access to ARVs combined with occurrence of side-effects after prolonged ARV therapy have led to searches for traditional medicines as alternative or complementary remedies to conventional HIV/AIDS management. Here we characterize a specific three-step traditional HIV/AIDS treatment regimen consisting of Cassia sieberiana root, Vitex doniana root, and Croton megalobotrys bark by combining qualitative interviews of traditional medical knowledge users in Botswana with in vitro HIV replication studies. Crude extracts from a total of seven medicinal plants were tested for in vitro cytotoxicity and inhibition of wild-type (NL4.3) and ARV-resistant HIV-1 replication in an immortalized GFP-reporter CD4+ T-cell line. C. sieberiana root, V. doniana root, and C. megalobotrys bark extracts inhibited HIV-1NL4.3 replication with dose-dependence and without concomitant cytotoxicity. C. sieberiana and V. doniana extracts inhibited HIV-1 replication by 50% at 84.8µg/mL and at 25µg/mL, respectively, while C. megalobotrys extracts inhibited HIV-1 replication by a maximum of 45% at concentrations as low as 0.05µg/mL. Extracts did not interfere with antiviral activities of licensed ARVs when applied in combination and exhibited comparable efficacies against viruses harboring major resistance mutations to licensed protease, reverse-transcriptase, or integrase inhibitors. We report for the first time a three-step traditional HIV/AIDS regimen, used alone or in combination with standard ARV regimens, where each step exhibited more potent ability to inhibit HIV replication in vitro. Our observations support the "reverse pharmacology" model where documented clinical experiences are used to identify natural products of therapeutic value. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

RNA interference inhibits yellow fever virus replication in vitro and in vivo.

PubMed

Pacca, Carolina C; Severino, Adriana A; Mondini, Adriano; Rahal, Paula; D'avila, Solange G P; Cordeiro, José Antonio; Nogueira, Mara Correa Lelles; Bronzoni, Roberta V M; Nogueira, Maurício L

2009-04-01

RNA interference (RNAi) is a process that is induced by double stranded RNA and involves the degradation of specific sequences of mRNA in the cytoplasm of the eukaryotic cells. It has been used as an antiviral tool against many viruses, including flaviviruses. The genus Flavivirus contains the most important arboviruses in the world, i.e., dengue (DENV) and yellow fever (YFV). In our study, we investigated the in vitro and in vivo effect of RNAi against YFV. Using stable cell lines that expressed RNAi against YFV, the cell lines were able to inhibit as much as 97% of the viral replication. Two constructions (one against NS1 and the other against E region of YFV genome) were able to protect the adult Balb/c mice against YFV challenge. The histopathologic analysis demonstrated an important protection of the central nervous system by RNAi after 10 days of viral challenge. Our data suggests that RNAi is a potential viable therapeutic weapon against yellow fever.

Fluoxetine Induces Proliferation and Inhibits Differentiation of Hypothalamic Neuroprogenitor Cells In Vitro

PubMed Central

Sousa-Ferreira, Lígia; Aveleira, Célia; Botelho, Mariana; Álvaro, Ana Rita; Pereira de Almeida, Luís; Cavadas, Cláudia

2014-01-01

A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells). Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides. PMID:24598761

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.

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.

Amperozide, a putative anti-psychotic drug: Uptake inhibition and release of dopamine in vitro in the rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eriksson, E.

1990-01-01

The effects of amperozide (a diphenylbutylpiperazinecarboxamide derivative) on the uptake and release of {sup 3}H-dopamine in vitro were investigated. Amperozide inhibited the amphetamine-stimulated release of dopamine from perfused rat striatal tissue in a dose-dependent manner. With 1 and 10 {mu}m amperozide there was significant inhibition of the amphetamine-stimulated release of dopamine, to 44 and 36 % of control. In contrast, 10 {mu}M amperozide significantly strengthened the electrically stimulated release of dopamine from perfused striatal slices. Amperozide 1-10 {mu}M had no significant effect on the potassium-stimulated release of dopamine, 10 {mu}M amperozide also slightly increased the basal release of {sup 3}H-dopaminemore » from perfused striatal tissue. These effects on various types of release are similar to those reported for uptake inhibitors. The uptake of dopamine in striatal tissue was inhibited by amperozide with IC{sub 50} values of 18 {mu}M for uptake in chopped tissue and 1.0 {mu}M for uptake in synaptosomes. Amperozide also inhibited the uptake of serotonin in synaptosomes from frontal cortex, IC{sub 50} = 0.32 {mu}M and the uptake of noradrenaline in cortical synaptosomes, IC{sub 50} = 0.78 {mu}M.« less

Ketoconazole inhibits the in vitro and in vivo metabolism of all-trans-retinoic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Wauwe, J.P.; Coene, M.C.; Goossens, J.

1988-05-01

Ketoconazole, an antifungal agent and inhibitor of certain mammalian cytochrome P-450-dependent enzymes, was studied for its effects on the in vitro and in vivo metabolism of all-trans-retinoic acid (RA). In vitro, ketoconazole (Ki = 0.75 microM) inhibited, in an apparently competitive manner, the cytochrome P-450-mediated metabolism to 4-hydroxy- and 4-keto-retinoic acids by hamster liver microsomes. In vivo, ketoconazole suppressed the formation of polar RA metabolites by normal rats dosed intrajugularly with 200 ng of (/sup 3/H)RA. After p.o. treatment with ketoconazole (2.5-40 mg/kg) given 1 hr before the (/sup 3/H)RA injection, the radioactivity extracted from the liver consisted of 25more » to 50% polar metabolites (control 66 +/- 1%) and 50 to 75% undegraded RA (control 34 +/- 1%) as evidenced by reverse-phase high-performance liquid chromatography. Time course experiments showed that ketoconazole's inhibitory effects lasted for 3 hr. Our data indicate the quantitative importance of the cytochrome P-450 enzymatic pathway in the biotransformation of RA. They also suggest that ketoconazole is capable of prolonging the biological half-life of RA and of improving the tissue levels of this compound.« less

An in vitro AChE inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS approach for screening and characterizing of AChE inhibitors from roots of Coptis chinensis Franch.

PubMed

Zhao, Hengqiang; Zhou, Siduo; Zhang, Minmin; Feng, Jinhong; Wang, Shanshan; Wang, Daijie; Geng, Yanling; Wang, Xiao

2016-02-20

In this study, an in vitro acetylcholinesterase (AChE) inhibition assay based on microplate reader combined with ultrafiltration high performance liquid chromatography-electrospray quadrupole time of flight mass (UF-HPLC-ESI-Q-TOF/MS) was developed for the rapid screening and identification of acetylcholinesterase inhibitors (AChEI) from roots of Coptis chinensis Franch. Incubation conditions such as enzyme concentration, incubation time, incubation temperature and co-solvent was optimized so as to get better screening results. Five alkaloids including columbamine, jatrorrhizine, coptisine, palmatine and berberine were found with AChE inhibition activity in the 80% ethanol extract of C. chinensis Franch. The screened compounds were identified by HPLC-DAD-ESI-Q-TOF/MS compared with the reference stands and literatures. The screened results were verified by in vitro AChE inhibition assays, palmatine showed the best AChE inhibitory activities with IC50 values of 36.6μM among the five compounds. Results of the present study indicated that the combinative method using in vitro AChE inhibition assay and UF-HPLC-ESI-Q-TOF/MS could be widely applied for rapid screening and identification of AChEI from complex TCM extract. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibition of the infectivity and inflammatory response of influenza virus by Arbidol hydrochloride in vitro and in vivo (mice and ferret).

PubMed

Wang, Yutao; Ding, Yuewen; Yang, Chunguang; Li, Runfeng; Du, Qiuling; Hao, Yanbing; Li, Zhengtu; Jiang, Haiming; Zhao, Jin; Chen, Qiaoyan; Yang, Zifeng; He, Zhanlong

2017-07-01

Influenza virus infections are the main contagious respiratory disease with high levels of morbidity and mortality worldwide. Antiviral drugs are indispensable for the prophylaxis and treatment of influenza and other respiratory viral infections. In this study, the Arbidol hydrochloride (ARB), which has been licensed in Russia and China, is used to investigate its anti-viral and anti-inflammatory efficacy in vitro and in vivo. The antiviral results in vitro showed that ARB had a better inhibition on Influenza virus A/PR/8/34 (H1N1), A/Guangdong/GIRD07/09 (H1N1), A/Aichi/2/68 (H3N2), A/HK/Y280/97 (H9N2) with IC 50 ranging from 4.4 to 12.1μM. The further mechanisms study demonstrated that ARB is able to inhibit hemagglutinin-mediated hemolysis at concentration of 3.91-15.63μg/mL. The anti-inflammatory efficacy in vitro indicated that IL-6, IP-10, MCP-1, RANTES and TNF-α levels were diminished by ARB at concentrations of 22.6 and 18.8μM. The in vivo results in mice model displayed that the survival rates of mice administered 25mg/mL and 45mg/mL ARB were 40% and 50% respectively. And also, ARB can inhibit the decrease of body weight at 45mg/mL and inhibit the increase of mice lung index at 25mg/mL and 45mg/mL comparing to virus group. In ferret model, the ARB-treated ferrets showed a fever that peaked at 2 dpi and gradually decreased beginning at 3 dpi while relatively high temperatures were observed until 4 dpi in the virus group. The ARB-treated group scored 0-1 in the activity level at 2 dpi and 3 dpi at all time points. The transcription levels of cytokines in the respiratory tract of ferrets were detected at 3 dpi. Several proinflammatory cytokines induced by influenza (IL-10, TNF-α, IL-8 and IL-6) were down-regulated by post-treatment with ARB. The histopathological results of ferret lung displayed that ARB can alleviate the influenza virus induced lung lesions. Our results clarified the activity of ARB in both suppressing virus propagation and modulating

Human Milk Mucin 1 and Mucin 4 Inhibit Salmonella enterica Serovar Typhimurium Invasion of Human Intestinal Epithelial Cells In Vitro123

PubMed Central

Liu, Bo; Yu, Zhuoteng; Chen, Ceng; Kling, David E.; Newburg, David S.

2012-01-01

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. PMID:22718031

In vitro investigation of anticancer and ACE-inhibiting activity, α-amylase and α-glucosidase inhibition, and antioxidant activity of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk.

PubMed

Ayyash, Mutamed; Al-Nuaimi, Amna K; Al-Mahadin, Suheir; Liu, Shao-Quan

2018-01-15

This study aimed to investigate in vitro the health-promoting benefits (anticancer activity, α-amylase and α-glucosidase inhibition, angiotensin-converting-enzyme (ACE)-inhibition, antioxidant and proteolytic activity) of camel milk fermented with indigenous probiotic strains of Lactobacillus spp., compared with fermented bovine milk. The three camel milk probiotic strains Lb. reuteri-KX881777, Lb. plantarum-KX881772, Lb. plantarum-KX881779 and a control strain Lb. plantarum DSM2468 were employed to ferment camel and bovine milks separately. The proteolytic and antioxidant activity of water soluble extracts (WSEs) from all fermented camel milks were higher than those of fermented bovine milk. α-Amylase inhibition of WSEs were >34% in both milk types fermented with all strains during storage periods, except the WSE of camel milk fermented by Lp.K772. The highest ACE-inhibition of the WSE from camel milk fermented by Lr.K777 was >80%. The proliferations of Caco-2, MCF-7 and HELA cells were more inhibited when treated with the WSE of fermented camel milk. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahalingam, Sharada, E-mail: mahalin2@illinois.edu; Gao, Liying, E-mail: lgao@uiuc.edu; Gonnering, Marni, E-mail: mgonne2@illinois.edu

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 folliclesmore » isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 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. - Highlights: • Equol exposure inhibits antral follicle growth. • Equol exposure increases follicle atresia. • Equol exposure inhibits sex steroid hormone levels. • Equol exposure inhibits mRNA levels of certain steroidogenic enzymes.« less

Naringin attenuates diabetic retinopathy by inhibiting inflammation, oxidative stress and NF-κB activation in vivo and in vitro.

PubMed

Liu, Lihua; Zuo, Zhongfu; Lu, Sijing; Liu, Aihua; Liu, Xuezheng

2017-07-01

Naringin, an essential flavonoid, inhibits inflammatory response and oxidative stress in diabetes. However, whether naringin has beneficial effects on diabetic retinopathy (DR) remains unknown. Streptozotocin (STZ, 65 mg/kg) was intraperitoneally injected into male rats (8 weeks old weighting 200-250 g) to establish diabetic model, then naringin (20, 40 or 80 mg/kg/day) was intraperitoneally injected into the diabetic rats for twelve weeks. Glial fibrillary acidic protein (GFAP) level, thickness of ganglion cell layer (GCL) and ganglion cell counts were assessed in diabetic retina in vivo . Naringin (50 μM) that significantly inhibited high glucose (HG, 25 mM)-induced cell proliferation was used to treat rat Muller cell line (rMC1) in vitro . Inflammatory response, oxidative stress and activation of nuclear factor kappa B (NF-κB) p65 were evaluated in retina in vivo and in rMC1 cells in vitro . Naringin alleviated DR symptoms as evidenced by the increased retinal ganglion cells and decreased GFAP level in rat retina. Naringin exhibited anti-inflammatory and antioxidative effects as confirmed by the down-regulated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and the up-regulated antioxidants, glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) in DR rats. Moreover, we found that naringin inhibited HG-induced proliferation, abnormal inflammatory response and oxidative stress in rMC1 cells. In addition, the enhanced nuclear translocation of NF-κB p65 in diabetic rat retina and HG-induced rMC1 cells was suppressed by naringin. Naringin attenuates inflammatory response, oxidative stress and NF-κB activation in experimental models of DR.

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

PubMed

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

2017-01-10

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.

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

Delamanid does not inhibit or induce cytochrome p450 enzymes in vitro.

PubMed

Shimokawa, Yoshihiko; Sasahara, Katsunori; Yoda, Noriaki; Mizuno, Katsuhiko; Umehara, Ken

2014-01-01

Delamanid is a new drug for the treatment of multidrug-resistant tuberculosis. Individuals who are co-infected with human immunodeficiency virus and Mycobacterium tuberculosis may require treatment with a number of medications that might interact significantly with the CYP enzyme system as inhibitors or inducers. It is therefore important to understand how drugs in development for the treatment of tuberculosis will affect CYP enzyme metabolism. The ability of delamanid to inhibit or induce CYP enzymes was investigated in vitro using human liver microsomes or human hepatocytes. Delamanid (100 µM) had little potential for mechanism-based inactivation on eight CYP isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Delamanid's metabolites were noted to inhibit the metabolism of some CYP isoforms, but these effects were observed only at metabolite concentrations that were well above those observed in human plasma during clinical trials. Delamanid (≤10 µM) did not induce CYP1A2, CYP2C9, and CYP3A4 activities in human hepatocytes, and there were no increases in CYP1A2, CYP2B6, CYP2C9, and CYP3A4 mRNA levels. Taken together, these data suggest that delamanid is unlikely to cause clinically relevant drug-drug interactions when co-administered with products that are metabolized by the CYP enzyme system.

Isoliquiritigenin Attenuates Adipose Tissue Inflammation in vitro and Adipose Tissue Fibrosis through Inhibition of Innate Immune Responses in Mice.

PubMed

Watanabe, Yasuharu; Nagai, Yoshinori; Honda, Hiroe; Okamoto, Naoki; Yamamoto, Seiji; Hamashima, Takeru; Ishii, Yoko; Tanaka, Miyako; Suganami, Takayoshi; Sasahara, Masakiyo; Miyake, Kensuke; Takatsu, Kiyoshi

2016-03-15

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.

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

PubMed Central

Challa, Azariyas A.; Vukmirovic, Milica; Blackmon, John; Stefanovic, Branko

2012-01-01

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis. PMID:22900077

A putative biomarker signature for clinically effective AKT inhibition: correlation of in vitro, in vivo and clinical data identifies the importance of modulation of the mTORC1 pathway.

PubMed

Cheraghchi-Bashi, Azadeh; Parker, Christine A; Curry, Ed; Salazar, Jean-Frederic; Gungor, Hatice; Saleem, Azeem; Cunnea, Paula; Rama, Nona; Salinas, Cristian; Mills, Gordon B; Morris, Shannon R; Kumar, Rakesh; Gabra, Hani; Stronach, Euan A

2015-12-08

Our identification of dysregulation of the AKT pathway in ovarian cancer as a platinum resistance specific event led to a comprehensive analysis of in vitro, in vivo and clinical behaviour of the AKT inhibitor GSK2141795. Proteomic biomarker signatures correlating with effects of GSK2141795 were developed using in vitro and in vivo models, well characterised for related molecular, phenotypic and imaging endpoints. Signatures were validated in temporally paired biopsies from patients treated with GSK2141795 in a clinical study. GSK2141795 caused growth-arrest as single agent in vitro, enhanced cisplatin-induced apoptosis in vitro and reduced tumour volume in combination with platinum in vivo. GSK2141795 treatment in vitro and in vivo resulted in ~50-90% decrease in phospho-PRAS40 and 20-80% decrease in fluoro-deoxyglucose (FDG) uptake. Proteomic analysis of GSK2141795 in vitro and in vivo identified a signature of pathway inhibition including changes in AKT and p38 phosphorylation and total Bim, IGF1R, AR and YB1 levels. In patient biopsies, prior to treatment with GSK2141795 in a phase 1 clinical trial, this signature was predictive of post-treatment changes in the response marker CA125. Development of this signature represents an opportunity to demonstrate the clinical importance of AKT inhibition for re-sensitisation of platinum resistant ovarian cancer to platinum.

Oral Multiple Sclerosis Drugs Inhibit the In vitro Growth of Epsilon Toxin Producing Gut Bacterium, Clostridium perfringens.

PubMed

Rumah, Kareem R; Vartanian, Timothy K; Fischetti, Vincent A

2017-01-01

There are currently three oral medications approved for the treatment of multiple sclerosis (MS). Two of these medications, Fingolimod, and Teriflunomide, are considered to be anti-inflammatory agents, while dimethyl fumarate (DMF) is thought to trigger a robust antioxidant response, protecting vulnerable cells during an MS attack. We previously proposed that epsilon toxin from the gut bacterium, Clostridium perfringens , may initiate newly forming MS lesions due to its tropism for blood-brain barrier (BBB) vasculature and central nervous system myelin. Because gut microbiota will be exposed to these oral therapies prior to systemic absorption, we sought to determine if these compounds affect C. perfringens growth in vitro . Here we show that Fingolimod, Teriflunomide, and DMF indeed inhibit C. perfringens growth. Furthermore, several compounds similar to DMF in chemical structure, namely α, β unsaturated carbonyls, also known as Michael acceptors, inhibit C. perfringens . Sphingosine, a Fingolimod homolog with known antibacterial properties, proved to be a potent C. perfringens inhibitor with a Minimal Inhibitory Concentration similar to that of Fingolimod. These findings suggest that currently approved oral MS therapies and structurally related compounds possess antibacterial properties that may alter the gut microbiota. Moreover, inhibition of C. perfringens growth and resulting blockade of epsilon toxin production may contribute to the clinical efficacy of these disease-modifying drugs.

The role of enzyme and substrate concentration in the evaluation of serum angiotensin converting enzyme (ACE) inhibition by enalaprilat in vitro.

PubMed

Weisser, K; Schloos, J

1991-10-09

The relationship between serum angiotensin converting enzyme (ACE) activity and concentration of the ACE inhibitor enalaprilat was determined in vitro in the presence of different concentrations (S = 4-200 mM) of the substrate Hip-Gly-Gly. From Henderson plots, a competitive tight-binding relationship between enalaprilat and serum ACE was found yielding a value of approximately 5 nM for serum ACE concentration (Et) and an inhibition constant (Ki) for enalaprilat of approximately 0.1 nM. A plot of reaction velocity (Vi) versus total inhibitor concentration (It) exhibited a non-parallel shift of the inhibition curve to the right with increasing S. This was reflected by apparent Hill coefficients greater than 1 when the commonly used inhibitory sigmoid concentration-effect model (Emax model) was applied to the data. Slopes greater than 1 were obviously due to discrepancies between the free inhibitor concentration (If) present in the assay and It plotted on the abscissa and could, therefore, be indicators of tight-binding conditions. Thus, the sigmoid Emax model leads to an overestimation of Ki. Therefore, a modification of the inhibitory sigmoid Emax model (called "Emax tight model") was applied, which accounts for the depletion of If by binding, refers to It and allows estimation of the parameters Et and IC50f (free concentration of inhibitor when 50% inhibition occurs) using non-linear regression analysis. This model could describe the non-symmetrical shape of the inhibition curves and the results for Ki and Et correlated very well with those derived from the Henderson plots. The latter findings confirm that the degree of ACE inhibition measured in vitro is, in fact, dependent on the concentration of substrate and enzyme present in the assay. This is of importance not only for the correct evaluation of Ki but also for the interpretation of the time course of serum ACE inhibition measured ex vivo. The non-linear model has some advantages over the linear Henderson

High-Throughput and Rapid Screening of Novel ACE Inhibitory Peptides from Sericin Source and Inhibition Mechanism by Using in Silico and in Vitro Prescriptions.

PubMed

Sun, Huaju; Chang, Qing; Liu, Long; Chai, Kungang; Lin, Guangyan; Huo, Qingling; Zhao, Zhenxia; Zhao, Zhongxing

2017-11-22

Several novel peptides with high ACE-I inhibitory activity were successfully screened from sericin hydrolysate (SH) by coupling in silico and in vitro approaches for the first time. Most screening processes for ACE-I inhibitory peptides were achieved through high-throughput in silico simulation followed by in vitro verification. QSAR model based predicted results indicated that the ACE-I inhibitory activity of these SH peptides and six chosen peptides exhibited moderate high ACE-I inhibitory activities (log IC 50 values: 1.63-2.34). Moreover, two tripeptides among the chosen six peptides were selected for ACE-I inhibition mechanism analysis which based on Lineweaver-Burk plots indicated that they behave as competitive ACE-I inhibitors. The C-terminal residues of short-chain peptides that contain more H-bond acceptor groups could easily form hydrogen bonds with ACE-I and have higher ACE-I inhibitory activity. Overall, sericin protein as a strong ACE-I inhibition source could be deemed a promising agent for antihypertension applications.

Naringin attenuates diabetic retinopathy by inhibiting inflammation, oxidative stress and NF-κB activation in vivo and in vitro

PubMed Central

Liu, Lihua; Zuo, Zhongfu; Lu, Sijing; Liu, Aihua; Liu, Xuezheng

2017-01-01

Objective(s): Naringin, an essential flavonoid, inhibits inflammatory response and oxidative stress in diabetes. However, whether naringin has beneficial effects on diabetic retinopathy (DR) remains unknown. Materials and Methods: Streptozotocin (STZ, 65 mg/kg) was intraperitoneally injected into male rats (8 weeks old weighting 200-250 g) to establish diabetic model, then naringin (20, 40 or 80 mg/kg/day) was intraperitoneally injected into the diabetic rats for twelve weeks. Glial fibrillary acidic protein (GFAP) level, thickness of ganglion cell layer (GCL) and ganglion cell counts were assessed in diabetic retina in vivo. Naringin (50 μM) that significantly inhibited high glucose (HG, 25 mM)-induced cell proliferation was used to treat rat Muller cell line (rMC1) in vitro. Inflammatory response, oxidative stress and activation of nuclear factor kappa B (NF-κB) p65 were evaluated in retina in vivo and in rMC1 cells in vitro. Results: Naringin alleviated DR symptoms as evidenced by the increased retinal ganglion cells and decreased GFAP level in rat retina. Naringin exhibited anti-inflammatory and antioxidative effects as confirmed by the down-regulated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and the up-regulated antioxidants, glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) in DR rats. Moreover, we found that naringin inhibited HG-induced proliferation, abnormal inflammatory response and oxidative stress in rMC1 cells. In addition, the enhanced nuclear translocation of NF-κB p65 in diabetic rat retina and HG-induced rMC1 cells was suppressed by naringin. Conclusion: Naringin attenuates inflammatory response, oxidative stress and NF-κB activation in experimental models of DR. PMID:28852447

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

Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro.

PubMed

Lambrecht, Jennifer A; Browne, Beth Ann; Downs, Diana M

2010-11-05

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

PubMed Central

Gandapu, Upendhar; Chaitanya, R. K.; Kishore, Golla; Reddy, Raju C.; Kondapi, Anand K.

2011-01-01

Background Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research. Methodology/Principal Findings In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI50 = 15.6 µM) is twice more toxic than nano-curcumin (GI50 = 32.5 µM), nano-curcumin (IC50<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC50 = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis. Conclusion Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness. PMID:21887247

Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro.

PubMed

Shin, Jin Soo; Jung, Eunhye; Kim, Meehyein; Baric, Ralph S; Go, Yun Young

2018-05-24

The Middle East respiratory syndrome-coronavirus (MERS-CoV), first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK), was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.

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. Copyright © 2012 Elsevier B.V. All rights reserved.

Growth-inhibiting effects of taxol on human liver cancer in vitro and in nude mice

PubMed Central

Yuan, Jin Hui; Zhang, Ru Ping; Zhang, Ru Gang; Guo, Li Xia; Wang, Xing Wang; Luo, Dan; Xie, Yong; Xie, Hong

2000-01-01

AIM: To investigate the effects of taxol on SMMC-7721 human hepatoma and its mechanisms. METHODS: In vitro cell growth was assessed by trypan blue exclusion method. Experimental hepatoma model was established by seeding SMMC-7721 cells subcutaneously into Balb/c (nu/nu) nude mice. In vivo tumor growth was determined by measurement of tumor diameter with Vernier calipers. The syntheses of DNA, RNA and protein were analyzed by incorporation of 3H-thymidine, 3H-uridine and 3H-leucine respectively. Using light and electron microscopes to observe the morphological changes of cells including mitosis and apoptosis. RESULTS: Taxol was effective against SMMC-7721 human hepatoma cell growth in the ranges of 2.5 nmol/L-10 nmol/L- with mitotic arrest and apoptosis in vitro. DNA, RNA and protein syntheses in cells were also obviously suppressed by in vitro treatment of taxol for 72 h. Taxol at 2.5 nmol/L reduced 3H-thymidine uptake to about 34% of the control value (P ＜ 0.05). Increasing the dose of taxol to 20 nmol/L resulted in a greater decrease in 3H-thymidine incorporation to 60% of the control value (P ＜ 0.01). At a concentration of 20 nmol/L, the 3H-uridine and 3H-leucine uptakes were reduced to 52% (P ＜ 0.05) and 63% (P ＜ 0.01), respectively. In vivo, taxol significantly inhibited SMMC-7721 tumor growth at 10 mg/kg, i.p. once daily for 10 d. A more than 90% decrease in tumor volume was observed by day 11 (P ＜ 0.01) similarly with mitotic arrest and cell apoptosis. CONCLUSION: Taxol has a marked anticancer activity in SMMC-7721 human hepatoma both in vitro and in nude mice. Its mechanisms might be associated with mitotic arrest, subsequently, apoptosis of the hepatoma cells. No obvious toxicity was observed with in vivo administration of taxol. PMID:11819558

Antisense oligonucleotides effectively inhibit the co-transcriptional splicing of a Candida group I intron in vitro and in vivo: Implications for antifungal therapeutics.

PubMed

Zhang, Libin; Leibowitz, Michael J; Zhang, Yi

2009-02-18

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3-P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

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 IC 50 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.

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.

Discovery of DNA dyes Hoechst 34580 and 33342 as good candidates for inhibiting amyloid beta formation: in silico and in vitro study

NASA Astrophysics Data System (ADS)

Thai, Nguyen Quoc; Tseng, Ning-Hsuan; Vu, Mui Thi; Nguyen, Tin Trung; Linh, Huynh Quang; Hu, Chin-Kun; Chen, Yun-Ru; Li, Mai Suan

2016-08-01

Combining Lipinski's rule with the docking and steered molecular dynamics simulations and using the PubChem data base of about 1.4 million compounds, we have obtained DNA dyes Hoechst 34580 and Hoechst 33342 as top-leads for the Alzheimer's disease. The binding properties of these ligands to amyloid beta (Aβ) fibril were thoroughly studied by in silico and in vitro experiments. Hoechst 34580 and Hoechst 33342 prefer to locate near hydrophobic regions with binding affinity mainly governed by the van der Waals interaction. By the Thioflavin T assay, it was found that the inhibition constant IC50 ≈ 0.86 and 0.68 μM for Hoechst 34580 and Hoechst 33342, respectively. This result qualitatively agrees with the binding free energy estimated using the molecular mechanic-Poisson Boltzmann surface area method and all-atom simulations with the AMBER-f99SB-ILDN force field and water model TIP3P. In addition, DNA dyes have the high capability to cross the blood brain barrier. Thus, both in silico and in vitro experiments have shown that Hoechst 34580 and 33342 are good candidates for treating the Alzheimer's disease by inhibiting Aβ formation.

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

N-acetylcysteine inhibits endothelial cell invasion and angiogenesis.

PubMed

Cai, T; Fassina, G; Morini, M; Aluigi, M G; Masiello, L; Fontanini, G; D'Agostini, F; De Flora, S; Noonan, D M; Albini, A

1999-09-01

The thiol N-acetylcysteine (NAC) is a chemopreventive agent that acts through a variety of mechanisms and can prevent in vivo carcinogenesis. We have previously shown that NAC inhibits invasion and metastasis of malignant cells as well as tumor take. Neovascularization is critical for tumor mass expansion and metastasis formation. We investigated whether a target of the anti-cancer activity of NAC could be the inhibition of the tumor angiogenesis-associated phenotype in vitro and in vivo using the potent angiogenic mixture of Kaposi's sarcoma cell products as a stimulus. Two endothelial (EAhy926 and human umbilical vein endothelial [HUVE]) cell lines were utilized in a panel of assays to test NAC ability in inhibiting chemotaxis, invasion, and gelatinolytic activity in vitro. NAC treatment of EAhy926 and HUVE cells in vitro dose-dependently reduced their ability to invade a reconstituted basement membrane, an indicator of endothelial cell activation. Invasion of HUVE cells was inhibited with an ID50 of 0.24 mM NAC, whereas inhibition of chemotaxis required a 10 fold higher doses, indicating that invasion is a preferential target. NAC inhibited the enzymatic activity and conversion to active forms of the gelatinase produced by endothelial cells. The matrigel in vivo assay was used for the evaluation of angiogenesis; NAC strongly inhibited neovascularization of the matrigel sponges in response to Kaposi's sarcoma cell products. NAC prevented angiogenesis while preserving endothelial cells, implying that it could be safely used as an anti-angiogenic treatment.

Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamaguchi, Kazuki; Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180; Feril, Loreto B., E-mail: ferilism@yahoo.com

2011-07-22

Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA,more » which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.« less

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis and in vitro reactivation study of isonicotinamide derivatives of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide as reactivators of Sarin and VX inhibited human acetylcholinesterase (hAChE).

PubMed

Karade, Hitendra N; Raviraju, G; Acharya, B N; Valiveti, Aditya Kapil; Bhalerao, Uma; Acharya, Jyotiranjan

2016-09-15

Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a-4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC50 values of these oximes were determined and correlated with the observed reactivation potential. Copyright © 2016 Elsevier Ltd. All rights reserved.

PRL-3 siRNA Inhibits the Metastasis of B16-BL6 Mouse Melanoma Cells In Vitro and In Vivo

PubMed Central

Qian, Feng; Li, Yu-Pei; Sheng, Xia; Zhang, Zi-Chao; Song, Ran; Dong, Wei; Cao, Shao-Xian; Hua, Zi-Chun; Xu, Qiang

2007-01-01

Phosphatase of regenerating liver-3 (PRL-3) has been proposed to promote the invasion of tumor cells to metastasis sites. However, the effect of PRL-3 on spontaneous metastasis has not been clearly demonstrated, and whether PRL-3 could become a new therapeutic target in malignant tumor is still unknown. In this study, we used PRL-3 siRNA as a molecular medicine to specifically reduce the expression of PRL-3 in B16-BL6 cells, a highly metastatic melanoma cell line. In vitro, PRL-3 siRNA significantly inhibited cell adhesion and migration, but had no effect on cell proliferation. In the spontaneous metastatic tumor model in vivo, PRL-3 siRNA treatment remarkably inhibited the proliferation of primary tumor, prevented tumor cells from invading the draining lymph nodes, and prolonged the life span of mice. Therefore, our results indicate that PRL-3 plays a critical role in promoting the whole process of spontaneous metastasis and tumor growth initiation, and that inhibiting PRL-3 will improve malignant tumor therapy. PMID:17592549

PRL-3 siRNA inhibits the metastasis of B16-BL6 mouse melanoma cells in vitro and in vivo.

PubMed

Qian, Feng; Li, Yu-Pei; Sheng, Xia; Zhang, Zi-Chao; Song, Ran; Dong, Wei; Cao, Shao-Xian; Hua, Zi-Chun; Xu, Qiang

2007-01-01

Phosphatase of regenerating liver-3 (PRL-3) has been proposed to promote the invasion of tumor cells to metastasis sites. However, the effect of PRL-3 on spontaneous metastasis has not been clearly demonstrated, and whether PRL-3 could become a new therapeutic target in malignant tumor is still unknown. In this study, we used PRL-3 siRNA as a molecular medicine to specifically reduce the expression of PRL-3 in B16-BL6 cells, a highly metastatic melanoma cell line. In vitro, PRL-3 siRNA significantly inhibited cell adhesion and migration, but had no effect on cell proliferation. In the spontaneous metastatic tumor model in vivo, PRL-3 siRNA treatment remarkably inhibited the proliferation of primary tumor, prevented tumor cells from invading the draining lymph nodes, and prolonged the life span of mice. Therefore, our results indicate that PRL-3 plays a critical role in promoting the whole process of spontaneous metastasis and tumor growth initiation, and that inhibiting PRL-3 will improve malignant tumor therapy.

Some indazoles reduced the activity of human serum paraoxonase 1, an antioxidant enzyme: in vitro inhibition and molecular modeling studies.

PubMed

Alım, Zuhal; Kılıç, Deryanur; Demir, Yeliz

2018-05-09

Paraoxonase 1 (PON1: EC 3.1.8.1) is a vital antioxidant enzyme against mainly atherosclerosis and many other diseases associated with oxidative stress. Thus, studies related to PON1 have an important place in the pharmacology. In this study, we aimed to evaluate the in vitro inhibition effects of some indazoles on the activity of human PON1. PON1 was purified from human serum with a specific activity of 5000 U/mg and 13.50% yield by using simple chromatographic methods. The indazoles showed K i values in a range of 26.0 ± 3.00-111 ± 31.0 μM against hPON1. All these indazoles exhibited competitive inhibition. In addition, molecular docking studies were performed in order to assess the probable binding mechanisms into the active site of hPON1. Molecular modeling studies confirmed our results. Inhibition of PON1 by indazoles supplies a verification to further consideration of limitation dosage of indazole molecule groups as drug.

Inhibition of duck hepatitis B virus replication by mimic peptides in vitro

PubMed Central

JIA, HONGYU; LIU, CHANGHONG; YANG, YING; ZHU, HAIHONG; CHEN, FENG; LIU, JIHONG; ZHOU, LINFU

2015-01-01

The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (P<0.05). The cytoplasmic DHBV-DNA content of the cells treated with mimic peptide 7 was lower than that in the other groups (P<0.05). In addition, the DHBV-DNA content of the nuclear fractions following the intracellular expression of mimic peptide 7 was significantly lower than that in the other groups (P<0.05). Mimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro. PMID:26640539

Inhibition of duck hepatitis B virus replication by mimic peptides in vitro.

PubMed

Jia, Hongyu; Liu, Changhong; Yang, Ying; Zhu, Haihong; Chen, Feng; Liu, Jihong; Zhou, Linfu

2015-11-01

The aim of the present study was to investigate the inhibitory effect of specific mimic peptides targeting duck hepatitis B virus polymerase (DHBVP) on duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Phage display technology (PDT) was used to screen for mimic peptides specifically targeting DHBVP and the associated coding sequences were determined using DNA sequencing. The selected mimic peptides were then used to treat primary duck hepatocytes infected with DHBV in vitro. Infected hepatocytes expressing the mimic peptides intracellularly were also prepared. The cells were divided into mimic peptide groups (EXP groups), an entecavir-treated group (positive control) and a negative control group. The medium was changed every 48 h. Following a 10-day incubation, the cell supernatants were collected. DHBV-DNA in the cellular nucleus, cytoplasm and culture supernatant was analyzed by quantitative polymerase chain reaction (qPCR). Eight mimic peptides were selected following three PDT screening rounds for investigation in the DHBV-infected primary duck hepatocytes. The qPCR results showed that following direct treatment with mimic peptide 2 or 7, intracellular expression of mimic peptide 2 or 7, or treatment with entecavir, the DHBV-DNA levels in the culture supernatant and cytoplasm of duck hepatocytes were significantly lower than those in the negative control (P<0.05). The cytoplasmic DHBV-DNA content of the cells treated with mimic peptide 7 was lower than that in the other groups (P<0.05). In addition, the DHBV-DNA content of the nuclear fractions following the intracellular expression of mimic peptide 7 was significantly lower than that in the other groups (P<0.05). Mimic peptides specifically targeting DHBVP, administered directly or expressed intracellularly, can significantly inhibit DHBV replication in vitro .

Oral Multiple Sclerosis Drugs Inhibit the In vitro Growth of Epsilon Toxin Producing Gut Bacterium, Clostridium perfringens

PubMed Central

Rumah, Kareem R.; Vartanian, Timothy K.; Fischetti, Vincent A.

2017-01-01

There are currently three oral medications approved for the treatment of multiple sclerosis (MS). Two of these medications, Fingolimod, and Teriflunomide, are considered to be anti-inflammatory agents, while dimethyl fumarate (DMF) is thought to trigger a robust antioxidant response, protecting vulnerable cells during an MS attack. We previously proposed that epsilon toxin from the gut bacterium, Clostridium perfringens, may initiate newly forming MS lesions due to its tropism for blood-brain barrier (BBB) vasculature and central nervous system myelin. Because gut microbiota will be exposed to these oral therapies prior to systemic absorption, we sought to determine if these compounds affect C. perfringens growth in vitro. Here we show that Fingolimod, Teriflunomide, and DMF indeed inhibit C. perfringens growth. Furthermore, several compounds similar to DMF in chemical structure, namely α, β unsaturated carbonyls, also known as Michael acceptors, inhibit C. perfringens. Sphingosine, a Fingolimod homolog with known antibacterial properties, proved to be a potent C. perfringens inhibitor with a Minimal Inhibitory Concentration similar to that of Fingolimod. These findings suggest that currently approved oral MS therapies and structurally related compounds possess antibacterial properties that may alter the gut microbiota. Moreover, inhibition of C. perfringens growth and resulting blockade of epsilon toxin production may contribute to the clinical efficacy of these disease-modifying drugs. PMID:28180112

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

PubMed Central

Laitala, T; Väänänen, H K

1994-01-01

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis.

PubMed

Harrison, Caroline A; Dalley, Andrew J; Mac Neil, Sheila

2005-01-01

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.

Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth

PubMed Central

2011-01-01

Introduction MicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is miR-21, which is consistently up-regulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets. Methods We used in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays. Results Anti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers (rs = -0.283, P = 0.005, Spearman's correlation analysis). Conclusions Knockdown of miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment. PMID:21219636

Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth.

PubMed

Yan, Li Xu; Wu, Qi Nian; Zhang, Yan; Li, Yang Yang; Liao, Ding Zhun; Hou, Jing Hui; Fu, Jia; Zeng, Mu Sheng; Yun, Jing Ping; Wu, Qiu Liang; Zeng, Yi Xin; Shao, Jian Yong

2011-01-10

MicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is miR-21, which is consistently up-regulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets. We used in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays. Anti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers (rs = -0.283, P = 0.005, Spearman's correlation analysis). Knockdown of miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment.

Evaluation of the in vitro/in vivo potential of five berries (bilberry, blueberry, cranberry, elderberry, and raspberry ketones) commonly used as herbal supplements to inhibit uridine diphospho-glucuronosyltransferase.

PubMed

Choi, Eu Jin; Park, Jung Bae; Yoon, Kee Dong; Bae, Soo Kyung

2014-10-01

In this study, we evaluated inhibitory potentials of popularly-consumed berries (bilberry, blueberry, cranberry, elderberry, and raspberry ketones) as herbal supplements on UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 in vitro. We also investigated the potential herb-drug interaction via UGT1A1 inhibition by blueberry in vivo. We demonstrated that these berries had only weak inhibitory effects on the five UGTs. Bilberry and elderberry had no apparent inhibitions. Blueberry weakly inhibited UGT1A1 with an IC50 value of 62.4±4.40 μg/mL and a Ki value of 53.1 μg/mL. Blueberry also weakly inhibited UGT2B7 with an IC50 value of 147±11.1 μg/mL. In addition, cranberry weakly inhibited UGT1A9 activity (IC50=458±49.7 μg/mL) and raspberry ketones weakly inhibited UGT2B7 activity (IC50=248±28.2 μg/mL). Among tested berries, blueberry showed the lowest IC50 value in the inhibition of UGT1A1 in vitro. However, the co-administration of blueberry had no effect on the pharmacokinetics of irinotecan and its active metabolite, SN-38, which was mainly eliminated via UGT1A1, in vivo. Our data suggests that these five berries are unlikely to cause clinically significant herb-drug interactions mediated via inhibition of UGT enzymes involved in drug metabolism. These findings should enable an understanding of herb-drug interactions for the safe use of popularly-consumed berries. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

In vitro inhibition of metabolism but not transport of gliclazide and repaglinide by Cree medicinal plant extracts.

PubMed

Cieniak, Carolina; Liu, Rui; Fottinger, Alexandra; Smiley, Sheila A M; Guerrero-Analco, Jose A; Bennett, Steffany A L; Haddad, Pierre S; Cuerrier, Alain; Saleem, Ammar; Arnason, John T; Foster, Brian C

2013-12-12

aglycone did not show significant effects on repaglinide metabolism, they demonstrated inhibition of gliclazide metabolism. The aglycone significantly affected levels of gliclazide and its metabolites. These studies demonstrate that the Cree plant extracts examined have the potential in vitro to cause drug interactions through effects on key metabolic enzymes. © 2013 Elsevier Ireland Ltd. All rights reserved.

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

PubMed Central

Gewirtz, A M; Calabretta, B; Rucinski, B; Niewiarowski, S; Xu, W Y

1989-01-01

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested

Caffeic and chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): a comparative study.

PubMed

Oboh, Ganiyu; Agunloye, Odunayo M; Adefegha, Stephen A; Akinyemi, Ayodele J; Ademiluyi, Adedayo O

2015-03-01

Chlorogenic acid is a major phenolic compound that forms a substantial part of plant foods and is an ester of caffeic acid and quinic acid. However, the effect of the structures of both chlorogenic and caffeic acids on their antioxidant and antidiabetic potentials have not been fully understood. Thus, this study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid with α-amylase and α-glucosidase (key enzymes linked to type 2 diabetes) activities in vitro. The inhibitory effect of the phenolic acids on α-amylase and α-glucosidase activities was evaluated. Thereafter, their antioxidant activities as typified by their 1,1-diphenyl-2 picrylhydrazyl radical scavenging ability and ferric reducing antioxidant properties were determined. The results revealed that both phenolic acids inhibited α-amylase and α-glucosidase activities in a dose-dependent manner (2-8 μg/mL). However, caffeic acid had a significantly (p<0.05) higher inhibitory effect on α-amylase [IC50 (concentration of sample causing 50% enzyme inhibition)=3.68 μg/mL] and α-glucosidase (IC50=4.98 μg/mL) activities than chlorogenic acid (α-amylase IC50=9.10 μg/mL and α-glucosidase IC50=9.24 μg/mL). Furthermore, both phenolic acids exhibited high antioxidant properties, with caffeic acid showing higher effects. The esterification of caffeic acid with quinic acid, producing chlorogenic acid, reduces their ability to inhibit α-amylase and α-glucosidase activities. Thus, the inhibition of α-amylase and α-glucosidase activities by the phenolic acids could be part of the possible mechanism by which the phenolic acids exert their antidiabetic effects.

JS-K has Potent Anti-Angiogenic Activity in vitro and Inhibits Tumor Angiogenesis in a Multiple Myeloma Model in vivo

PubMed Central

Kaur, Gurmeet; Kiziltepe, Tanyel; Anderson, Kenneth C.; Kutok, Jeffery L.; Jia, Lee; Boucher, Kenneth M.; Saavedra, Joseph E.; Keefer, Larry K.; Shami, Paul J.

2009-01-01

Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in multiple cancers. We have designed a prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent anti-neoplastic activity. We studied the effect of JS-K on angiogenesis. JS-K inhibited the proliferation of HUVEC’s with a 50% inhibitory concentration (IC50) of 0.432, 0.466, and 0.505 µM at 24, 48, and 72 hours, respectively. In the cord formation assay, JS-K led to a decrease in the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 µM, respectively. JS-K inhibited cell migration at 5 hours using VEGF as a chemoattractant. Migration inhibition occurred with an IC50 of 0.493 µM. In the chick aortic ring assay using VEGF or FGF-b for vessel growth stimulation, 0.5 µM JS-K completely inhibited vessel growth. JS-K inhibited tumor angiogenesis in vivo in NIH III mice implanted subcutaneously with OPM1 multiple myeloma cells. JS-K is a potent inhibitor of angiogenesis in vitro and tumor vessel growth in vivo. As such, it establishes a new class of anti-neoplastic agents that target the malignant cells directly as well as their microenvironment. PMID:20723011

L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

PubMed

Huang, Hongbiao; Liu, Ningning; Guo, Haiping; Liao, Siyan; Li, Xiaofen; Yang, Changshan; Liu, Shouting; Song, Wenbin; Liu, Chunjiao; Guan, Lixia; Li, Bing; Xu, Li; Zhang, Change; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

2012-01-01

L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21(cip1) gene, mRNA and protein in cancer cells but not p27(kip1); (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1) gene but not p27(kip1) detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

L-Carnitine Is an Endogenous HDAC Inhibitor Selectively Inhibiting Cancer Cell Growth In Vivo and In Vitro

PubMed Central

Liao, Siyan; Li, Xiaofen; Yang, Changshan; Liu, Shouting; Song, Wenbin; Liu, Chunjiao; Guan, Lixia; Li, Bing; Xu, Li; Zhang, Change; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

2012-01-01

L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21cip1 gene, mRNA and protein in cancer cells but not p27kip1; (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21cip1 gene but not p27kip1 detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance. PMID:23139833

Anti-Müllerian hormone inhibits activation and growth of bovine ovarian follicles in vitro and is localized to growing follicles.

PubMed

Yang, M Y; Cushman, R A; Fortune, J E

2017-05-01

Does anti-Müllerian hormone (AMH) inhibit activation (initiation of growth) of primordial follicles and attenuate the growth of primary follicles in cattle, an excellent animal model for human ovarian follicular development? AMH inhibited activation of bovine primordial follicles and attenuated the growth of activated follicles in vitro. In mice null mutant for AMH, the pool of primordial follicles is depleted prematurely and AMH inhibits follicle activation in vitro. Results of studies with human ovarian tissue in vitro were inconsistent. Our previous work provided indirect evidence that AMH inhibits follicle activation in bovine ovaries. Pieces of fetal bovine ovarian cortex (2 pieces/culture well), obtained during mid or late pregnancy, were cultured in control medium or with graded doses of AMH for 2, 10 or 12 days. Effects of treatment on follicle activation and growth were determined by histological morphometry; follicles in every 20th histological section were staged (primordial or primary), counted, and measured. In addition, AMH was immunolocalized in bovine ovaries obtained at various times during pregnancy (n = 20 ovaries). Bovine fetal ovaries at mid or late gestation were obtained at a commercial abattoir. Pieces of ovarian cortex were cultured without or with AMH and fixed for histological morphometry on Day 0 and at the end of culture. Treatments were applied to duplicate cultures from each of two or three fetuses. In 12-day cultures, addition of AMH was delayed until the third day. Histological analysis provided information about the types, numbers and sizes of follicles in cortical pieces before and after treatments. Ovaries obtained during the second and third trimesters were assessed for the presence of AMH by immunohistochemistry. AMH (100-500 ng/ml) inhibited follicle activation in response to an activator (insulin) in ovarian cortical pieces from fetal ovaries in late gestation. Dose-dependent inhibitory effects on the diameters of primary

Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression.

PubMed

Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

2012-07-15

Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer.

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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Icotinib enhances lung cancer cell radiosensitivity in vitro and in vivo by inhibiting MAPK/ERK and AKT activation.

PubMed

Fu, Yonghong; Zhang, Sen; Wang, Dongjie; Wang, Jing

2018-05-16

Icotinib hydrochloride is a small epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that was developed by Chinese scientists. While clinical trials have revealed its efficacy in the treatment of lung cancer, very little is known about its role in enhancing radiosensitivity. In this study, we investigated the effectiveness of Icotinib in enhancing lung cancer cell radiosensitivity and have detailed its underlying molecular mechanism. The lung cancer cell line H1650 was pretreated with or without Icotinib for 24 hours before radiation, and clonogenic survival assay was performed. Cell apoptosis was also analyzed by flow cytometry, while western blotting was performed to examine the activation of EGFR and its downstream kinases in H1650 cells after Icotinib and radiation treatment. Furthermore, a xenograft animal model was established to evaluate the radiosensitivity of Icotinib in vivo and to confirm its mechanism. Our results demonstrate that pretreatment with Icotinib reduced clonogenic survival after radiation, inhibited EGFR activation, and increased radiation-induced apoptosis in H1650 cells. The phosphorylation of protein kinase B (AKT), extracellular regulated protein kinase 1/2 (ERK1/2), and EGFR was inhibited after Icotinib and radiation combination treatment in vitro and in vivo compared with individual treatments. Combination treatment also affected the expression of the DNA repair protein H2A histone family member X (γ-H2AX). In conclusion, our results reveal that Icotinib enhances radiosensitivity in lung cancers in vitro and in vivo and the mechanism of this may involve blocking the EGFR-AKT and MAPK-ERK pathways and limiting DNA repair. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

SCY-635, a Novel Nonimmunosuppressive Analog of Cyclosporine That Exhibits Potent Inhibition of Hepatitis C Virus RNA Replication In Vitro ▿ †

PubMed Central

Hopkins, Sam; Scorneaux, Bernard; Huang, Zhuhui; Murray, Michael G.; Wring, Stephen; Smitley, Craig; Harris, Richard; Erdmann, Frank; Fischer, Gunter; Ribeill, Yves

2010-01-01

SCY-635 is a novel nonimmunosuppressive cyclosporine-based analog that exhibits potent suppression of hepatitis C virus (HCV) replication in vitro. SCY-635 inhibited the peptidyl prolyl isomerase activity of cyclophilin A at nanomolar concentrations but showed no detectable inhibition of calcineurin phosphatase activity at concentrations up to 2 μM. Metabolic studies indicated that SCY-635 did not induce the major cytochrome P450 enzymes 1A2, 2B6, and 3A4. SCY-635 was a weak inhibitor and a poor substrate for P-glycoprotein. Functional assays with stimulated Jurkat cells and stimulated human peripheral blood mononuclear cells indicated that SCY-635 is a weaker inhibitor of interleukin-2 secretion than cyclosporine. A series of two-drug combination studies was performed in vitro. SCY-635 exhibited synergistic antiviral activity with alpha interferon 2b and additive antiviral activity with ribavirin. SCY-635 was shown to be orally bioavailable in multiple animal species and produced blood and liver concentrations of parent drug that exceeded the 50% effective dose determined in the bicistronic con1b-derived replicon assay. These results suggest that SCY-635 warrants further investigation as a novel therapeutic agent for the treatment of individuals who are chronically infected with HCV. PMID:19933795

Autophagy inhibition sensitizes WYE-354-induced anti-colon cancer activity in vitro and in vivo.

PubMed

Wang, Lijun; Zhu, Yun-Rong; Wang, Shaowei; Zhao, Song

2016-09-01

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 are frequently dysregulated in human colon cancers. In the present study, we evaluated the potential anti-colon cancer cell activity by a novel mTORC1/2 dual inhibitor WYE-354. We showed that WYE-354 was anti-survival and anti-proliferative when adding to primary (patient-derived) and established (HCT-116, HT-29, Caco-2, LoVo, and DLD-1 lines) colon cancer cells. In addition, WYE-354 treatment activated caspase-dependent apoptosis in the colon cancer cells. Mechanistically, WYE-354 blocked mTORC1 and mTORC2 activation. Meanwhile, it also induced autophagy activation in the colon cancer cells. Autophagy inhibitors (bafilomycin A1 and 3-methyladenine), or shRNA-mediated knockdown of autophagy elements (Beclin-1 and ATG-5), remarkably sensitized WYE-354-mediated anti-colon cancer cell activity in vitro. Further studies showed that WYE-354 administration inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Importantly, its activity in vivo was further potentiated with co-administration of the autophagy inhibitor 3-MA. Phosphorylations of Akt (Ser-473) and S6 were also decreased in WYE-354-treated HT-29 xenografts. Together, these pre-clinical results demonstrate the potent anti-colon cancer cell activity by WYE-354, and its activity may be further augmented with autophagy inhibition.

An easily accessible sulfated saccharide mimetic inhibits in vitro human tumor cell adhesion and angiogenesis of vascular endothelial cells

PubMed Central

Marano, Grazia; Gronewold, Claas; Frank, Martin; Merling, Anette; Kliem, Christian; Sauer, Sandra; Wiessler, Manfred; Frei, Eva

2012-01-01

Summary Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell’s microenvironment resulting in an increased malignancy. Schmidt’s imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethyl)furan as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis. The most active compound, (4-{[(β-D-galactopyranosyl)oxy]methyl}furan-3-yl)methyl hydrogen sulfate (GSF), inhibited the activation of matrix-metalloproteinase-2 (MMP-2) as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM) proteins, fibrinogen and fibronectin. In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(β-D-galactopyranosyl)oxy]methyl}furan (BGF) nor methyl β-D-galactopyranoside nor 3,4-bis(hydroxymethyl)furan, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin αvβ3 was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site. These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis

The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro.

PubMed

Tian, Yuwei; Wang, Hui; Li, Bing; Ke, Mengyun; Wang, Jing; Dou, Jie; Zhou, Changlin

2013-11-01

The 30-amino acid antimicrobial peptide Cbf-K16 is a cathelicidin-BF (BF-30) Lys16 mutant derived from the snake venom of Bungarus fasciatus. Our previous study found that BF-30 selectively inhibited the proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, but had a negligible effect on human lung cells. In the present study, it was demonstrated for the first time that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells in vitro, with low toxicity to normal cells. The half-maximal inhibitory concentrations (IC50) of Cbf-K16 against H460 human non-small cell lung carcinoma cells and mouse Lewis lung cancer cells were only 16.5 and 10.5 µM, respectively, which were much less compared to that of BF-30 (45 and 40.3 µM). Data using a transmission electron microscope (TEM) assay showed that, at 20 and 40 µM, Cbf-K16 induced the rupture of the cytoplasmic membrane, which was consistent with data obtained from lactate dehydrogenase (LDH) release assays. The LDH release increased from 17.8 to 52.9% as the duration and dosage of Cbf-K16 increased. Annexin V-fluorescein and propidium iodide staining assays indicated that there were no obvious apoptotic effects at the different dosages and times tested. In H460 cells, the rate of genomic DNA binding increased from 51.9 to 86.8% as the concentration of Cbf-K16 increased from 5 to 10 µM. These data indicate that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells via cytoplasmic membrane permeabilization and DNA binding, rather than apoptosis. Although Cbf-K16 displayed significant cytotoxic activity (40 µM) against tumor cells, in splenocytes no significant inhibitory effect was observed and hemolysis was only 5.6%. These results suggest that Cbf-K16 is a low-toxicity anti-lung cancer drug candidate.

Synthesis and in-vitro reactivation screening of imidazolium aldoximes as reactivators of sarin and VX-inhibited human acetylcholinesterase (hAChE).

PubMed

Sharma, Rahul; Gupta, Bhanushree; Sahu, Arvind Kumar; Acharya, Jyotiranjan; Satnami, Manmohan L; Ghosh, Kallol K

2016-11-25

Post-treatment of organophosphate (OP) poisoning involves the application of oxime reactivator as an antidote. Structurally different oximes are widely studied to examine their kinetic and mechanistic behavior against OP-inhibited cholinesterase enzyme. A series of structurally related 1,3-disubstituted-2-[(hydroxyiminomethyl)alkyl]imidazolium halides (5a-5e, 9a-9c) were synthesized and further evaluated for their in-vitro reactivation ability to reactivate sarin- and VX-inhibited human acetylcholinesterase (hAChE). The observed results were compared with the reactivation efficacy of standard reactivators; 2-PAM, obidoxime and HI-6. Amongst the synthesized oximes, 5a, 9a and 9b were found to be most potent reactivators against sarin-inhibited hAChE while in case of VX only 9a exhibited comparable reactivity with 2-PAM. Incorporation of pyridinium ring to the imidazole ring resulted in substantial increase in the reactivation strength of prepared reactivator. Physicochemical properties of synthesized reactivators have also been evaluated. Copyright © 2016. Published by Elsevier Ireland Ltd.

Inhibition of metastatic potential of B16-F10 melanoma cell line in vivo and in vitro by biflorin.

PubMed

Andrade Carvalho, Adriana; da Costa, Patrícia Marçal; Da Silva Souza, Luciana Gregório; Lemos, Telma Leda G; Alves, Ana Paula Negreiros Nunes; Pessoa, Cláudia; de Moraes, Manoel Odorico

2013-08-14

The aim of this study was to determine the antimetastatic potential of biflorin using in vivo and in vitro approaches. Biflorin was isolated from Capraria biflora collected in Fortaleza, Ceará, Brazil. Adhesion, migration and invasion assays were performed to avail of the antimetastatic potential of this quinone. Experimental metastasis was performed to avail of the antimetastatic potential of bilflorin using in vivo assay. Treatment with biflorin (25 and 50mg/kg/day) was shown to be effective in reducing B16-F10 melanoma metastasis in C57BL/6 mice. The administration of biflorin at 25mg/kg/day intraperitoneally inhibited the formation of metastases by about 57% compared to untreated control animals. When the animals were treated with 50mg/kg/day intraperitoneally, there was a 71% decrease in the number of lung metastases. Morphological assays showed the presence of hemosiderin and erythrocytes in the lung parenchyma, indicating the occurrence of hemorrhage, probably a side effect of biflorin. Biflorin at non-toxic concentrations (0.5, 1.0 and 1.5g/mL) was tested directly on B16-F10 cells in vitro, and it inhibited cell adhesion to type I collagen and cell motility using the wound-healing assay. These data suggest that biflorin has a promising antimetastatic potential, as shown by its anti-adhesion, anti-migration and anti-invasion properties against a metastatic melanoma cell line. However, further studies are essential to elucidate its mechanism of action. Copyright © 2013 Elsevier Inc. All rights reserved.

Extracts from the edible seaweed, Ascophyllum nodosum, inhibit lipase activity in vitro: contributions of phenolic and polysaccharide components.

PubMed

Austin, Ceri; Stewart, Derek; Allwood, J William; McDougall, Gordon J

2018-01-24

A polyphenol-rich extract (PRE) from the edible seaweed, Ascophyllum nodosum, inhibited pancreatic lipase activity in an oil-based turbidimetric assay with an IC 50 of 200 μg gallic acid equivalents (GAE) perassay) [∼230 μg DW] whereas the known inhibitor, Orlistat, gave an IC 50 at 0.4 μg per assay. A phlorotannin-enriched fraction (TRF) purified from the PRE was more potent with an IC 50 = 60 μg GAE per assay (∼65 μg DW). When the assay was started by the addition of lipase, both Orlistat and TRF were much less effective which suggests that pre-incubation of enzyme and inhibitor improved inhibition. Based on phenol content, water extracts from Ascophyllum were more potent lipase inhibitors than PRE (IC 50 ∼ 150 μg GAE per assay). However, this was equivalent to ∼580 μg DW and these extracts contained polysaccharides (e.g. alginate content = 110 μg mL -1 ) which may also contribute to inhibition. Indeed, a polysaccharide-enriched fraction obtained by ethanol precipitation gave an IC 50 of 1000 μg DW which was equivalent to 130 μg GAE and 420 μg alginate per assay. Therefore a >3 fold increase in alginate content did not markedly improve inhibition. Re-precipitation increased alginate content and reduced polyphenol content but lipase inhibition was markedly reduced (i.e. IC 50 at ∼1100 μg DW per assay, 700 μg alginate and 25 μg GAE). Purifying the polysaccharide fraction by ion exchange removed all phenolics but the IC 50 increased to >2500 μg DW, equivalent to >1970 μg alginate per assay. In conclusion, polysaccharides and phlorotannins may inhibit lipase in an additive fashion, with phlorotannins apparently more effective in vitro. However, interactions between these components may be important when food products containing this edible seaweed are consumed.

Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress.

PubMed

Masud Alam, Md; Kariya, Ryusho; Kawaguchi, Azusa; Matsuda, Kouki; Kudo, Eriko; Okada, Seiji

2016-10-01

Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.

In vitro α-glucosidase inhibition, antioxidant, anticancer, and antimycobacterial properties of ethyl acetate extract of Aegle tamilnadensis Abdul Kader (Rutaceae) leaf.

PubMed

R, Pratap Chandran; S, Nishanth Kumar; S, Manju; S, Abdul Kader; B S, Dileep Kumar

2015-01-01

The present study was aimed to investigate in vitro α-glucosidase inhibition, antioxidant, anticancer, and antimycobacterial activities of the ethyl acetate extract of A. tamilnadensis leaves. The extract recorded strong α-glucosidase inhibition with an IC50 value of 100 μg/ml. The antioxidant potential of the extract was evaluated by nitric oxide radical inhibition, lipid peroxidation inhibition, ferric thiocyanate, and ABTS radical scavenging assay, and the extract recorded significant antioxidant activity. The ferric thiocyanate activity of extract was superior to butylated hydroxyl anisol (BHA), the standard antioxidant agent. The anticancer activity of the extract was evaluated against (1) breast cancer cell lines (MDAM B-231), (2) cervical cancer cell lines (HeLa), and (3) lung cancer cell line (A 549) using MTT assay, and significant activity was recorded against A 549 with an IC50 value of 64 μg/ml. Further studies on the morphology, acridine orange/ethidium bromide staining, and cell cycle analysis by flow cytometry confirm the extract-induced apoptosis in A 549. This extract also recorded significant anti-tuberculosis activity against Mycobacterium smegmatis. The current study suggests that the ethyl acetate extract of A. tamilnadensis is a potential source of natural α-glucosidase inhibitor and antioxidant for protection as well as prevention of life-threatening diseases like cancer.

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.

Net Expression Inhibits the Growth of Pancreatic Ductal Adenocarcinoma Cell PL45 In Vitro and In Vivo

PubMed Central

Li, Baiwen; Wan, Xinjian; Zhu, Qi; Li, Lei; Zeng, Yue; Hu, Duanmin; Qian, Yueqin; Lu, Lungen; Wang, Xingpeng; Meng, Xiangjun

2013-01-01

Pancreatic ductal adenocarcinoma has a poor prognosis due to late diagnosis and a lack of effective therapeutic options. Thus, it is important to better understand its molecular mechanisms and to develop more effective treatments for the disease. The ternary complex factor Net, which exerts its strong inhibitory function on transcription of proto-oncogene gene c-fos by forming ternary complexes with a second transcription factor, has been suspected of being involved in pancreatic cancer and other tumors biology. In this study, we found that the majority of pancreatic ductal adenocarcinoma tissues and cell lines had weak or no expression of Net, whereas significantly high level of Net expression occurred in paired adjacent normal tissues we studied. Furthermore, using in vitro and in vivo model systems, we found that overexpression of Net inhibited cell growth and survival and induced cell apoptosis in human pancreatic ductal adenocarcinoma cell PL45; the mechanisms by which Net inhibited the cell cycle progression were mainly through P21-Cyclin D1/CDK4 Pathway. Our data thus suggested that Net might play an important role in pancreatic carcinogenesis, possibly by acting as a tumor suppressor gene. PMID:23469073

Inhibition of MAO by fractions and constituents of hypericum extract.

PubMed

Bladt, S; Wagner, H

1994-10-01

The inhibition of monoamine oxidase (MAO) by six fractions from hypericum extract and three characteristic constituents (as pure substances) were analyzed in vitro and ex vivo to study the antidepressive mechanism of action. Rat brain homogenates were used as the in vitro model, while the ex vivo analysis was performed after intraperitoneal application of the test substances to albino rats. Massive inhibition of MAO-A could be shown with the total extract and all fractions only at the concentration of 10(-3) mol/L. At 10(-4) mol/L, one fraction rich in flavonoides showed an inhibition of 39%, and all other fractions demonstrated less than 25% inhibition. Using pure hypericin as well as in all ex vivo experiments, no relevant inhibiting effects could be shown. From the results it can be concluded that the clinically proven antidepressive effect of hypericum extract cannot be explained in terms of MAO inhibition.

Pigment epithelium derived factor inhibits the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro.

PubMed

Sun, Yanmei; Che, Xuan; Zhu, Libo; Zhao, Mengdan; Fu, Guofang; Huang, Xiufeng; Xu, Hong; Hu, Fuqiang; Zhang, Xinmei

2012-01-01

Angiogenesis is a prerequisite for the formation and development of endometriosis. Pigment epithelium derived factor (PEDF) is a natural inhibitor of angiogenesis. We previously demonstrated a reduction of PEDF in the peritoneal fluid, serum and endometriotic lesions from women with endometriosis compared with women without endometriosis. Here, we aim to investigate the inhibitory effect of PEDF on human endometriotic cells in vivo and in vitro. We found that PEDF markedly inhibited the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro by up-regulating PEDF expression and down-regulating vascular endothelial growth factor (VEGF) expression. Moreover, apoptotic index was significantly increased in endometriotic lesions in vivo and endometriotic stromal cells in vitro when treated with PEDF. In mice treated with PEDF, decreased microvessel density labeled by Von Willebrand factor but not by α-Smooth Muscle Actin was observed in endometriotic lesions. And it showed no increase in PEDF expression of the ovary and uterus tissues. These findings suggest that PEDF gene therapy may be a new treatment for endometriosis.

Pigment Epithelium Derived Factor Inhibits the Growth of Human Endometrial Implants in Nude Mice and of Ovarian Endometriotic Stromal Cells In Vitro

PubMed Central

Sun, Yanmei; Che, Xuan; Zhu, Libo; Zhao, Mengdan; Fu, Guofang; Huang, Xiufeng; Xu, Hong; Hu, Fuqiang; Zhang, Xinmei

2012-01-01

Angiogenesis is a prerequisite for the formation and development of endometriosis. Pigment epithelium derived factor (PEDF) is a natural inhibitor of angiogenesis. We previously demonstrated a reduction of PEDF in the peritoneal fluid, serum and endometriotic lesions from women with endometriosis compared with women without endometriosis. Here, we aim to investigate the inhibitory effect of PEDF on human endometriotic cells in vivo and in vitro. We found that PEDF markedly inhibited the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro by up-regulating PEDF expression and down-regulating vascular endothelial growth factor (VEGF) expression. Moreover, apoptotic index was significantly increased in endometriotic lesions in vivo and endometriotic stromal cells in vitro when treated with PEDF. In mice treated with PEDF, decreased microvessel density labeled by Von Willebrand factor but not by α-Smooth Muscle Actin was observed in endometriotic lesions. And it showed no increase in PEDF expression of the ovary and uterus tissues. These findings suggest that PEDF gene therapy may be a new treatment for endometriosis. PMID:23028859

JS-K has potent anti-angiogenic activity in vitro and inhibits tumour angiogenesis in a multiple myeloma model in vivo.

PubMed

Kiziltepe, Tanyel; Anderson, Kenneth C; Kutok, Jeffery L; Jia, Lee; Boucher, Kenneth M; Saavedra, Joseph E; Keefer, Larry K; Shami, Paul J

2010-01-01

Glutathione S-transferases (GSTs) play an important role in multidrug resistance and are upregulated in multiple cancers. We have designed a prodrug class that releases nitric oxide on metabolism by GST. O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antineoplastic activity. We studied the effect of JS-K on angiogenesis in human umbilical vein endothelial cells (HUVECs), OPM1 multiple myeloma cells, chick aortic rings and in mice. JS-K inhibited the proliferation of HUVECs with a 50% inhibitory concentration (IC50) of 0.432, 0.466 and 0.505 microm at 24, 48 and 72 h, respectively. In the cord formation assay, JS-K led to a decrease in the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 microm, respectively. JS-K inhibited cell migration at 5 h using VEGF as a chemoattractant. Migration inhibition occurred with an IC50 of 0.493 microm. In the chick aortic ring assay using VEGF or FGF-2 for vessel growth stimulation, 0.5 microm JS-K completely inhibited vessel growth. JS-K inhibited tumour angiogenesis in vivo in NIH III mice implanted subcutaneously with OPM1 multiple myeloma cells. JS-K is a potent inhibitor of angiogenesis in vitro and tumour vessel growth in vivo. As such, it establishes a new class of antineoplastic agent that targets the malignant cells directly as well as their microenvironment.

Hypoglycemic effect of basil (Ocimum basilicum) aqueous extract is mediated through inhibition of α-glucosidase and α-amylase activities: an in vitro study.

PubMed

El-Beshbishy, Ha; Bahashwan, Sa

2012-02-01

The present study investigated the in vitro hypoglycemic activity of basil (Ocimum basilicum) aqueous extract. Preliminary phytochemical screening of the extract revealed the presence of reducing sugars, cardiac glycosides, tannins, saponins, glycosides, flavonoids and steroids. The total polyphenols content (TPC), flavonoids content (FC), percentage diphenylpicrylhydrazyl (DPPH( · )) radical inhibition and total antioxidant status (TAS) were estimated. The FC was 41 ± 2.2 rutin/g dry extract, the TPC was 146 ± 5.26 mg catechin/g dry extract and the TAS was 5.12 ± 0.7 mmol/L. The %DPPH( · ) free radical inhibition was 60%, 54%, 49% and 43%, respectively, for different extract concentrations; 20, 18.2, 16.3 and 14.5 mg/ml, respectively. The extract elicited significant dose-dependent pattern against rat intestinal sucrase (RIS; IC(50) = 36.72 mg/ml), rat intestinal maltase (RIM; IC(50) = 21.31 mg/ml) and porcine pancreatic α-amylase (PPA; IC(50) = 42.50 mg/ml) inhibitory activities. The inhibition was greater against maltase compared with sucrase. These effects may be attributed to the high TPC and FC levels. The linear regression analysis revealed strong significant positive correlations between %DPPH( · ) radical inhibition and each of %RIS, %RIM and %PPA inhibiting activity. Also, strong significant positive correlations between %RIS and either %RIM or %PPA inhibition activity were observed. We concluded therefore that basil aqueous extract via antioxidant and possibly α-glucosidase and α-amylase inhibiting activities, offered positive benefits to control diabetes.

Toxicity of mycotoxins for the rat pulmonary macrophage in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sorenson, W.G.; Gerberick, G.F.; Lewis, D.M.

1986-04-01

The presence of mycotoxins in grains is well documented. Workers in grain handling occupations are commonly exposed to grain dust aerosols. Work in our laboratory has shown that T-2 toxin is highly toxic to rat alveolar macrophages in vitro, causing loss of viability, release of radiolabeled chromium, inhibition of macromolecular synthesis, inhibition of phagocytosis, and inhibition of macrophage activation. Similarly, patulin caused a significant release of radiolabeled chromium, decrease in ATP levels, significant inhibition of protein and RNA synthesis, and inhibition of phagocytosis. The data show that both T-2 toxin and patulin are highly toxic to rat alveolar macrophages inmore » vitro. The data further suggest that the presence of these mycotoxins in airborne respirable dust might present a hazard to exposed workers.« less

Inhibition of osteopontin suppresses in vitro and in vivo angiogenesis in endometrial cancer.

PubMed

Du, Xue-lian; Jiang, Tao; Sheng, Xiu-gui; Gao, Rong; Li, Qing-shui

2009-12-01

Osteopontin (OPN) has been found to play an important role in tumor angiogenesis in recent years. Our previous studies have shown that OPN is overexpressed in tumor-associated human endometrial endothelial cells (HEECs) isolated from tissue samples of patients with endometrial cancer. In the present study, we aimed to further determine the role of OPN in endometrial cancer-associated angiogenesis. We knock down OPN expression in HEECs and human endometrial cancer Ishikawa (ISK) cells using the small interference RNA method, and then evaluate the effects of OPN on endometrial cancer-associated angiogenesis by in vivo mouse studies and in vitro assays. Our results revealed that proliferative activity of HEECs and ISK cells in vitro was not affected by transfection with the siOPN-RNA (P>0.05). Inhibition of OPN expression in HEECs reduced the cell migration, with the percentage of repaired area of 36.32+/-2.88 vs. 8.54+/-1.13 (P=0.007). HEEC/siOPN and ISK/siOPN demonstrated 67.4% and 51.2% decreased invasiveness compared with controls, respectively (P<0.05). The number of branched points per well was obviously lower in HEEC/siOPN than that in HEEC/Control (32.46+/-17.10 vs. 53.15+/-15.44, P=0.021). Furthermore, ISK cells transfected with OPN siRNA formed smaller tumor in mice and led to a lower microvessel density, i.e., angiogenesis, in transplanted tumors of mice than scrambled siRNA controls (12.88+/-7.14 vs. 28.42+/-9.69 vessels per HPF, P=0.019). These data confirm the positive role of OPN in endometrial cancer-associated angiogenesis and might be of great benefit for finding rational approach in endometrial cancer therapy.

Rapamycin Inhibits Human Laryngotracheal Stenosis–derived Fibroblast Proliferation, Metabolism, and Function in Vitro

PubMed Central

Namba, Daryan R.; Ma, Garret; Samad, Idris; Ding, Dacheng; Pandian, Vinciya; Powell, Jonathan D.; Horton, Maureen R.; Hillel, Alexander T.

2015-01-01

Objective To determine if rapamycin inhibits the growth, function, and metabolism of human laryngotracheal stenosis (LTS)–derived fibroblasts. Study Design Controlled in vitro study. Setting Tertiary care hospital in a research university. Subjects and Methods Fibroblasts isolated from biopsies of 5 patients with laryngotracheal stenosis were cultured. Cell proliferation, histology, gene expression, and cellular metabolism of LTS-derived fibroblasts were assessed in 4 conditions: (1) fibroblast growth medium, (2) fibroblast growth medium with dimethylsulfoxide (DMSO), (3) fibroblast growth medium with 10−10 M (low-dose) rapamycin dissolved in DMSO, and (4) fibroblast growth medium with 10−9 M (high-dose) rapamycin dissolved in DMSO. Results The LTS fibroblast count and DNA concentration were reduced after treatment with high-dose rapamycin compared to DMSO (P = .0007) and normal (P = .0007) controls. Collagen I expression decreased after treatment with high-dose rapamycin versus control (P = .0051) and DMSO (P = .0093) controls. Maximal respiration decreased to 68.6 pMoles of oxygen/min/10 mg/protein from 96.9 for DMSO (P = .0002) and 97.0 for normal (P = .0022) controls. Adenosine triphosphate (ATP) production decreased to 66.8 pMoles from 88.1 for DMSO (P = .0006) and 83.3 for normal (P = .0003) controls. Basal respiration decreased to 78.6 pMoles from 108 for DMSO (P = .0002) and 101 for normal (P = .0014) controls. Conclusions Rapamycin demonstrated an anti-fibroblast effect by significantly reducing the proliferation, metabolism, and collagen deposition of human LTS fibroblast in vitro. Rapamycin significantly decreased oxidative phosphorylation of LTS fibroblasts, suggesting at a potential mechanism for the reduced proliferation and differentiation. Furthermore, rapamycin’s anti-fibroblast effects indicate a promising adjuvant therapy for the treatment of laryngotracheal stenosis. PMID:25754184

Inhibition of rabbit platelet activation in vitro by antagonists of platelet-activating factor (PAF)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cox, C.P.; Wood, K.L.

1986-03-05

The authors used washed, (/sup 3/H)serotonin-labeled rabbit platelets to study the in vitro aggregation and secretion responses induced by graded doses of PAF in the presence or absence of specific antagonists of PAF. These antagonists included CV-3988, L-652,731, triazolam and alprazolam. Platelets were pretreated with either an antagonist or the appropriate diluent for 60 sec prior to the addition of PAF (2 x 10/sup -10/ to 2 x 10/sup -7/ M). Aggregation was monitored continuously and recorded as the height of the aggregation tracing at 60 sec post-PAF. Secretion of (/sup 3/H)-serotonin was measured in a sample of the plateletsmore » removed at 60 sec post-PAF. When 2 x 10/sup -10/ M PAF was used as the stimulus, the concentration of antagonist needed for 50% inhibition (IC/sub 50/) of secretion was obtained at 0.05 ..mu..M, 0.15 ..mu..M, 0.6 ..mu..M and 2.5 ..mu..M, respectively, for L-652,731, CV-3988, triazolam and alprazolam. The corresponding IC/sub 50/ for aggregation was obtained at 0.2 ..mu..M, 0.1 ..mu..M, 1.5 ..mu..M and 6.5 ..mu..M, respectively. The inhibitory effects of these antagonists could be overcome by increasing the dose of PAF used. Although all of the antagonists were capable of completely inhibiting platelet aggregation and secretion, L-652,731 was the most potent PAF antagonist on a molar basis.« less

Effect of CO2 laser on root caries inhibition around composite restorations: an in vitro study.

PubMed

de Melo, Jociana Bandeira; Hanashiro, Fernando Seishim; Steagall, Washington; Turbino, Miriam Lacalle; Nobre-dos-Santos, Marinês; Youssef, Michel Nicolau; de Souza-Zaroni, Wanessa Christine

2014-03-01

The aim of the present study was to investigate the in vitro effect of CO2 laser on the inhibition of root surface demineralization around composite resin restorations. For this purpose, 30 blocks obtained from human molar roots were divided into three groups: group 1 (negative control), cavity prepared with cylindrical diamond bur + acid etching + adhesive + composite resin restoration; group 2, cavity prepared with cylindrical diamond bur + CO2 laser (5.0 J/cm(2)) + acid etching + adhesive + composite resin; and group 3, cavity prepared with cylindrical diamond bur + CO2 laser (6.0 J/cm(2)) + acid etching + adhesive + composite resin. After this procedure, the blocks were submitted to thermal and pH cycling. Root surface demineralization around the restorations was measured by microhardness analysis. The hardness results of the longitudinally sectioned root surface were converted into percentage of mineral volume, which was used to calculate the mineral loss delta Z (ΔZ). The percentage of mineral volume, ΔZ, and the percentage of demineralization inhibition of the groups were statistically analyzed by using analysis of variance and Tukey-Kramer test. The percentage of mineral volume was higher in the irradiated groups up to 80 μm deep. The ΔZ was significantly lower in the irradiated groups than in the control group. The percentage of reduction in demineralization ranged from 19.73 to 29.21 in position 1 (50 μm), and from 24.76 to 26.73 in position 2 (100 μm), when using 6 and 5 J/cm(2), respectively. The CO2 laser was effective in inhibiting root demineralization around composite resin restorations.

Panax notoginseng saponins (PNS) inhibits breast cancer metastasis.

PubMed

Wang, Peiwei; Cui, Jingang; Du, Xiaoye; Yang, Qinbo; Jia, Chenglin; Xiong, Minqi; Yu, Xintong; Li, Li; Wang, Wenjian; Chen, Yu; Zhang, Teng

2014-07-03

Panax notoginseng (Burkill) F.H. Chen (Araliaceae) has been extensively used as a therapeutic agent to treat a variety of diseases. Panax notoginseng saponins (PNS) consist of major therapeutically active components of Panax notoginseng. PNS inhibit the growth of a variety of tumor cells in vitro and in vivo. The aim of the study is to investigate the effects and underlying mechanisms of PNS on breast cancer metastasis. 4T1 cell, a highly metastatic mouse breast carcinoma cell line, was utilized for in vitro and in vivo assays. In vitro assays were first performed to examine the effects of PNS on 4T1 cell viability, migration and invasion, respectively. Real-time PCR analyses were also performed to examine the effects of PNS on the expression of genes associated with tumor metastasis. The effect of PNS on 4T1 tumor cell metastasis was further assessed in spontaneous and experimental metastasis models in vivo. PNS treatment exhibited a dose-dependent effect on impairing 4T1 cell viability in vitro. However, when examined at a lower dose that did not affect cell viability, the migration and invasion of 4T1 cell was remarkably inhibited in vitro. Meanwhile, PNS treatment led to upregulated expression of genes known to inhibit metastasis and downregulated expression of genes promoting metastasis in cultured 4T1 cells. These results suggested a selective effect of PNS on 4T1 migration and invasion. This hypothesis was further addressed in 4T1 metastasis models in vivo. The results showed that the lung metastasis was significantly inhibited by PNS treatment in both spontaneous and experimental metastasis models. Taken together, our results demonstrated an inhibitory effect of PNS on 4T1 tumor metastasis, warranting further evaluation of PNS as a therapeutic agent for treating breast cancer metastasis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Conifer flavonoid compounds inhibit detoxification enzymes and synergize insecticides.

PubMed

Wang, Zhiling; Zhao, Zhong; Cheng, Xiaofei; Liu, Suqi; Wei, Qin; Scott, Ian M

2016-02-01

Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1(st) instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

Synthesis and evaluation of hetero- and homo-dimers of ribosome-targeting antibiotics: Antimicrobial activity, in vitro inhibition of translation, and drug resistance

PubMed Central

Berkov-Zrihen, Yifat; Green, Keith D.; Labby, Kristin J.; Feldman, Mark; Garneau-Tsodikova, Sylvie; Fridman, Micha

2013-01-01

In this study, we describe the synthesis of a full set of homo- and hetero-dimers of three intact structures of different ribosome-targeting antibiotics: tobramycin, clindamycin, and chloramphenicol. Several aspects of the biological activity of the dimeric structures were evaluated including antimicrobial activity, inhibition of in vitro bacterial protein translation, and the effect of dimerization on the action of several bacterial resistance mechanisms that deactivate tobramycin and chloramphenicol. This study demonstrates that covalently linking two identical or different ribosome-targeting antibiotics may lead to (i) a broader spectrum of antimicrobial activity, (ii) improved inhibition of bacterial translation properties compared to that of the parent antibiotics, and (iii) reduction in the efficacy of some drug-modifying enzymes that confer high levels of resistance to the parent antibiotics from which the dimers were derived. PMID:23786357

Inhibition of cyclooxygenase-independent platelet aggregation by sodium salicylate.

PubMed

Violi, F; Alessandri, C; Praticò, D; Guzzo, A; Ghiselli, A; Balsano, F

1989-06-15

The effect of acetylsalicylic acid (ASA) on platelet aggregation (PA) and thromboxane A2 (TxA2) formation was investigated in vitro and ex vivo after 1 g or 300 mg ASA administration to healthy subjects. 50-100 microM ASA inhibited PA by single aggregating agent such as platelet aggregating factor (PAF) or epinephrine and reduced to less than or equal to 5% of control platelet TxB2 formation, but did not influence PA by epinephrine plus PAF. The latter was inhibited by increasing ASA concentration. In samples incubated with 100 microM ASA and stimulated with epinephrine plus PAF, PA could be inhibited by the addition of 100-300 microM sodium salicylate. After 300 mg-1 g ASA administration to healthy subjects, the inhibition of PA by epinephrine plus PAF was more marked by highest doses of ASA. This study suggests that aspirin inhibits PA with a cyclooxygenase-independent mechanism; this effect is mediated, at least in vitro, by salicylic acid.

A semi-quantitative translational pharmacology analysis to understand the relationship between in vitro ENT1 inhibition and the clinical incidence of dyspnoea and bronchospasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenbrier Ribeiro, Lyn, E-mail: Lyn.Rosenbrierrib

Adenosine contributes to the pathophysiology of respiratory disease, and adenosine challenge leads to bronchospasm and dyspnoea in patients. The equilibrative nucleoside transporter 1 (ENT1) terminates the action of adenosine by removal from the extracellular environment. Therefore, it is proposed that inhibition of ENT1 in respiratory disease patients leads to increased adenosine concentrations, triggering bronchospasm and dyspnoea. This study aims to assess the translation of in vitro ENT1 inhibition to the clinical incidence of bronchospasm and dyspnoea in respiratory disease, cardiovascular disease and healthy volunteer populations. Four marketed drugs with ENT1 activity were assessed; dipyridamole, ticagrelor, draflazine, cilostazol. For each patientmore » population, the relationship between in vitro ENT1 [{sup 3}H]-NBTI binding affinity (K{sub i}) and [{sup 3}H]-adenosine uptake (IC{sub 50}) to the incidence of: (1) bronchospasm/severe dyspnoea; (2) tolerated dyspnoea and; (3) no adverse effects, was evaluated. A high degree of ENT1 inhibition (≥ 13.3x K{sub i}, ≥ 4x IC{sub 50}) associated with increased incidence of bronchospasm/severe dyspnoea for patients with respiratory disease only, whereas a lower degree of ENT1 inhibition (≥ 0.1x K{sub i}, ≥ 0.05x IC{sub 50}) associated with a tolerable level of dyspnoea in both respiratory and cardiovascular disease patients. ENT1 inhibition had no effect in healthy volunteers. Furthermore, physicochemical properties correlative with ENT1 binding were assessed using a set of 1625 diverse molecules. Binding to ENT1 was relatively promiscuous (22% compounds K{sub i} < 1 μM) especially for neutral or basic molecules, and greater incidence tracked with higher lipophilicity (clogP > 5). This study rationalises inclusion of an assessment of ENT1 activity during early safety profiling for programs targeting respiratory disorders. - Highlights: • ENT1 inhibition causes bronchospasm and severe dyspnoea in

PDE4 inhibition reduces neointima formation and inhibits VCAM-1 expression and histone methylation in an Epac-dependent manner.

PubMed

Lehrke, Michael; Kahles, Florian; Makowska, Anna; Tilstam, Pathricia V; Diebold, Sebastian; Marx, Judith; Stöhr, Robert; Hess, Katharina; Endorf, Elizabeth B; Bruemmer, Dennis; Marx, Nikolaus; Findeisen, Hannes M

2015-04-01

Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation following vascular injury. In this study we have investigated the effects of specific PDE4 inhibition with roflumilast on SMC proliferation and inflammatory activation in vitro and neointima formation following guide wire-induced injury of the femoral artery in mice in vivo. In vitro, roflumilast did not affect SMC proliferation, but diminished TNF-α induced expression of the vascular cell adhesion molecule 1 (VCAM-1). Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 expression. Consistently, the reduction of VCAM-1 expression was rescued following inhibition of Epac. TNF-α induced NFκB p65 translocation and VCAM-1 promoter activity were not altered by roflumilast in SMCs. However, roflumilast treatment and Epac activation repressed the induction of the activating epigenetic histone mark H3K4me2 at the VCAM-1 promoter, while PKA activation showed no effect. Furthermore, HDAC inhibition blocked the inhibitory effect of roflumilast on VCAM-1 expression. Both, roflumilast and Epac activation reduced monocyte adhesion to SMCs in vitro. Finally, roflumilast treatment attenuated femoral artery intima-media ratio by more than 50% after 4weeks. In summary, PDE4 inhibition regulates VCAM-1 through a novel Epac-dependent mechanism, which involves regulatory epigenetic components and reduces neointima formation following vascular injury. PDE4 inhibition and Epac activation might represent novel approaches for the treatment of vascular diseases, including atherosclerosis and in-stent restenosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

miR-34a inhibits the in vitro cell proliferation and migration in human esophageal cancer.

PubMed

Shi, Hui; Zhou, Shengluan; Liu, Junhua; Zhu, Jun; Xue, Jianhua; Gu, Luo; Chen, Yijiang

2016-05-01

Increasing studies demonstrate that reduced expression of miR-34a is involved in the initiation and progression of cancers, and it has been characterized as a tumor suppressor in various types of cancers. In present study, we investigated the expression and role of miR-34a in esophageal cancer. qRT-PCR assays were performed to analyze the expression of miR-34a in human esophageal cancer tissues and adjacent esophageal tissues. CCK8 assay, flow cytometry analysis and in vitro migration assays were performed to analyze the role of miR-34a in human esophageal cancer cell. MSP assay was performed to analyze the DNA methylation of the miR-34a promoter. The expression of miR-34a was down-regulated in human esophageal cancer tissues. miR-34a ectopic expression affected esophageal cancer cells survival, proliferation and capabilities of migration in vitro. p53 status was not correlated with miR-34a. Subsequently, aberrant DNA methylation of the miR-34a promoter was found in human esophageal cancer, and 5-AZA-dC inhibited DNA methylation of the miR-34a promoter. our data showed that miR-34a acted as a tumor suppressor in human esophageal cancer. Copyright © 2016. Published by Elsevier GmbH.

CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

PubMed

He, Xing-Xing; Zhang, Yu-Nan; Yan, Jun-Wei; Yan, Jing-Jun; Wu, Qian; Song, Yu-Hu

2016-01-01

The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

Plumbagin inhibits cytokinesis in Bacillus subtilis by inhibiting FtsZ assembly--a mechanistic study of its antibacterial activity.

PubMed

Bhattacharya, Anusri; Jindal, Bhavya; Singh, Parminder; Datta, Anindya; Panda, Dulal

2013-09-01

The assembly of FtsZ plays a central role in construction of the cytokinetic Z-ring that orchestrates bacterial cell division. A naturally occurring naphthoquinone, plumbagin, is known to exhibit antibacterial properties against several types of bacteria. In this study, plumbagin was found to perturb formation of the Z-ring in Bacillus subtilis 168 cells and to cause elongation of these cells without an apparent effect on nucleoid segregation, indicating that it may inhibit FtsZ assembly. Furthermore, it bound to purified B. subtilis FtsZ (BsFtsZ) with a dissociation constant of 20.7 ± 5.6 μM, and inhibited the assembly and GTPase activity of BsFtsZ in vitro. Interestingly, plumbagin did not inhibit either the assembly or GTPase activity of Escherichia coli FtsZ (EcFtsZ) in vitro. Using docking analysis, a putative plumbagin-binding site on BsFtsZ was identified, and the analysis indicated that hydrophobic interactions and hydrogen bonds predominate. Based on the in silico analysis, two variants of BsFtsZ, namely D199A and V307R, were constructed to explore the binding interaction of plumbagin and BsFtsZ. The effects of plumbagin on the assembly and GTPase activity of the variant BsFtsZ proteins in vitro indicated that the residues D199 and V307 may be involved in the binding of plumbagin to BsFtsZ. The results suggest that plumbagin inhibits bacterial proliferation by inhibiting the assembly of FtsZ, and provide insight into the binding site of plumbagin on BsFtsZ, which may help in the design of potent FtsZ-targeted antibacterial agents. © 2013 FEBS.

Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro.

PubMed

Roelofs, Maarke J E; Temming, A Roberto; Piersma, Aldert H; van den Berg, Martin; van Duursen, Majorie B M

2014-01-01

Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-)androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO), fluconazole (FLUC), flusilazole (FLUS), hexaconazole (HEXA), myconazole (MYC), penconazole (PEN), prochloraz (PRO), tebuconazole (TEBU), triadimefon (TRIA), and triticonazole (TRIT) were examined using murine Leydig (MA-10) cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T) secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC 50 = 12.4 μM) or TEBU (IC 50 = 2.4 μM) in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS) formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC 50 s ranging from 10.7 to 71.5 μM) and effect potencies (REPs) were calculated relative to the known AR antagonist flutamide (FLUT). FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61) and MYC the least potent (REP = 0.03) AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human) risk assessment of this class of compounds.

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

Imatinib mesylate inhibits Leydig cell tumor growth: evidence for in vitro and in vivo activity.

PubMed

Basciani, Sabrina; Brama, Marina; Mariani, Stefania; De Luca, Gabriele; Arizzi, Mario; Vesci, Loredana; Pisano, Claudio; Dolci, Susanna; Spera, Giovanni; Gnessi, Lucio

2005-03-01

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.

Tocotrienol inhibits proliferation of human Tenon's fibroblasts in vitro: a comparative study with vitamin E forms and mitomycin C.

PubMed

Meyenberg, Alexander; Goldblum, David; Zingg, Jean-Marc; Azzi, Angelo; Nesaretnam, Kalanithi; Kilchenmann, Monika; Frueh, Beatrice E

2005-12-01

To evaluate the potential of the vitamin E compound alpha-tocotrienol as antifibrotic agent in vitro. Using human Tenon's capsule fibroblast cultures, the antiproliferative and cytotoxic effects of the different vitamin E forms alpha-tocopherol, alpha-tocopheryl acetate, alpha-tocopheryl succinate and alpha-tocotrienol were compared with those of mitomycin C. To mimic subconjunctival and regular oral application in vivo, exposure time of serum-stimulated and serum-restimulated fibroblasts (SF and RF, respectively) to vitamin E forms was set at 6 days. Cultures were only exposed for 5 min to mitomycin C due to its known acute toxicity and to mimic the short-time intraoperative administration. Proliferation (expressed as % of control) was determined by DNA content quantification on days 2, 4 and 6, whereas cytotoxicity was assessed by cell morphology and glucose 6-phosphate dehydrogenase (G6PD) release after 24 h. alpha-Tocopherol and alpha-tocopheryl acetate stimulated growth of SF, but not RF. Reduction of fibroblast content by alpha-tocopheryl succinate was accompanied by increased G6PD release and necrosis. Contrary to alpha-tocopheryl succinate, 50 microM or repeatedly 20 microM of alpha-tocotrienol significantly inhibited proliferation without causing cellular toxicity (maximal effect: 46.8%). RF were more sensitive to this effect than SF. Mitomycin C 100-400 microg/ml showed a stronger antiproliferative effect than alpha-tocotrienol (maximal effect: 13.8%). Morphologic characteristics of apoptosis were more commonly found under treatment with mitomycin C. Of the vitamin E forms tested, only alpha-tocotrienol significantly inhibited growth at non-toxic concentrations. In this in vitro study, antiproliferative effects of mitomycin C were stronger than those of alpha-tocotrienol.

In Vitro Inhibition of Leishmania Attachment to Sandfly Midguts and LL-5 Cells by Divalent Metal Chelators, Anti-gp63 and Phosphoglycans.

PubMed

Soares, Rodrigo Pedro; Altoé, Ellen Cristina Félix; Ennes-Vidal, Vítor; da Costa, Simone M; Rangel, Elizabeth Ferreira; de Souza, Nataly Araújo; da Silva, Vanderlei Campos; Volf, Petr; d'Avila-Levy, Claudia Masini

2017-07-01

Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous and visceral leishmaniasis, respectively. Several aspects of the vector-parasite interaction involving gp63 and phosphoglycans have been individually assayed in different studies. However, their role under the same experimental conditions was not studied yet. Here, the roles of divalent metal chelators, anti-gp63 antibodies and purified type I phosphoglycans (PGs) were evaluated during in vitro parasite attachment to the midgut of the vector. Parasites were treated with divalent metal chelators or anti-gp63 antibodies prior to the interaction with Lutzomyia longipalpis/Lutzomyia intermedia midguts or sand fly LL-5 cells. In vitro binding system was used to examine the role of PG and gp63 in parallel. Treatment with divalent metal chelators reduced Le. infantum adhesion to the Lu. longipalpis midguts. The most effective compound (Phen) inhibited the binding in both vectors. Similar results were observed in the interaction between both Leishmania species and the cell line LL-5. Finally, parallel experiments using anti-gp63-treated parasites and PG-incubated midguts demonstrated that both approaches substantially inhibited attachment in the natural parasite-vector pairs Le. infantum/Lu. longipalpis and Le. braziliensis/Lu. intermedia. Our results suggest that gp63 and/or PG are involved in parasite attachment to the midgut of these important vectors. Copyright © 2017 Elsevier GmbH. All rights reserved.

Antiplasmodial, beta-haematin inhibition, antitrypanosomal and cytotoxic activity in vitro of novel 4-aminoquinoline 2-imidazolines.

PubMed

Musonda, Chitalu C; Yardley, Vanessa; de Souza, Renata C Carvalho; Ncokazi, Kanyile; Egan, Timothy J; Chibale, Kelly

2008-12-07

A novel series of 4-aminoquinoline-containing 2-imidazolines were synthesized via a one-pot 3-component condensation reaction of amine, aldehyde and isocyanoacetate. The products were obtained in high yield as well as purity and were evaluated directly against two strains of Plasmodium falciparum and Trypanosoma brucei. Compound was the most active across all parasites with ED(50) = 3.3 nM against a chloroquine (CQ)-sensitive 3D7 strain, ED(50) = 33 nM against a CQ-resistant K1 strain and ED(50) = 70 nM against T. brucei. Several compounds were able to inhibit formation of beta-haematin in vitro, suggesting haemozoin formation in the malaria parasite as a possible target. On the other hand, evaluation against a human KB cell line revealed that the compounds were generally non-cytotoxic to the host cells.

Inhibition of vascular smooth muscle cell proliferation and migration in vitro and neointimal hyperplasia in vivo by adenoviral-mediated atrial natriuretic peptide delivery.

PubMed

Larifla, Laurent; Déprez, Isabelle; Pham, Isabelle; Rideau, Dominique; Louzier, Vanessa; Adam, Micheline; Eloit, Marc; Foucan, Lydia; Adnot, Serge; Teiger, Emmanuel

2012-07-01

Vascular smooth muscle cell (VSMC) proliferation and migration are important components of the remodeling process in atherosclerosis or following angioplasty. Atrial natriuretic peptide (ANP) inhibits the growth of VSMCs in vitro but this effect has not been proven in vivo. In the present study, we examined the effects of local overexpression of ANP following gene transfer on in vitro VSMC proliferation and migration and in vivo neointimal formation in a rat carotid artery model of vascular injury. ANP gene transfer was performed using a recombinant adenovirus containing the ANP cDNA controlled by the Rous sarcoma virus (RSV) long terminal repeat (Ad-RSV-ANP). A recombinant adenovirus expressing the RSV-controlled β-galactosidase gene (Ad-RSV-β-gal) was used as the control. Rat VSMC culture was used for in vitro studies. In the in vivo experiments, carotid arteries were analyzed after balloon injury and local infusion of the viral solution. VSMCs transfected by Ad-RSV-ANP produced a significant amount of ANP detected by immunoreactive assay and accumulated about 6.5 times more cGMP than the viral control. VSMC proliferation stimulated with 10% fetal calf serum was reduced by 31% and migration by 25%. Fourteen days after injury, neointimal formation and the intima/media ratio were reduced by 25% and 28%, respectively, in the Ad-RSV-ANP-treated group compared to the control group. The present study demonstrates the efficacy of recombinant adenovirus Ad-RSV-ANP with respect to inhibiting rat VSMC proliferation and migration. Our findings also provide evidence that ANP is implicated in the modulation of vascular remodeling following endothelial injury. Copyright © 2012 John Wiley & Sons, Ltd.

Cannabinoids Inhibit T-cells via Cannabinoid Receptor 2 in an in vitro Assay for Graft Rejection, the Mixed Lymphocyte Reaction

PubMed Central

Robinson, Rebecca Hartzell; Meissler, Joseph J.; Breslow-Deckman, Jessica M.; Gaughan, John; Adler, Martin W.; Eisenstein, Toby K.

2013-01-01

Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. This study tested the capacity of Δ9-tetrahydrocannabinol (Δ9-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation. Both CB2-selective agonists and Δ9-THC significantly suppressed the MLR in a dose dependent fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2-selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3+ cells to these compounds completely inhibited their action in a reconstituted MLR. Further, the CB2-selective agonists completely inhibited proliferation of purified T-cells activated by anti-CD3 and anti-CD28 antibodies. T-cell function was decreased by the CB2 agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly decreased in the cannabinoid treated cells. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients. PMID:23824763

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.

Inhibition of the β-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus

PubMed Central

Lefebvre, Anne-Laure; Le Moigne, Vincent; Bernut, Audrey; Veckerlé, Carole; Compain, Fabrice; Herrmann, Jean-Louis; Kremer, Laurent

2017-01-01

ABSTRACT Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M. abscessus produces the broad-spectrum β-lactamase BlaMab. We determine whether inhibition of BlaMab by avibactam improves the activity of imipenem against M. abscessus. The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of BlaMab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (<0.1% survival), with 3.2- and 4.3-log10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of BlaMab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding BlaMab was induced (20-fold) in the infected macrophages. Inhibition of BlaMab by avibactam improved the efficacy of imipenem against M. abscessus in vitro, in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of BlaMab, since the production of the β-lactamase is inducible in macrophages. PMID:28096155

Inhibition of the β-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus.

PubMed

Lefebvre, Anne-Laure; Le Moigne, Vincent; Bernut, Audrey; Veckerlé, Carole; Compain, Fabrice; Herrmann, Jean-Louis; Kremer, Laurent; Arthur, Michel; Mainardi, Jean-Luc

2017-04-01

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M abscessus produces the broad-spectrum β-lactamase Bla Mab We determine whether inhibition of Bla Mab by avibactam improves the activity of imipenem against M. abscessus The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of Bla Mab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (<0.1% survival), with 3.2- and 4.3-log 10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of Bla Mab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding Bla Mab was induced (20-fold) in the infected macrophages. Inhibition of Bla Mab by avibactam improved the efficacy of imipenem against M. abscessus in vitro , in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of Bla Mab , since the production of the β-lactamase is inducible in macrophages. Copyright © 2017 American Society for Microbiology.

A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

PubMed

Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

2014-03-01

Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer. © 2013 UICC.

Myostatin inhibits porcine intramuscular preadipocyte differentiation in vitro.

PubMed

Sun, W X; Dodson, M V; Jiang, Z H; Yu, S G; Chu, W W; Chen, J

2016-04-01

This study assessed the effect of myostatin on adipogenesis by porcine intramuscular preadipocytes. Intramuscular preadipocytes were isolated from the longissimus dorsi muscle of newborn pigs. Myostatin inhibited intramuscular preadipocyte differentiation in a dose-dependent manner. Myostatin treatment during preadipocyte differentiation significantly (P < 0.05) inhibited the expression of the adipogenic marker genes CCAAT/enhancer-binding protein β, CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, fatty acid-binding protein, and adiponectin. Myostatin also significantly (P < 0.05) reduced the release of glycerol and decreased both adipose triglyceride lipase and hormone-sensitive lipase expression in intramuscular adipocytes. Our study suggests that myostatin acts as an extrinsic regulatory factor in regulating intramuscular adipogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition of DOR prevents remifentanil induced postoperative hyperalgesia through regulating the trafficking and function of spinal NMDA receptors in vivo and in vitro.

PubMed

Wang, Chunyan; Li, Yize; Wang, Haiyun; Xie, Keliang; Shu, Ruichen; Zhang, Linlin; Hu, Nan; Yu, Yonghao; Wang, Guolin

2015-01-01

Several studies have demonstrated that intraoperative remifentanil infusions have been associated with opioid-induced hyperalgesia (OIH). Activation of delta opioid receptor (DOR) and augmentation of N-methyl-d-aspartate (NMDA) receptor expression and function may play an important role in the development of OIH. The aim of this study was to investigate whether DOR inhibition could prevent remifentanil-induced hyperalgesia via regulating spinal NMDA receptor expression and function in vivo and in vitro. A rat model of remifentanil-induced postoperative hyperalgesia was performed with the DOR agonist deltorphin-deltorphin II or the DOR antagonist naltrindole injected intrathecally 10 min before remifentanil infusion. Mechanical and thermal hyperalgesia were measured at -24h, 2, 6, 24 and 48 h after remifentanil infusion. Western blot was applied to detect the membrane and total expression of DOR and NMDA receptor subunits (NR1, NR2A and NR2B) in spinal cord L4-L6 segments. In addition, whole-cell patch-clamp recording was used to investigate the effect of DOR inhibition on NMDA receptor-induced current in spinal cord slices in vitro. We found that membrane trafficking of DOR, NR1 and NR2B subunits in the spinal cord increased after remifentanil administration and surgery. The DOR antagonist naltrindole could attenuate mechanical and thermal hyperalgesia without affecting baseline nociceptive threshold, reduce membrane expression of DOR and decrease the membrane and total expressions of NR1 and NR2B subunits. Furthermore, the amplitude and the frequency of NMDA receptor-induced current were significantly increased by remifentanil incubation in neurons of the dorsal horn, which was reversed by the application of naltrindole. The above results indicate that inhibition of DOR could significantly inhibit remifentanil-induced hyperalgesia via modulating the total protein level, membrane trafficking and function of NMDA receptors in the dorsal horn of spinal cord

Noncalcemic 20-hydroxyvitamin D3 inhibits human melanoma growth in in vitro and in vivo models

PubMed Central

Kim, Tae-Kang; Yang, Chuan He; Pfeffer, Lawrence M.; Tuckey, Robert C.; Slominski, Andrzej T.

2017-01-01

A novel pathway of vitamin D3 (D3) metabolism, initiated by C20-hydroxylation of D3 by CYP11A1, has been confirmed to operate in vivo. Its major product, 20(OH)D3, exhibits antiproliferative activity in vitro comparable to that of 1,25(OH)2D3, but is noncalcemic in mice and rats. To further characterize the antimelanoma activity of 20(OH)D3, we tested its effect on colony formation of human melanoma cells in monolayer culture and anchorage-independent growth in soft agar. The migratory capabilities of the cells and cell-cell and cell-extracellular matrix interactions were also evaluated using transwell cell migration and spheroid toxicity assays. To assess the antimelanoma activity of 20(OH)D3 in vivo, age-matched immunocompromised mice were subcutaneously implanted with luciferase-labelled SKMel-188 cells and were randomly assigned to be treated with either 20(OH)D3 or vehicle (n=10 per group). Tumor size was measured with caliper and live bioimaging methods, and overall health condition expressed as a total body score scale. The following results were observed: (i) 20(OH)D3 inhibited colony formation both in monolayer and soft agar conditions, (ii) 20(OH)D3 inhibited melanoma cells in both transwell migration and spheroid toxicity assays, and (iii) 20(OH)D3 inhibited melanoma tumor growth in immunocompromised mice without visible signs of toxicity. However, although the survival rate was 90% in both groups, the total body score was higher in the treatment group compared to control group (2.8 vs. 2.55). In conclusion, 20(OH)D3, an endogenously produced secosteroid, is an excellent candidate for further preclinical testing as an antimelanoma agent. PMID:28039464

Benzenediamine analog FC-99 inhibits TLR2 and TLR4 signaling in peritoneal macrophage in vitro.

PubMed

Yang, Liu; Dou, Huan; Song, Yuxian; Hou, Yayi

2016-01-01

Inflammatory bowel disease (IBD) is an inflammatory disorder, characterized by abnormally increased expression of Toll-like receptors TLR2 and TLR4 in the colon and increased pro-inflammatory cytokine production by macrophages. In the present study, we explored the effect of FC-99, a novel benzenediamine analog, on dextran sulfate sodium (DSS)-induced mouse colitis and investigated its potential mechanism. The results revealed that FC-99 improved the colon morphology and the clinical parameters in DSS-induced mouse colitis. FC-99 inhibited the increase of DSS-induced T helper cells (Th) 1 and Th17 and enhanced the number of regulatory T cells (Treg) in mesenteric lymph nodes (MLN), but had no effect on Th2 cells. FC-99 also suppressed the DSS-induced secretion of interleukin (IL)-1β, IL-6, and the tumor necrosis factor (TNF)-α in the colon and hindered the infiltration of macrophages into colon lamina propria. Flow cytometric analysis also confirmed that FC-99 reduced CD11b(+)F4/80(+) colon macrophages, and down-regulated TNF-α level in situ. Moreover, FC-99 inhibited concentration-dependently the expression of TNF-α and IL-6 in vitro from mouse peritoneal macrophages, which were induced by TLR ligands: PamCSK4 and peptidoglycan (PGN, TLR2 ligand) as well as LPS (TLR4 ligand). Of note, FC-99 also suppressed the activation of TLR2 and TLR4 signaling pathways and the downstream nuclear factor-κB (NF-κB) in the DSS-induced mouse colitis. FC-99 improved the condition of DSS-induced mouse colitis by inhibiting the activation of TLR2 and TLR4 signaling pathways in macrophage. These results suggest that FC-99 may be developed as a new therapeutic drug for IBD. Copyright © 2015 Elsevier Inc. All rights reserved.

Structure-function studies on hsp47: pH-dependent inhibition of collagen fibril formation in vitro.

PubMed Central

Thomson, C A; Ananthanarayanan, V S

2000-01-01

Hsp47, a 47 kDa heat shock protein whose expression level parallels that of collagen, has been regarded as a collagen-specific molecular chaperone. Studies from other laboratories have established the association of Hsp47 with the nascent as well as the triple-helical procollagen molecule in the endoplasmic reticulum and its dissociation from procollagen in the Golgi. One of several roles suggested for Hsp47 in collagen biosynthesis is the prevention of aggregation of procollagen in the endoplasmic reticulum. However, no experimental evidence has been available to verify this suggestion. In the present study we have followed the aggregation of mature triple-helical collagen molecules into fibrils by using turbidimetric measurements in the absence and presence of Hsp47. In the pH range 6-7, fibril formation of type I collagen, as monitored by turbidimetry, proceeds with a lag of approx. 10 min and levels off by approx. 60 min. The addition of Hsp47 at pH 7 effectively inhibits fibril formation at and above a 1:1 molar ratio of Hsp47 to triple-helical collagen. This inhibition is markedly pH-dependent, being significantly diminished at pH 6. CD and fluorescence spectral data of Hsp47 in the pH range 4.2-7.4 reveal a significant alteration in its structure at pH values below 6.2, with a decrease in alpha-helix and an increase in beta-structure. This conformational change is likely to be the basis of the decreased binding of Hsp47 to collagen in vitro at pH 6.3 as well as its inability to inhibit collagen fibril formation at this pH. Our results also provide a functional assay for Hsp47 that can be used in studies on collagen and Hsp47 interactions. PMID:10903151

Curcumin Inhibits Tau Aggregation and Disintegrates Preformed Tau Filaments in vitro.

PubMed

Rane, Jitendra Subhash; Bhaumik, Prasenjit; Panda, Dulal

2017-01-01

The pathological aggregation of tau is a common feature of most of the neuronal disorders including frontotemporal dementia, Parkinson's disease, and Alzheimer's disease. The inhibition of tau aggregation is considered to be one of the important strategies for treating these neurodegenerative diseases. Curcumin, a natural polyphenolic molecule, has been reported to have neuroprotective ability. In this work, curcumin was found to bind to adult tau and fetal tau with a dissociation constant of 3.3±0.4 and 8±1 μM, respectively. Molecular docking studies indicated a putative binding site of curcumin in the microtubule-binding region of tau. Using several complementary techniques, including dynamic light scattering, thioflavin S fluorescence, 90° light scattering, electron microscopy, and atomic force microscopy, curcumin was found to inhibit the aggregation of tau. The dynamic light scattering analysis and atomic force microscopic images revealed that curcumin inhibits the oligomerization of tau. Curcumin also disintegrated preformed tau oligomers. Using Far-UV circular dichroism, curcumin was found to inhibit the β-sheets formation in tau indicating that curcumin inhibits an initial step of tau aggregation. In addition, curcumin inhibited tau fibril formation. Furthermore, the effect of curcumin on the preformed tau filaments was analyzed by atomic force microscopy, transmission electron microscopy, and 90° light scattering. Curcumin treatment disintegrated preformed tau filaments. The results indicated that curcumin inhibited the oligomerization of tau and could disaggregate tau filaments.

Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yang; Pang, Xiaoyan; Dong, Mei

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 obesitymore » 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.« less

Polysaccharide peptides from COV-1 strain of Coriolus versicolor inhibit tolbutamide 4-hydroxylation in the rat in vitro and in vivo.

PubMed

Yeung, John H K; Chan, Siu-Lung; Or, Penelope M Y

2006-08-01

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. In this study, the effects of whole PSP extract and water extract of PSP on 4-hydroxylation of tolbutamide were investigated in rat liver microsomes in vitro and in vivo in the rat. Both the whole PSP extract and the water soluble fraction (0.5-20 microM) decreased the metabolism of tolbutamide to 4-hydroxytolbutamide in vitro. Enzyme kinetics studies showed that PSP inhibited tolbutamide 4-hydroxylase activity in a competitive, concentration-dependent manner. The whole PSP extract had a Ki value of 12.6 microM and IC50 at 18.4 microM, while the water extract had a Ki value of 6.9 microM and IC50 at 9.8 microM. Sulphaphenazole, a specific human CYP2C9 inhibitor, showed a Ki value of 30.8 microM and IC50 at 44.0 microM in the test system. In the pharmacokinetic studies in vivo, acute PSP (4 micromol/kg, i.p.) treatment did not produce significant changes in tolbutamide clearance, but produced a decrease in the Cinitial (7.4%) and an increase in the Vd (7.4%). Sub-chronic pre-treatment of PSP (1-2 micromol/kg/day, i.p.) for three days did not affect the clearance and AUC of tolbutamide, but the Cinitial was decreased, together with increases in the T1/2, and Vd. The formation of 4-hydroxytolbutamide in vivo was decreased in both acute and sub-chronic studies. Taken together, this study demonstrated the PSP can inhibit tolbutamide 4-hydroxylation both in vitro and in vivo. Despite the fact that CYP isoforms that metabolise tolbutamide are different between rat and human liver due to different catalytic characteristics, and rat studies may not be directly extrapolatable to man, the concomitant use of PSP with other CYP2C substrates should be carefully monitored.

Dihydroartiminisin inhibits the growth and metastasis of epithelial ovarian cancer.

PubMed

Wu, Buchu; Hu, Ke; Li, Shu; Zhu, Jing; Gu, Liying; Shen, Haoran; Hambly, Brett D; Bao, Shisan; Di, Wen

2012-01-01

Dihydroartiminisin (DHA), the active component of a Chinese herb (Artemisia annua), has been utilised as an anti-malarial drug since ancient China. DHA has also been shown to inhibit proliferation of cancer in vitro. However, the capacity of DHA to inhibit the development of ovarian cancer is still unclear. The adhesion, invasion, and migration of human ovarian cancer cell line (HO8910PM) was determined following DHA treatment in vitro, using Matrigel coated plate, transwell membrane chamber, and wound healing models, respectively. A mouse ovarian cancer model was established by orthotopic inoculation of HO8910PM cell line in nude mice. The growth and metastasis in vivo was determined 8 weeks post-implantation in response to DHA treatment. The expression of phosphorylated focal adhesion kinase (pFAK) and matrix metalloproteinases (MMP-2 and MMP-9) was evaluated using Western blotting. The expression of Von Willebrand factor (vWF) and infiltration of macrophages were determined, using immunohistochemistry. DHA inhibits ovarian cancer cell proliferation, adhesion, migration and invasion in vitro in a dose-dependent manner, consistent with decreased expression of pFAK and MMP-2, but not MMP-9. DHA inhibited metastasis significantly in vivo, associated with reduced vWF expression and macrophage infiltration. In conclusion, DHA inhibits the development of ovarian cancer, in part via down-regulating pFAK, MMP-2, vWF and macrophage infiltration.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yu-Jie; Guo, Yan; Yang, Qing

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 inmore » 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

Selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of EGFR expression.

PubMed

Dai, Yumin; Hogan, Shelly; Schmelz, Eva M; Ju, Young H; Canning, Corene; Zhou, Kequan

2011-01-01

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC(50) = 4.8 μg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus.

PubMed

Hong, S K; Matsumoto, A; Horinouchi, S; Beppu, T

1993-01-01

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

Combinatorial Effects of Aromatic 1,3-Disubstituted Ureas and Fluoride on In vitro Inhibition of Streptococcus mutans Biofilm Formation.

PubMed

Kaur, Gurmeet; Balamurugan, P; Uma Maheswari, C; Anitha, A; Princy, S Adline

2016-01-01

Dental caries occur as a result of disequilibrium between acid producing pathogenic bacteria and alkali generating commensal bacteria within a dental biofilm (dental plaque). Streptococcus mutans has been reported as a primary cariogenic pathogen associated with dental caries. Emergence of multidrug resistant as well as fluoride resistant strains of S. mutans due to over use of various antibiotics are a rising problem and prompted the researchers worldwide to search for alternative therapies. In this perspective, the present study was aimed to screen selective inhibitors against ComA, a bacteriocin associated ABC transporter, involved in the quorum sensing of S. mutans. In light of our present in silico findings, 1,3-disubstituted urea derivatives which had better affinity to ComA were chemically synthesized in the present study for in vitro evaluation of S. mutans biofilm inhibition. The results revealed that 1,3-disubstituted urea derivatives showed good biofilm inhibition. In addition, synthesized compounds exhibited potent synergy with a very low concentration of fluoride (31.25-62.5 ppm) in inhibiting the biofilm formation of S. mutans without affecting the bacterial growth. Further, the results were supported by confocal laser scanning microscopy. On the whole, from our experimental results we conclude that the combinatorial application of fluoride and disubstituted ureas has a potential synergistic effect which has a promising approach in combating multidrug resistant and fluoride resistant S. mutans in dental caries management.

Flavonoids from Litsea japonica Inhibit AGEs Formation and Rat Lense Aldose Reductase In Vitro and Vessel Dilation in Zebrafish.

PubMed

Lee, Ik-Soo; Kim, Yu Jin; Jung, Seung-Hyun; Kim, Joo-Hwan; Kim, Jin Sook

2017-02-01

In our ongoing efforts to identify effective naturally sourced agents for the treating of diabetic complications, two new ( 1 and 2 ) and 11 known phenolic compounds ( 3 - 13 ) were isolated from an 80 % ethanol extract of Litsea japonica leaves. The structures of the new compounds were established by spectroscopic and chemical studies. These isolates ( 1 - 13 ) were subjected to an in vitro bioassay evaluating their inhibitory activity on advanced glycation end products formation and rat lens aldose reductase activity. Of the compounds evaluated, the flavonoids ( 3, 4, 6 - 8, 11 , and 12 ) markedly inhibited advanced glycation end products formation, with IC 50 values of 7.4-72.0 µM, compared with the positive control, aminoguanidine (IC 50  = 975.9 µM). In the rat lens aldose reductase assay, consistent with the inhibition of advanced glycation end products formation, the flavonoids ( 3, 4, 6 - 8, 11 , and 12 ) exhibited considerable inhibition of rat lens aldose reductase activity, with IC 50 values of 1.1-12.5 µM. In addition, the effects of kaempferol ( 4 ) and tiliroside ( 7 ) on the dilation of hyaloid-retinal vessels induced by high glucose in larval zebrafish were investigated. Only kaempferol significantly reduced the diameters of high glucose-induced hyaloid-retinal vessels, by 52.2 % at 10 µM, compared with those in the high glucose-treated control group. Georg Thieme Verlag KG Stuttgart · New York.

Characterization of plasma cholinesterase from the White stork (Ciconia ciconia) and its in vitro inhibition by anticholinesterase pesticides.

PubMed

Oropesa, Ana-Lourdes; Gravato, Carlos; Sánchez, Susana; Soler, Francisco

2013-11-01

Blood plasma cholinesterase (ChE) activity is a sensitive biomarker of exposure to organophosphorus (OP) and carbamate (CB) insecticides in vertebrates. Several studies indicate that more than one ChE form may be present in blood of birds. In this study the predominant ChE activity (acetylcholinesterase - AChE- or butyrylcholinesterase - BChE-), the range of ChE activity as well as ChE age-dependent changes in non-exposed individuals of White stork (Ciconia ciconia) have been established. The in vitro sensitivity of ChE to OP and CB insecticides such as paraoxon-methyl, carbofuran and carbaryl was also investigated. Plasma ChE was characterised using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylcholine and butyrylcholine and the predominant enzymatic activity in plasma of White storks was BChE. Normal plasma BChE activity in White stork was 0.32±0.01μmol/min/ml for adults and 0.28±0.03μmol/min/ml for juveniles. So, the age had no significant effect on the range of BChE activity. The study on the in vitro inhibitory potential of tested anticholinesterase pesticides on plasma ChE activity revealed that paraoxon-methyl is the most potent inhibitor followed by carbofuran and finally by carbaryl. The percentage of in vitro plasma ChE inhibition was observed to be similar between adults and juveniles. Copyright © 2013 Elsevier Inc. All rights reserved.

Combination of flavonoids from Oroxylum indicum seed extracts and acarbose improves the inhibition of postprandial blood glucose: In vivo and in vitro study.

PubMed

Zhang, Bo-Wei; Sang, Yuan-Bin; Sun, Wen-Long; Yu, He-Shui; Ma, Bai-Ping; Xiu, Zhi-Long; Dong, Yue-Sheng

2017-07-01

The combined effect of Oroxylum indicum seed extracts (OISE) or major flavonoids from OISE and acarbose on reducing postprandial blood glucose (PBG) levels was investigated in vitro and in vivo. In vitro, the IC 50 values of OISE and baicalein against α-glucosidase were 43.4±0.731μgmL -1 and 25.9±0.412μgmL -1 respectively. A combination of acarbose with OISE or baicalein synergistically inhibited rat intestinal α-glucosidase. The combination index (CI) values for acarbose with OISE ranged from 0.33 to 0.75, suggesting a synergistic but not additive effect. OISE was determined to be a non-competitive inhibitor of maltose-hydrolyzing activity. In vivo, OISE were administered to normoglycemic and diabetic mice, either alone or in combination with acarbose. At doses between 50 and 200mgkg -1 , OISE enhanced the efficacy of acarbose by up to 5-fold. These results demonstrated that OISE enhances the efficacy of acarbose in vivo, and that the combination of OISE and acarbose displayed a synergistic effect in vitro. Therefore, OISE can be used to design dietary supplements to treat diabetes. Copyright © 2017. Published by Elsevier Masson SAS.

Progesterone inhibits the in vitro L3/L4 molting process in Haemonchus contortus.

PubMed

Gutiérrez-Amézquita, R A; Morales-Montor, J; Muñoz-Guzmán, M A; Nava-Castro, K E; Ramírez-Álvarez, H; Cuenca-Verde, C; Moreno-Mendoza, N A; Cuéllar-Ordaz, J A; Alba-Hurtado, F

2017-12-15

We evaluated the direct effects of progesterone on the morphology, maturation and behavior of Haemonchus contortus larvae in vitro. The presence and location of possible progesterone receptors in these larvae were also determined. The addition of 8ng/mL of progesterone to larval cultures over 10days reduced larval enlargement, while the addition of 160ng/mL of the hormone increased the enlargement. Up to 62% and 65% of the H. contortus larvae molted from third-stage larvae (L3) to fourth-stage larvae (L4) when cultured in RPMI-1640 media without hormone for 5 and 10days, respectively. The addition of different progesterone concentrations (1, 8, 16, 80 and 160ng/mL) to the larval cultures significantly inhibited the molting process within the same periods. The addition of 8ng/mL or higher progesterone concentrations to the cultures significantly increased larval motility (p<0.05) compared with unstimulated larvae. Flow cytometry showed the expression of progesterone receptors (P4-R) in 15% of the cells from newly isolated H. contortus larvae. When the larvae were cultured for 5days in the presence of the hormone, the percentage of P 4 -R+ cells remained the same. In contrast, unstimulated larvae showed a significant reduction in the number of P 4 -R+ cells. Using confocal microscopy, a greater concentration of P 4 -Rs was immunolocated in the anterior portion of the alimentary tract of the larvae, suggesting that the cells in this region are targeted by the hormone. The results of the present study show that H. contortus larvae have possible P 4 -Rs and respond to this hormone by inhibiting their molting process, thereby suggesting the participation of progesterone in the larval arrest phenomenon. Copyright © 2017 Elsevier B.V. All rights reserved.

Decorin-loaded poly lactic-co-glycolic acid nanoparticles modified by anti-alpha fetoprotein antibody: preparation, proliferation inhibition and induced apoptosis effects on HepG2 cells in vitro.

PubMed

Yang, Qiaoli; Wang, Shuyue; Wang, Yuan; Qu, Yane; Xue, Jun; Mi, Yang; Wang, Yanhong; Luo, Xuguang; Deng, Zhihua; Wang, Guiqin

2017-06-01

Decorin (DCN) is a negative regulatory factor for the growth of cancer cells and can inhibit the proliferation, metastasis of cancer cells and angiogenesis in cancer tissues. The aims of this study were to prepare the nanoparticles consisting of DCN and poly lactic-co-glycolic acid (PLGA) modified by anti-alpha fetoprotein (AFP) monoclonal antibody (mAb) and to examine the conventional physical properties, the in-vitro release of DCN and the targeting effect of these nanoparticles on HepG2 cells. The encapsulated plasmid was slowly and steadily released from the nanoparticles. The targeted PLGA nanoparticles were initiatively taken in HepG2 cells high-efficiently. According to the results of RT-PCR, DCN gene in AFPmAb-PLGA-rhDCN nanoparticles can be expressed in HepG2 cells successfully. These nanoparticles significantly inhibited the proliferation of HepG2 cells and induced apoptosis. The mRNA expression of Bcl-2 gene in the AFPmAb-PLGA-rhDCN-treated groups appeared significantly to decrease and the caspase-3 gene had the opposite trend as compared with that of control group (P < 0.01). These studies revealed that these nanoparticles were capable of specifically targeting the HepG2 cells and inhibiting the proliferation and they induce apoptosis of HepG2 cells in vitro, which was in a dose- and time-dependent manner. © 2017 Royal Pharmaceutical Society.

Soy Pulp Extract Inhibits Angiotensin I-Converting Enzyme (ACE) Activity In Vitro: Evidence for Its Potential Hypertension-Improving Action.

PubMed

Nishibori, Naoyoshi; Kishibuchi, Reina; Morita, Kyoji

2017-05-04

Soy pulp, called "okara" in Japanese, is known as a by-product of the production of bean curd (tofu), and expected to contain a variety of biologically active substances derived from soybean. However, the biological activities of okara ingredients have not yet been fully understood, and the effectiveness of okara as a functional food seems necessary to be further evaluated. Then the effect of okara extract on angiotensin I-converting enzyme (ACE) activity was examined in vitro, and the extract was shown to cause the inhibition of ACE activity in a manner depending on its concentration. Kinetic analysis indicated that this enzyme inhibition was accompanied by an increase in the Km value without any change in Vmax. Further studies suggested that putative inhibitory substances contained in the extract might be heat stable and dialyzable, and recovered mostly in the peptide fraction obtained by a spin-column separation and a high performance liquid chromatography (HPLC) fractionation. Therefore, the extract was speculated to contain small-size peptides responsible for the inhibitory effect of okara extract on ACE activity, and could be expected to improve the hypertensive conditions by reducing the production of hypertensive peptide.

Cannabidiol inhibits angiogenesis by multiple mechanisms

PubMed Central

Solinas, M; Massi, P; Cantelmo, AR; Cattaneo, MG; Cammarota, R; Bartolini, D; Cinquina, V; Valenti, M; Vicentini, LM; Noonan, DM; Albini, A; Parolaro, D

2012-01-01

BACKGROUND AND PURPOSE Several studies have demonstrated anti-proliferative and pro-apoptotic actions of cannabinoids on various tumours, together with their anti-angiogenic properties. The non-psychoactive cannabinoid cannabidiol (CBD) effectively inhibits the growth of different types of tumours in vitro and in vivo and down-regulates some pro-angiogenic signals produced by glioma cells. As its anti-angiogenic properties have not been thoroughly investigated to date, and given its very favourable pharmacological and toxicological profile, here, we evaluated the ability of CBD to modulate tumour angiogenesis. EXPERIMENTAL APPROACH Firstly, we evaluated the effect of CBD on human umbilical vein endothelial cell (HUVEC) proliferation and viability – through [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and FACS analysis – and in vitro motility – both in a classical Boyden chamber test and in a wound-healing assay. We next investigated CBD effects on different angiogenesis-related proteins released by HUVECs, using an angiogenesis array kit and an ELISA directed at MMP2. Then we evaluated its effects on in vitro angiogenesis in treated HUVECs invading a Matrigel layer and in HUVEC spheroids embedded into collagen gels, and further characterized its effects in vivo using a Matrigel sponge model of angiogenesis in C57/BL6 mice. KEY RESULTS CBD induced HUVEC cytostasis without inducing apoptosis, inhibited HUVEC migration, invasion and sprouting in vitro, and angiogenesis in vivo in Matrigel sponges. These effects were associated with the down-modulation of several angiogenesis-related molecules. CONCLUSIONS AND IMPLICATIONS This study reveals that CBD inhibits angiogenesis by multiple mechanisms. Its dual effect on both tumour and endothelial cells supports the hypothesis that CBD has potential as an effective agent in cancer therapy. PMID:22624859

Cannabidiol inhibits angiogenesis by multiple mechanisms.

PubMed

Solinas, M; Massi, P; Cantelmo, A R; Cattaneo, M G; Cammarota, R; Bartolini, D; Cinquina, V; Valenti, M; Vicentini, L M; Noonan, D M; Albini, A; Parolaro, D

2012-11-01

Several studies have demonstrated anti-proliferative and pro-apoptotic actions of cannabinoids on various tumours, together with their anti-angiogenic properties. The non-psychoactive cannabinoid cannabidiol (CBD) effectively inhibits the growth of different types of tumours in vitro and in vivo and down-regulates some pro-angiogenic signals produced by glioma cells. As its anti-angiogenic properties have not been thoroughly investigated to date, and given its very favourable pharmacological and toxicological profile, here, we evaluated the ability of CBD to modulate tumour angiogenesis. Firstly, we evaluated the effect of CBD on human umbilical vein endothelial cell (HUVEC) proliferation and viability - through [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and FACS analysis - and in vitro motility - both in a classical Boyden chamber test and in a wound-healing assay. We next investigated CBD effects on different angiogenesis-related proteins released by HUVECs, using an angiogenesis array kit and an ELISA directed at MMP2. Then we evaluated its effects on in vitro angiogenesis in treated HUVECs invading a Matrigel layer and in HUVEC spheroids embedded into collagen gels, and further characterized its effects in vivo using a Matrigel sponge model of angiogenesis in C57/BL6 mice. CBD induced HUVEC cytostasis without inducing apoptosis, inhibited HUVEC migration, invasion and sprouting in vitro, and angiogenesis in vivo in Matrigel sponges. These effects were associated with the down-modulation of several angiogenesis-related molecules. This study reveals that CBD inhibits angiogenesis by multiple mechanisms. Its dual effect on both tumour and endothelial cells supports the hypothesis that CBD has potential as an effective agent in cancer therapy. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Inhibition of gluconeogenesis by Malmea depressa root.

PubMed

Andrade-Cetto, Adolfo

2011-09-01

Malmea depressa is traditionally used in the Mayan communities of southeastern Mexico to treat type 2 diabetes. A root bark infusion is being taken throughout the day, between meals. The aim of this study was to determine whether an ethanolic extract of Malmea depressa would reduce hepatic glucose production by targeting gluconeogenesis. The effects of the plant extract on gluconeogenesis (in vivo) and the activity of GL-6-P (in vitro) were examined. The plant extract was analyzed by HPLC to confirm its phytochemical composition. The inhibition of gluconeogenesis was tested in vivo by performing a pyruvate tolerance test in n5-STZ after an 18-h fasting period. The extracts effect on glucose-6-phosphatase activity were assayed in vitro with intact rat liver microsomes. Using HPLC-DAD we confirmed that the phytochemical compositions of the tested extract were similar to those previously reported. We proved that the ethanolic extract of the root bark of Malmea depressa dose-dependently inhibits a glucose peak. Furthermore, the gluconeogenesis inhibition was confirmed in vitro using a pyruvate test. The results suggest that administration of Malmea depressa can improve glycemic control by blocking hepatic glucose production, especially in the fasting state. These data support its traditional use as an infusion consumed continually throughout the day. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Nanoencapsulation of dietary flavonoid fisetin: Formulation and in vitro antioxidant and α-glucosidase inhibition activities.

PubMed

Sechi, Mario; Syed, Deeba N; Pala, Nicolino; Mariani, Alberto; Marceddu, Salvatore; Brunetti, Antonio; Mukhtar, Hasan; Sanna, Vanna

2016-11-01

The bioactive flavonoid fisetin (FS) is a diet-derived antioxidant that is being increasingly investigated for its health-promoting effects. Unfortunately, the poor physicochemical and pharmacokinetic properties affect and limit the clinical application. In this study, novel polymeric nanoparticles (NPs), based on Poly-(ε-caprolactone) (PCL) and PLGA-PEG-COOH, encapsulating FS were formulated as suitable oral controlled release systems. Results showed NPs having a mean diameter of 140-200nm, and a percent loading of FS ranging from 70 to 82%. In vitro release studies revealed that NPs are able to protect and preserve the release of FS in gastric simulated conditions, also controlling the release in the intestinal medium. Moreover, the DPPH and ABTS scavenging capacity of FS, as well as α-glucosidase inhibition activity, that resulted about 20-fold higher than commercial Acarbose, were retained during nanoencapsulation process. In summary, our developed NPs can be proposed as an attractive delivery system to control the release of antioxidant and anti-hyperglycemic FS for nutraceutical and/or therapeutic application. Copyright © 2016 Elsevier B.V. All rights reserved.

In silico and in vitro inhibition of cytochrome P450 3A by synthetic stilbenoids.

PubMed

Basheer, Loai; Schultz, Keren; Guttman, Yelena; Kerem, Zohar

2017-12-15

Inhibition of cytochrome P450 3A4 (CYP3A4), the major drug metabolizing enzyme, by dietary compounds has recently attracted increased attention. Evaluating the potency of the many known inhibitory compounds is a tedious and time consuming task, yet it can be achieved using computing tools. Here, CDOCKER and Glide served to design model inhibitors in order to characterize molecular features of an inhibitor. Assessing nitro-stilbenoids, both approaches suggested nitrostilbene to be a weaker inhibitor of CYP3A4 than resveratrol, and stronger than dimethoxy-nitrostilbene. Nitrostilbene and resveratrol, but not dimethoxy-nitrostilbene, engage electrostatic interactions in the enzyme cavity, and with the haem. In vitro assessment of the inhibitory capacity supported the in silico predictions, suggesting that evaluating the electrostatic interactions of a compound with the prosthetic group allows the prediction of inhibitory potency. Since both programs yielded related results, it is suggested that for CYP3A4, computing tools may allow rapid identification of potent dietary inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Curcumin synergizes with resveratrol to inhibit colon cancer.

PubMed

Majumdar, Adhip P N; Banerjee, Sanjeev; Nautiyal, Jyoti; Patel, Bhaumik B; Patel, Vaishali; Du, Jianhua; Yu, Yingjie; Elliott, Althea A; Levi, Edi; Sarkar, Fazlul H

2009-01-01

Development and progression of many malignancies, including colorectal cancer, are associated with activation of multiple signaling pathways. Therefore, inhibition of these signaling pathways with noncytotoxic natural products represents a logical preventive and/or therapeutic approach for colon cancer. Curcumin and resveratrol, both of which inhibit the growth of transformed cells and colon carcinogenesis, were selected to examine whether combining them would be an effective preventive and/or therapeutic strategy for colon cancer. Indeed, the combination of curcumin and resveratrol was found to be more effective in inhibiting growth of p53-positive (wt) and p53-negative colon cancer HCT-116 cells in vitro and in vivo in SCID xenografts of colon cancer HCT-116 (wt) cells than either agent alone. Analysis by Calcusyn software showed synergism between curcumin and resveratrol. The inhibition of tumors in response to curcumin and/or resveratrol was associated with the reduction in proliferation and stimulation of apoptosis accompanied by attenuation of NF-kappaB activity. In vitro studies have further demonstrated that the combinatorial treatment caused a greater inhibition of constitutive activation of EGFR and its family members as well as IGF-1R. Our current data suggest that the combination of curcumin and resveratrol could be an effective preventive/therapeutic strategy for colon cancer.

IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohshima-Hosoyama, Sachiko; Hosoyama, Tohru; Nelon, Laura D.

2010-09-03

Research highlights: {yields} Igf1r is overexpressed and activated in a Sonic Hedgehog driven model of medulloblastoma. {yields} Picropodophyllin targets and abrogates IGF signaling in medulloblastoma. {yields} Picropodophyllin inhibits medulloblastoma tumor cell growth by induction of apoptosis. -- Abstract: The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression.more » We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.« less

Two Lactobacillus Species Inhibit the Growth and α-Toxin Production of Clostridium perfringens and Induced Proinflammatory Factors in Chicken Intestinal Epithelial Cells in Vitro

PubMed Central

Guo, Shuangshuang; Liu, Dan; Zhang, Beibei; Li, Zhui; Li, Yehan; Ding, Binying; Guo, Yuming

2017-01-01

Clostridium perfringens is the causative pathogen of avian necrotic enteritis. Lactobacillus spp. are well-characterized probiotics with anti-microbial and immune-modulatory activities. In the present study, we investigated the effects of L. acidophilus and L. fermentum on the growth, α-toxin production and inflammatory responses of C. perfringens. In in vitro culture experiments, both lactobacilli inhibited the growth of C. perfringens (P < 0.01), accompanied with a decrease in pH (P < 0.01). Supernatants from lactobacilli cultures also suppressed the growth of C. perfringens during 24 h of incubation (P < 0.01), but this inhibitory effect disappeared after 48 h. Both lactobacilli decreased the α-toxin production of C. perfringens (P < 0.01) without influencing its biomass, and even degraded the established α-toxin (P < 0.01). Lower environmental pH reduced the α-toxin production as well (P < 0.01). Preincubation with L. acidophilus decreased the attachment of C. perfringens to cells (P < 0.01) with the cell cytotoxicity being unaffected. Both lactobacilli pretreatment reduced the up-regulation of proinflammatory factors, peptidoglycan (PGN) receptors and nuclear factor kappa B (NF-κB) p65 in C. perfringens-challenged chicken intestinal epithelial cells (P < 0.05). In conclusion, L. acidophilus and L. fermentum inhibited the pathological effects of C. perfringens in vitro conditions. PMID:29118744

Direct factor IXa inhibition with the RNA-aptamer pegnivacogin reduces platelet reactivity in vitro and residual platelet aggregation in patients with acute coronary syndromes.

PubMed

Staudacher, Dawid L; Putz, Vera; Heger, Lukas; Reinöhl, Jochen; Hortmann, Marcus; Zelenkofske, Steven L; Becker, Richard C; Rusconi, Christopher P; Bode, Christoph; Ahrens, Ingo

2017-04-01

Residual platelet reactivity is a predictor of poor prognosis in patients with acute coronary syndromes (ACSs) undergoing percutaneous coronary intervention. Thrombin is a major platelet activator and upon initiation of the coagulation cascade, it is subsequently produced downstream of factor IXa, which itself is known to be increased in ACS. Pegnivacogin is a novel RNA-aptamer based factor IXa inhibitor featuring a reversal agent, anivamersen. We hypothesized that pegnivacogin could reduce platelet reactivity. Whole blood samples from healthy volunteers were incubated in vitro in the presence and absence of pegnivacogin and platelet reactivity was analysed. In addition, platelet aggregometry was performed in blood samples from ACS patients in the RADAR trial featuring the intravenous administration of pegnivacogin as well as reversal by anivamersen. In vitro, pegnivacogin significantly reduced adenosine diphosphate-induced CD62P-expression (100% vs. 89.79±4.04%, p=0.027, n=9) and PAC-1 binding (100% vs. 83.02±4.08%, p=0.010, n=11). Platelet aggregation was reduced (97.71±5.30% vs. 66.53±9.92%, p=0.013, n=10) as evaluated by light transmission aggregometry. In the presence of the RNA-aptamer reversal agent anivamersen, neither CD62P-expression nor platelet aggregation was attenuated. In patients with ACS treated with aspirin and clopidogrel, residual platelet aggregation was significantly reduced 20 min after intravenous bolus of 1 mg/kg pegnivacogin (100% versus 43.21±8.23%, p=0.020). Inhibition of factor IXa by pegnivacogin decreases platelet activation and aggregation in vitro. This effect was negated by anivamersen. In ACS patients, platelet aggregation was significantly reduced after intravenous pegnivacogin. An aptamer-based anticoagulant inhibiting factor IXa therefore might be a promising antithrombotic strategy in ACS patients.

SRC family kinase (SFK) inhibition reduces rhabdomyosarcoma cell growth in vitro and in vivo and triggers p38 MAP kinase-mediated differentiation

PubMed Central

Casini, Nadia; Forte, Iris Maria; Mastrogiovanni, Gianmarco; Pentimalli, Francesca; Angelucci, Adriano; Festuccia, Claudio; Tomei, Valentina; Ceccherini, Elisa; Di Marzo, Domenico; Schenone, Silvia; Botta, Maurizio; Giordano, Antonio; Indovina, Paola

2015-01-01

Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS. PMID:25762618

Ferrocene-based guanidine derivatives: in vitro antimicrobial, DNA binding and docking supported urease inhibition studies.

PubMed

Gul, Rukhsana; Rauf, Muhammad Khawar; Badshah, Amin; Azam, Syed Sikander; Tahir, Muhammad Nawaz; Khan, Azim

2014-10-06

Some novel ferrocenyl guanidines 1-8 were synthesized and characterized by different spectroscopic methods, elemental analysis and single crystal X-rays diffraction techniques. The crystallographic studies revealed that the existence of the strong non-bonding interactions facilitate these molecules to interact with biological macro-molecules like DNA that described to inherit good biological activities. The DNA interaction studies carried out by cyclic voltammetry (CV) and UV-visible spectroscopy are in close agreement with the binding constants (K) (0.79-5.4) × 10(5) (CV) and (0.72-5.1) × 10(5) (UV-vis). The shift in peak potential, current and absorption maxima of the studied ferrocenyl guanidines in the presence of DNA revealed that CV coupled with UV-vis spectroscopy could provide an opportune to characterize metal-based compounds-DNA interaction mechanism, a prerequisite for the design of new anticancer agents and understanding the molecular basis of their action. The compounds 1-8 have been screened for their antibacterial, antifungal and urease inhibition potency. A concurrent in silico study has also been applied on ferrocene moiety impregnated guanidines 1-8 to identify most active compounds having for inhibiting the activity of urease (pdb id 3LA4). Most of the compounds were found as potent inhibitors of urease and the compound 1 was found to be the most active with an IC50 of 16.83 ± 0.03 μM. The docking scores are in close agreement with the in vitro obtained IC50 values of inhibitors 1-8. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Forskolin-stimulated vasopressin and oxytocin release from the rat hypothalamo-neurohypophysial system in vitro is inhibited by melatonin.

PubMed

Roszczyk, Magdalena; Juszczak, Marlena

2014-01-01

Previous in vivo and in vitro studies have shown that melatonin changes vasopressin (AVP) and oxytocin (OT) secretion from the rat neurohypophysis. Additionally, melatonin is known to inhibit the forskolin-induced (forskolin is a strong adenylyl cyclase - AC activator) increase in cAMP accumulation in the rat pituitary. To determine whether the possible response of vasopressinergic and/or oxytocinergic neurones to melatonin could be mediated through a cAMP-dependent mechanism, the effect of different concentrations of melatonin (i.e. 10-11, 10-9, 10-7, 10-5 and 10-3 M) on forskolin-stimulated AVP and OT release from the rat hypothalamo-neurohypophysial (H-NH) system was studied in vitro. Male rats served as donors of the H-NH explants, which were placed in 1 mLof normal Krebs-Ringer fluid (nKRF), heated to 37oC and constantly gassed with carbogen (95% O2 and 5% CO2). The H-NH explants were incubated successively in nKRF {fluid B1} and incubation fluid as B1 enriched with an appropriate concentration of melatonin, i.e. 10-11 - 10-3 M and/or forskolin (at a concentration of 10-5 M) or their vehicles (0.1% ethanol or DMSO) {fluid B2}. After 20 min incubation in fluid B1 and next B2, the media were collected and immediately frozen before AVP and OT estimation by the RIA. The AVP and OT secretion was determined by using B2/B1 ratio for each H-NH explant. We have demonstrated that the highly effective AC activator - forskolin significantly stimulated both AVP and OT release from isolated rat H-NH system. Such an effect of forskolin was reduced by melatonin at concentrations of 10-9, 10-7 and 10-5 M. The strongest effect was exerted by this hormone at a concentration of 10-7 M, which inhibited not only forskolin-stimulated, but also basal, AVP and OT release. On the contrary, the highest studied concentration (i.e. 10-3 M) of melatonin stimulated both AVP and OT basal release, but when forskolin was present in the medium melatonin at such a concentration remained inactive

Inhibition of catalase activity in vitro by diesel exhaust particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

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{supmore » 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.« less

Violacein, an indole-derived purple-colored natural pigment produced by Janthinobacterium lividum, inhibits the growth of head and neck carcinoma cell lines both in vitro and in vivo.

PubMed

Masuelli, Laura; Pantanella, Fabrizio; La Regina, Giuseppe; Benvenuto, Monica; Fantini, Massimo; Mattera, Rosanna; Di Stefano, Enrica; Mattei, Maurizio; Silvestri, Romano; Schippa, Serena; Manzari, Vittorio; Modesti, Andrea; Bei, Roberto

2016-03-01

Violacein (VIO; 3-[1,2-dihydro-5-(5-hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ylidene]-1,3-dihydro-2H-indol-2-one), an indole-derived purple-colored pigment, produced by a limited number of Gram-negative bacteria species, including Chromobacterium violaceum and Janthinobacterium lividum, has been demonstrated to have anti-cancer activity, as it interferes with survival transduction signaling pathways in different cancer models. Head and neck carcinoma (HNC) represents the sixth most common and one of the most fatal cancers worldwide. We determined whether VIO was able to inhibit head and neck cancer cell growth both in vitro and in vivo. We provide evidence that VIO treatment of human and mouse head and neck cancer cell lines inhibits cell growth and induces autophagy and apoptosis. In fact, VIO treatment increased PARP-1 cleavage, the Bax/Bcl-2 ratio, the inhibition of ERK1 and ERK2 phosphorylation, and the expression of light chain 3-II (LC3-II). Moreover, VIO was able to induce p53 degradation, cytoplasmic nuclear factor kappa B (NF-κB) accumulation, and reactive oxygen species (ROS) production. VIO induced a significant increase in ROS production. VIO administration was safe in BALB/c mice and reduced the growth of transplanted salivary gland cancer cells (SALTO) in vivo and prolonged median survival. Taken together, our results indicate that the treatment of head and neck cancer cells with VIO can be useful in inhibiting in vivo and in vitro cancer cell growth. VIO may represent a suitable tool for the local treatment of HNC in combination with standard therapies.

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

Resveratrol Inhibits Development of Experimental Endometriosis In Vivo and Reduces Endometrial Stromal Cell Invasiveness In Vitro1

PubMed Central

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

2010-01-01

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. PMID:20844278

Lithium inhibits tumor lymphangiogenesis and metastasis through the inhibition of TGFBIp expression in cancer cells

PubMed Central

Maeng, Yong-Sun; Lee, Rina; Lee, Boram; Choi, Seung-il; Kim, Eung Kweon

2016-01-01

Metastasis is the main cause of mortality in cancer patients. Although there are many anti-cancer drugs targeting tumor growth, anti-metastatic agents are rarely developed. Angiogenesis and lymphangiogenesis are crucial for cancer progression; in particular, lymphangiogenesis is pivotal for metastasis in cancer. Here we report that lithium inhibits colon cancer metastasis by blocking lymphangiogenesis. Lithium reduces the expression of transforming growth factor-β-induced protein (TGFBIp) in colon cancer cells by inhibiting Smad3 phosphorylation via GSK3β inactivation. Moreover, lithium inhibits lymphatic endothelial cell migration, which is increased upon TGFBIp expression in tumor cells. Lithium had no significant effect on SW620 tumor growth in vitro and in vivo; however, it inhibited lymphangiogenesis in tumors. In tumor xenografts model, lithium was found to prevent metastasis to the lungs, liver, and lymph nodes by inhibiting TGFBIp-induced tumor lymphangiogenesis. Collectively, our findings demonstrate a novel role of lithium in the inhibition of colon cancer metastasis by blocking TGFBIp expression, and thereby TGFBIp-induced lymphangiogenesis, in primary tumors. PMID:26857144

Mink parvoviruses and interferons: in vitro studies.

PubMed Central

Wiedbrauk, D L; Bloom, M E; Lodmell, D L

1986-01-01

Although interferons can inhibit the replication of a number of viruses, little is known about their ability to inhibit parvovirus replication. Therefore, in vitro experiments were done to determine if Aleutian disease virus and mink enteritis virus, two autonomously replicating mink parvoviruses, induced interferon, were sensitive to the effects of interferon, or inhibited the production of interferon. The results indicated that these parvoviruses neither induced nor were sensitive to the effects of interferon. Furthermore, preexisting parvovirus infections did not inhibit poly(I).poly(C)-induced interferon production. This independence from the interferon system may, therefore, be a general property of the autonomously replicating parvoviruses. PMID:2431162

[Effect of S1PR2 inhibition on epithelial ovarian cancer SKOV3 cell proliferation in vitro and in vivo].

PubMed

Dai, L; Liu, Y X; Xie, L; Di, W

2018-02-25

Objective: To study the effect and mechanism of S1PR2 inhibition on epithelial ovarian cancer SKOV3 cell proliferation in vitro and in vivo. Methods: (1) A pair of S1PR2 gene small interference RNA (siRNA) , namely si-S1PR2, and a pair of negative control siRNA were designed. Western blot methods were used to detect the silence efficiency of the S1PR2 in the si-S1PR2 group, blank control group and negative control group. (2) Study in vitro: the experiment included three groups, namely si-S1PR2 group, blank control group and negative control group. Cell counting kit-8 (CCK-8) assay was used to detect the proliferation inhibition rates of the transfected cells. The cell cycles of the transfected cells were measured by flow cytometry. Western blot was used to detect the levels of phosph-extracellular regulated protein kinase 1/2 (p-ERK1/2) of the transfected cells. (3) Study in vivo:to establish intraperitoneal transplantation models, 8 mice in each group were intraperitoneally injected with 5×10(6) SKOV3 cells. Phosphate buffered saline (PBS) or JTE-013 were administered into mice twice per week starting on day 7 after the injection of the cancer cells. Twenty-eight days after nude mice intraperitoneal injection with JTE-013 or PBS, the mice were sacrificed and the number and the weight of visible tumors were calculated. Results: (1) The results of western blot showed that the relative S1PR2 protein expression levels were 0.24±0.04 in the si-S1PR2 group, which was lower than that in the blank control group (1.10±0.14, P< 0.01) and negative control group (1.07±0.13, P< 0.01) . (2) The results of CCK-8 assay indicated that after transfected for 24, 48 and 72 hours, the proliferation inhibition rate of si-S1PR2 group were respectively (26.6±3.3) %, (35.0±3.4) %, and (34.0±2.8) %, significantly lower than those in the blank control group (all 0; all P< 0.01) and negative control group [ (1.7±0.9) %, (2.5±0.5) %,and (2.4±1.1) % respectively; all P< 0.01]. The

A critical evaluation of the experimental design of studies of mechanism based enzyme inhibition, with implications for in vitro-in vivo extrapolation.

PubMed

Ghanbari, F; Rowland-Yeo, K; Bloomer, J C; Clarke, S E; Lennard, M S; Tucker, G T; Rostami-Hodjegan, A

2006-04-01

The published literature on mechanism based inhibition (MBI) of CYPs was evaluated with respect to experimental design, methodology and data analysis. Significant variation was apparent in the dilution factor, ratio of preincubation to incubation times and probe substrate concentrations used, and there were some anomalies in the estimation of associated kinetic parameters (k(inact), K(I), r). The impact of the application of inaccurate values of k(inact) and K(I) when extrapolating to the extent of inhibition in vivo is likely to be greatest for those compounds of intermediate inhibitory potency, but this also depends on the fraction of the net clearance of substrate subject to MBI and the pre-systemic and systemic exposure to the inhibitor. For potent inhibitors, the experimental procedure is unlikely to have a material influence on the maximum inhibition. Nevertheless, the bias in the values of the kinetic parameters may influence the time for recovery of enzyme activity following re-synthesis of the enzyme. Careful attention to the design of in vitro experiments to obtain accurate kinetic parameters is necessary for a reliable prediction of different aspects of the in vivo consequences of MBI. The review calls for experimental studies to quantify the impact of study design in studies of MBI, with a view to better harmonisation of protocols.

Instant coffee extract with high chlorogenic acids content inhibits hepatic G-6-Pase in vitro, but does not reduce the glycaemia.

PubMed

Bassoli, Bruna Kempfer; Cassolla, Priscila; Borba-Murad, Glaucia Regina; Constantin, Jorgete; Salgueiro-Pagadigorria, Clairce Luzia; Bazotte, Roberto Barbosa; de Souza, Helenir Medri

2015-06-01

Coffee is the main source of chlorogenic acid in the human diet, and it contains several chlorogenic acid isomers, of which the 5-caffeoylquinic acid (5-CQA) is the predominant isomer. Because there are no available data about the action of chlorogenic acids from instant coffee on hepatic glucose-6-phosphatase (G-6-Pase) activity and blood glucose levels, these effects were investigated in rats. The changes on G-6-Pase activity and liver glucose output induced by 5-CQA were also investigated. Instant coffee extract with high chlorogenic acids content (37.8%) inhibited (p < 0.05) the G-6-Pase activity of the hepatocyte microsomal fraction in a dose-dependent way (up to 53), but IV administration of this extract did not change the glycaemia (p > 0.05). Similarly, 5-CQA (1 mM) reduced (p < 0.05) the activity of microsomal G-6-Pase by about 40%, but had no effect (p > 0.05) on glucose output arising from glycogenolysis in liver perfusion. It was concluded that instant coffee extract with high content of chlorogenic acids inhibited hepatic G-6-Pase in vitro, but failed to reduce the glycaemia probably because the coffee chlorogenic acids did not reach enough levels within the hepatocytes to inhibit the G-6-Pase and reduce the liver glucose output. Copyright © 2015 John Wiley & Sons, Ltd.

Macrophage migration inhibition test in untreated syphilis.

PubMed

Bowszyc, J

1975-01-01

Two modifications of macrophage migration inhibition test, one of George and Vaughan and the other one of Svejcar, were performed on a total of 78 cases of untreated syphilis at various stages. As specific antigens were used: Treponema Pallidum ultrasonate and cardiolipin. Inhibition of migration was observed in 87 percent patients with primary syphillis and in all patients with late and late congenital syphilis. After improving and standardisation of the technique of Treponema Pallidum antigen-the migration inhibition test may be recommended as a specific in vitro-test for detection of cell mediated immunity in syphilis.

Design of a Drug-in-Adhesive Transdermal Patch for Risperidone: Effect of Drug-Additive Interactions on the Crystallization Inhibition and In Vitro/In Vivo Correlation Study.

PubMed

Weng, Wei; Quan, Peng; Liu, Chao; Zhao, Hanqing; Fang, Liang

2016-10-01

The purpose of this work was to develop and design an appropriate drug-in-adhesive patch for transdermal delivery of risperidone (RISP). Various formulation factors were investigated by in vitro permeation study using excised rabbit skin. Increasing the drug concentration in the pressure sensitive adhesive (PSA) was used to enhance the drug permeation. To overcome the high crystallization tendency of the patch, several crystallization inhibitors such as PVP, PEG, and surfactants and fatty acids were evaluated by microscopy study. The mechanism of crystallization inhibition was investigated by differential scanning calorimetry, nuclear magnetic resonance spectrometer, and FT-IR studies. RISP and its active metabolite were determined after topical application of the optimized transdermal patch, and the in vivo pharmacokinetic parameters were compared with the intravenous administration group. The microscopy study indicated that fatty acid greatly inhibited the crystallization of RISP in PSA. The inhibition was attributed to the drug-additive interaction between amino group of RISP and the carboxyl group of fatty acid which was further confirmed by (1)H-NMR and FT-IR studies. The optimal permeation profile was obtained with the patches containing 5% RISP and 5% oleic acid in Duro-Tak(®) 87-2287. The in vivo pharmacokinetic study exhibited a sustained absorption and metabolism profile and well correlated with the in vitro permeation data. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

PubMed

Tate, Courtney M; Mc Entire, Jacquelyn; Pallini, Roberto; Vakana, Eliza; Wyss, Lisa; Blosser, Wayne; Ricci-Vitiani, Lucia; D'Alessandris, Quintino Giorgio; Morgante, Liliana; Giannetti, Stefano; Larocca, Luigi Maria; Todaro, Matilde; Benfante, Antonina; Colorito, Maria Luisa; Stassi, Giorgio; De Maria, Ruggero; Rowlinson, Scott; Stancato, Louis

2015-01-01

Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

Correlation between in vitro and in vivo concentration–effect relationships of naproxen in rats and healthy volunteers

PubMed Central

Huntjens, Dymphy R H; Spalding, David J M; Danhof, Meindert; Della Pasqua, Oscar E

2006-01-01

Understanding the mechanisms underlying the analgesic effect of new cyclooxygenase inhibitors is essential to identify dosing requirements in early stages of drug development. Accurate extrapolation to humans of in vitro and in vivo findings in preclinical species is needed to optimise dosing regimen in inflammatory conditions. The current investigation characterises the inhibition of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) by naproxen in vitro and in vivo in rat and human blood. The inhibition of PGE2 in the absence or presence of increasing concentrations of naproxen (10−8–10−1 M) was measured by ex vivo whole blood stimulation with LPS, whereas inhibition of TXB2 was measured in serum following blood clotting. In further experiments, inhibition of PGE2 and TXB2 levels was also assessed ex vivo in animals treated with naproxen (2.5, 10, 25 mg kg−1). Subsequently, pharmacokinetic (PK)/pharmacodynamics (PD) modelling of in vitro and in vivo data was performed using nonlinear mixed effects in NONMEM (V). Inhibition of PGE2 and TXB2 was characterised by a sigmoid Emax model. The exposure–response relationships in vitro and in vivo were of the same order of magnitude in both species. IC80 estimates obtained in vitro were similar for PGE2 inhibition (130.8±11 and 131.9±19 10−6 M, mean±s.d. for humans and rats, respectively), but slightly different for TXB2 inhibition (103.9±15 and 151.4±40 10−6 M, mean±s.d. for humans and rats, respectively, P< 0.05). These differences, however, may not be biologically relevant. The results confirm the value of exposure–effect relationships determined in vitro as a means to predict the pharmacological activity in vivo. This analysis also highlights the need to parameterise concentration–effect relationships in early drug development, as indicated by the estimates of IC80 for PGE2 and TXB2 inhibition. PMID:16682968

Inhibition of Oxidative Stress and Lipid Peroxidation by Anthocyanins from Defatted Canarium odontophyllum Pericarp and Peel Using In Vitro Bioassays

PubMed Central

Khoo, Hock Eng; Azlan, Azrina; Ismail, Amin; Abas, Faridah; Hamid, Muhajir

2014-01-01

Canarium odontophyllum, also known as CO, is a highly nutritious fruit. Defatted parts of CO fruit are potent sources of nutraceutical. This study aimed to determine oxidative stress and lipid peroxidation effects of defatted CO pericarp and peel extracts using in vitro bioassays. Cell cytotoxic effect of the CO pericarp and peel extracts were also evaluated using HUVEC and Chang liver cell lines. The crude extracts of defatted CO peel and pericarp showed cytoprotective effects in t-BHP and 40% methanol-induced cell death. The crude extracts also showed no toxic effect to Chang liver cell line. Using CD36 ELISA, NAD+ and LDL inhibition assays, inhibition of oxidative stress were found higher in the crude extract of defatted CO peel compared to the pericarp extract. Hemoglobin and LDL oxidation assays revealed both crude extracts had significantly reduced lipid peroxidation as compared to control. TBARS values among defatted CO pericarp, peel, and cyanidin-3-glucoside showed no significant differences for hemoglobin and LDL oxidation assays. The protective effects of defatted CO parts, especially its peel is related to the presence of high anthocyanin that potentially offers as a pharmaceutical ingredient for cardioprotection. PMID:24416130

Aspirin induces apoptosis in vitro and inhibits tumor growth of human hepatocellular carcinoma cells in a nude mouse xenograft model

PubMed Central

HOSSAIN, MOHAMMAD AKBAR; KIM, DONG HWAN; JANG, JUNG YOON; KANG, YONG JUNG; YOON, JEONG-HYUN; MOON, JEON-OK; CHUNG, HAE YOUNG; KIM, GI-YOUNG; CHOI, YUNG HYUN; COPPLE, BRYAN L.; KIM, NAM DEUK

2012-01-01

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells, including colon, prostate, breast and leukemia. Among them, aspirin, a classical NSAID, shows promise in cancer therapy in certain types of cancers. We hypothesized that aspirin might affect the growth of liver cancer cells since liver is the principal site for aspirin metabolism. Therefore, we investigated the effects of aspirin on the HepG2 human hepatocellular carcinoma cell line in vitro and the HepG2 cell xenograft model in BALB/c nude mice. We found that treatment with aspirin inhibited cell growth and induced apoptosis involving both extrinsic and intrinsic pathways as measured by DNA ladder formation, alteration in the Bax/Bcl-2 ratio, activation of the caspase activities and related protein expressions. In vivo antitumor activity assay also showed that aspirin resulted in significant tumor growth inhibition compared to the control. Oral administration of aspirin (100 mg/kg/day) caused a significant reduction in the growth of HepG2 tumors in nude mice. These findings suggest that aspirin may be used as a promising anticancer agent against liver cancer. PMID:22179060

Structure-Activity–Dependent Regulation of Cell Communication by Perfluorinated Fatty Acids using in Vivo and in Vitro Model Systems

PubMed Central

Upham, Brad L.; Park, Joon-Suk; Babica, Pavel; Sovadinova, Iva; Rummel, Alisa M.; Trosko, James E.; Hirose, Akihiko; Hasegawa, Ryuichi; Kanno, Jun; Sai, Kimie

2009-01-01

Background Perfluoroalkanoates, [e.g., perfluorooctanoate (PFOA)], are known peroxisome proliferators that induce hepatomegaly and hepatocarcinogenesis in rodents, and are classic non-genotoxic carcinogens that inhibit in vitro gap-junctional intercellular communication (GJIC). This inhibition of GJIC is known to be a function of perfluorinated carbon lengths ranging from 7 to 10. Objectives The aim of this study was to determine if the inhibition of GJIC by PFOA but not perfluoropentanoate (PFPeA) observed in F344 rat liver cells in vitro also occurs in F344 rats in vivo and to determine mechanisms of PFOA dysregulation of GJIC using in vitro assay systems. Methods We used an incision load/dye transfer technique to assess GJIC in livers of rats exposed to PFOA and PFPeA. We used in vitro assays with inhibitors of cell signaling enzymes and antioxidants known to regulate GJIC to identify which enzymes regulated PFOA-induced inhibition of GJIC. Results PFOA inhibited GJIC and induced hepatomegaly in rat livers, whereas PFPeA had no effect on either end point. Serum biochemistry of liver enzymes indicated no cytotoxic response to these compounds. In vitro analysis of mitogen-activated protein kinase (MAPK) indicated that PFOA, but not PFPeA, can activate the extracellular receptor kinase (ERK). Inhibition of GJIC, in vitro, by PFOA depended on the activation of both ERK and phosphatidylcholine-specific phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism. Conclusions The in vitro analysis of GJIC, an epigenetic marker of tumor promoters, can also predict the in vivo activity of PFOA, which dysregulated GJIC via ERK and PC-PLC. PMID:19440492

A tiered approach to evaluate an iodine recycling inhibition ...

EPA Pesticide Factsheets

The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces thyroid hormone synthesis, which leads to insufficiency in tissues and subsequent negative developmental consequences. Iodide recycling by IYD is especially critical for low iodine diets and low iodine environments, including most freshwater ecosystems. We developed a putative adverse outcome pathway for IYD inhibition in amphibians and evaluated IYD inhibition with a tiered approach: 1) development of an in vitro IYD enzyme inhibition assay for chemical screening of compounds of interest to the US EPA, 2) ex vivo thyroid culture to establish thyroglobulin iodination as a biomarker of IYD inhibition, and 3) in vivo bioassays to characterize an organismal adverse outcome and test essentiality of IYD activity. An in vitro colorimetric assay was developed to measure activity of recombinant human IYD enzyme in a 96-well format, establishing the feasibility of medium to high throughput screening of chemicals for IYD inhibition. In ex vivo thyroid culture studies, thyroxine (T4), monoiodotyrosine (MIT), and diiodotyrosine (DIT) were quantified in individual thyroid glands and the media using a ultrahigh performance LC-MS/MS. In vivo exposure of developing Xenopus laevis to a suspected IYD inhibitor (3-L-nitro-tyrosine) resulted in markedly delayed metamorphosis and glandular

Quercetin protects rat dorsal root ganglion neurons against high glucose-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition.

PubMed

Shi, Yue; Liang, Xiao-chun; Zhang, Hong; Wu, Qun-li; Qu, Ling; Sun, Qing

2013-09-01

To examine the effects of quercetin, a natural antioxidant, on high glucose (HG)-induced apoptosis of cultured dorsal root ganglion (DRG) neurons of rats. DRG neurons exposed to HG (45 mmol/L) for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species (ROS) level and apoptosis were determined. The expression of NF-кB, IкBα, phosphorylated IкBα and Nrf2 was examined using RT PCR and Western blot assay. The expression of hemeoxygenase-1 (HO-1), IL-6, TNF-α, iNOS, COX-2, and caspase-3 were also examined. HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway. Co-treatment with quercetin (2.5, 5, and 10 mmol/L) dose-dependently decreased HG-induced caspase-3 activation and apoptosis. Quercetin could directly scavenge ROS and significantly increased the expression of Nrf-2 and HO-1 in DRG neurons. Quercetin also dose-dependently inhibited the NF-κB signaling pathway and suppressed the expression of iNOS, COX-2, and proinflammatory cytokines IL-6 and TNF-α. Quercetin protects rat DRG neurons against HG-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition, thus may be beneficial for the treatment of diabetic neuropathy.

Design, characterization, and in vitro cellular inhibition and uptake of optimized genistein-loaded NLC for the prevention of posterior capsular opacification using response surface methodology.

PubMed

Zhang, Wenji; Li, Xuedong; Ye, Tiantian; Chen, Fen; Sun, Xiao; Kong, Jun; Yang, Xinggang; Pan, Weisan; Li, Sanming

2013-09-15

This study was to design an innovative nanostructured lipid carrier (NLC) for drug delivery of genistein applied after cataract surgery for the prevention of posterior capsular opacification. NLC loaded with genistein (GEN-NLC) was produced with Compritol 888 ATO, Gelucire 44/14 and Miglyol 812N, stabilized by Solutol(®) HS15 by melt emulsification method. A 2(4) central composite design of 4 independent variables was performed for optimization. Effects of drug concentration, Gelucire 44/14 concentration in total solid lipid, liquid lipid concentration, and surfactant concentration on the mean particle size, polydispersity index, zeta potential and encapsulation efficiency were investigated. Analysis of variance (ANOVA) statistical test was used to assess the optimization. The optimized GEN-NLC showed a homogeneous particle size of 90.16 nm (with PI=0.33) of negatively charged surface (-25.08 mv) and high encapsulation efficiency (91.14%). Particle morphology assessed by TEM revealed a spherical shape. DSC analyses confirmed that GEN was mostly entrapped in amorphous state. In vitro release experiments indicated a prolonged and controlled genistein release for 72 h. In vitro growth inhibition assay showed an effective growth inhibition of GEN-NLCs on human lens epithelial cells (HLECs). Preliminary cellular uptake test proved a enhanced penetration of genistein into HLECs when delivered in NLC. Copyright © 2013 Elsevier B.V. All rights reserved.

Inhibition of in vitro leukotriene B4 biosynthesis in human neutrophil granulocytes and docking studies of natural quinones.

PubMed

Landa, Premysl; Kutil, Zsofia; Temml, Veronika; Malik, Jan; Kokoska, Ladislav; Widowitz, Ute; Pribylova, Marie; Dvorakova, Marcela; Marsik, Petr; Schuster, Daniela; Bauer, Rudolf; Vanek, Tomas

2013-01-01

Quinones are compounds frequently contained in medicinal plants used for the treatment of inflammatory diseases. Therefore, the impact of plant-derived quinones on the arachidonic acid metabolic pathway is worthy of investigation. In this study, twenty-three quinone compounds of plant origin were tested in vitro for their potential to inhibit leukotriene B4 (LTB4) biosynthesis in activated human neutrophil granulocytes with 5-lipoxygenase (5-LOX) activity. The benzoquinones primin (3) and thymohydroquinone (4) (IC50 = 4.0 and 4.1 microM, respectively) showed activity comparable with the reference inhibitor zileuton (1C50 = 4.1 microM). Moderate activity was observed for the benzoquinone thymoquinone (2) (1C50 = 18.2 microM) and the naphthoquinone shikonin (1) (IC50 = 24.3 microM). The anthraquinone emodin and the naphthoquinone plumbagin (5) displayed only weak activities (IC50 > 50 microM). The binding modes of the active compounds were further evaluated in silico by molecular docking to the human 5-LOX crystal structure. This process supports the biological data and suggested that, although the redox potential is responsible for the quinone's activity on multiple targets, in the case of 5-LOX the molecular structure plays a vital role in the inhibition. The obtained results suggest primin as a promising compound for the development of dual COX-2/5-LOX inhibitors.

Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

PubMed Central

Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

2015-01-01

Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

Antioxidant activity, inhibition of nitric oxide overproduction, and in vitro antiproliferative effect of maple sap and syrup from Acer saccharum.

PubMed

Legault, Jean; Girard-Lalancette, Karl; Grenon, Carole; Dussault, Catherine; Pichette, André

2010-04-01

Antioxidant activity, inhibition of nitric oxide (NO) overproduction, and antiproliferative effect of ethyl acetate extracts of maple sap and syrup from 30 producers were evaluated in regard to the period of harvest in three different regions of Québec, Canada. Oxygen radical absorbance capacity (ORAC) values of maple sap and syrup extracts are, respectively, 12 +/- 6 and 15 +/- 5 micromol of Trolox equivalents (TE)/mg. The antioxidant activity was also confirmed by a cell-based assay. The period of harvest has no statistically significant incidence on the antioxidant activity of both extracts. The antioxidant activity of pure maple syrup was also determined using the ORAC assay. Results indicate that the ORAC value of pure maple syrup (8 +/- 2 micromol of TE/mL) is lower than the ORAC value of blueberry juice (24 +/- 1 micromol of TE/mL) but comparable to the ORAC values of strawberry (10.7 +/- 0.4 micromol of TE/mL) and orange (10.8 +/- 0.5 micromol of TE/mL) juices. Maple sap and syrup extracts showed to significantly inhibit lipopolysaccharide-induced NO overproduction in RAW264.7 murine macrophages. Maple syrup extract was significantly more active than maple sap extract, suggesting that the transformation of maple sap into syrup increases NO inhibition activity. The highest NO inhibition induced by the maple syrup extracts was observed at the end of the season. Moreover, darker maple syrup was found to be more active than clear maple syrup, suggesting that some colored oxidized compounds could be responsible in part for the activity. Finally, maple syrup extracts (50% inhibitory concentration = 42 +/- 6 microg/mL) and pure maple syrup possess a selective in vitro antiproliferative activity against cancer cells.

Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE) for 5α-reductase isoform II inhibition using a cell-free in vitro test system.

PubMed

Pais, Pilar; Villar, Agustí; Rull, Santiago

2016-01-01

The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen - 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE) has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive - some have shown significant results, and others have not - possibly the result of varying bioactivities of the SPEs used in the studies. To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE), an inhibitor of the 5α-reductase isoenzyme type II. The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 μg/mL), SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%-75% inhibition of 5α-reductase type II. SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The bioactivity of SPSE corresponds favorably to that reported for the hexane extract used in a large number of positive BPH clinical trials, as well as to finasteride

Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE) for 5α-reductase isoform II inhibition using a cell-free in vitro test system

PubMed Central

Pais, Pilar; Villar, Agustí; Rull, Santiago

2016-01-01

Background The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen – 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE) has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive – some have shown significant results, and others have not – possibly the result of varying bioactivities of the SPEs used in the studies. Purpose To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE), an inhibitor of the 5α-reductase isoenzyme type II. Materials and methods The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. Results By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 μg/mL), SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%–75% inhibition of 5α-reductase type II. Conclusion SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The bioactivity of SPSE corresponds favorably to that reported for the hexane extract used in a large

Xanthine oxidase inhibiting effects of noni (Morinda citrifolia) fruit juice.

PubMed

Palu, Afa; Deng, Shixin; West, Brett; Jensen, Jarakae

2009-12-01

Morinda citrifolia L. (noni), family Rubiaceae, has been used in Polynesia for over 2000 years for its reputed health benefits, one of which is its therapeutic effects on gout (langa e hokotanga hui). However, its healing mechanism has not been elucidated. This study showed that in an in vitro bioassay that Tahitian Noni Juice (TNJ) inhibited xanthine oxidase (XO) concentration dependently. Concentrations of 1, 5 and 10 mg/mL of TNJ inhibited XO by 11%, 113% and 148%, respectively, with an IC50 of 3.8 mg compared with an IC50 of 2.4 microm for allopurinol. Noni fruit juice concentrate (NFJC) also inhibited XO concentration dependently. Concentrations of 1 and 5 mg/mL NFJC inhibited XO in vitro by 184% and 159%, respectively. A 0.1 mg/mL methanol extract (NFJME) from the fractionation of noni fruit puree inhibited XO by 64%. It was elucidated that the noni fruit juice inhibitory effect on XO enzymes is the mechanism by which noni ameliorates gout and gout-like diseases. Further, the results also support the traditional usage of noni in the treatment of gout. Copyright (c) 2009 John Wiley & Sons, Ltd.

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

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.

Biodegradable Hydrophilic Polyurethane PEGU25 Loading Antimicrobial Peptide Bmap-28: A Sustained-release Membrane Able to Inhibit Bacterial Biofilm Formation in Vitro

PubMed Central

Wang, Jianzhong; Liu, Qinyu; Tian, Ye; Jian, Zhongyu; Li, Hong; Wang, Kunjie

2015-01-01

Catheter-related infection makes up a large part of hospital infection and contributes 80% to all nosocomial urological infection, costing hundreds of millions dollar per year for treatment. Biodegradable hydrophilic material incorporating antibiotic substance is a promising way to prevent catheter-related infection. And antimicrobial peptide seems an optimal drug for its desirable antibiotic effect. In the current research, we produced a new kind of antibiotic material by incorporating antimicrobial peptide Bmap-28 with polyurethane PEGU25 and tested its effect on Proteus mirabilis in vitro. Compared with the control group, PEGU25 membrane incorporating Bmap-28 had a significant lower bacteria load after co-cultured with the Proteus mirabilis. And its antibiotic effect could be observed throughout the whole 7-day test. Also the Bmap-28 membrane could delay catheter obstruction caused by encrustation. Our findings reveal that PEGU25 incorporating Bmap-28 can well inhibit bacterial biofilm formation of common pathogens for catheter-related urinary tract infection in vitro, which makes it a promising antibiotic material for medical tubes for urology. PMID:25727362

Lactucaxanthin - a potential anti-diabetic carotenoid from lettuce (Lactuca sativa) inhibits α-amylase and α-glucosidase activity in vitro and in diabetic rats.

PubMed

Gopal, Sowmya Shree; Lakshmi, Magisetty Jhansi; Sharavana, Gurunathan; Sathaiah, Gunaseelan; Sreerama, Yadahally N; Baskaran, Vallikannan

2017-03-22

Intestinal and pancreatic α-amylase and α-glucosidase inhibitors offer an approach to lower the levels of post-prandial hyperglycemia through the control of dietary starch breakdown in digestion. This study hypothesized that lactucaxanthin (Lxn) in lettuce (Lactuca sativa) inhibits the activity of α-amylase and α-glucosidase. In this study, the interaction of Lxn with α-amylase and α-glucosidase in silico and its inhibitory effect on these enzymes were studied using in vitro and STZ-induced diabetic rat models. Lxn was isolated from lettuce with 96% purity confirmed by HPLC and LCMS. The in silico analysis showed that Lxn has a lower binding energy (-6.05 and -6.34 kcal mol -1 ) with α-amylase and α-glucosidase compared to their synthetic inhibitors, acarbose (-0.21 kcal mol -1 ) and miglitol (-2.78 kcal mol -1 ), respectively. In vitro α-amylase and α-glucosidase inhibition assays revealed that Lxn had IC 50 values of 435.5 μg mL -1 and 1.84 mg mL -1 , but acarbose has values of 2.5 and 16.19 μg mL -1 . The in vivo results showed an increased activity for α-amylase and α-glucosidase in the intestine (4.7 and 1.30 fold, p < 0.05) and pancreas (1.3 and 1.48 fold, p < 0.05) of STZ induced diabetic rats compared to normal rats. Whereas the activity decreased (p < 0.05) in the Lxn fed diabetic rats, except for the intestinal α-glucosidase activity (1.69 ± 0.12 PNP per min per mg protein). This was confirmed by the low blood glucose level (239.4 ± 18.2 mg dL -1 ) in diabetic rats fed Lxn compared to the diabetic group (572.2 ± 30.5 mg dL -1 , p < 0.05). Lxn significantly inhibited (p < 0.05) the activity of α-amylase and α-glucosidase and could be of medical and nutritional relevance in the treatment of diabetes.

Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies

PubMed Central

2012-01-01

Background Activated mast cells are present within airway smooth muscle (ASM) bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide), long-acting β2-agonists (salmeterol, formoterol) and thiazolidinediones (ciglitazone, rosiglitazone) on CXCL10 production by ASM cells (ASMC) from people with and without asthma were investigated in vitro. Methods Confluent serum-deprived cells were treated with the agents before and during cytokine stimulation for 0-24 h. CXCL10 protein/mRNA, IκB-α levels and p65 activity were measured using ELISA, RT PCR, immunoblotting and p65 activity assays respectively. Data were analysed using ANOVA followed by Fisher’s post-hoc test. Results Fluticasone and/or salmeterol at 1 and 100 nM inhibited CXCL10 release induced by IL-1β and TNF-α, but not IFNγ or all three cytokines (cytomix). The latter was also not affected by budesonide and formoterol. In asthmatic ASMC low salmeterol, but not formoterol, concentrations increased cytomix-induced CXCL10 release and at 0.01 nM enhanced NF-κB activity. Salmeterol 0.1nM together with fluticasone 0.1 and 10 nM still increased CXCL10 release. The thiazolidinediones ciglitazone and rosiglitazone (at 25 and 100 μM) inhibited cytomix-induced CXCL10 release but these inhibitory effects were not prevented by the PPAR-g antagonist GW9662. Ciglitazone did not affect early NF-κB activity and CXCL10 mRNA production. Conclusions Thus the thiazolidinediones inhibited asthmatic ASMC CXCL10 release under conditions when common asthma therapies were ineffective or enhanced it. They may provide an alternative strategy to reduce mast cell-ASM interactions and restore normal airway physiology in asthma. PMID:23034049

Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies.

PubMed

Seidel, Petra; Alkhouri, Hatem; Lalor, Daniel J; Burgess, Janette K; Armour, Carol L; Hughes, J Margaret

2012-10-04

Activated mast cells are present within airway smooth muscle (ASM) bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide), long-acting β2-agonists (salmeterol, formoterol) and thiazolidinediones (ciglitazone, rosiglitazone) on CXCL10 production by ASM cells (ASMC) from people with and without asthma were investigated in vitro. Confluent serum-deprived cells were treated with the agents before and during cytokine stimulation for 0-24 h. CXCL10 protein/mRNA, IκB-α levels and p65 activity were measured using ELISA, RT PCR, immunoblotting and p65 activity assays respectively. Data were analysed using ANOVA followed by Fisher's post-hoc test. Fluticasone and/or salmeterol at 1 and 100 nM inhibited CXCL10 release induced by IL-1β and TNF-α, but not IFNγ or all three cytokines (cytomix). The latter was also not affected by budesonide and formoterol. In asthmatic ASMC low salmeterol, but not formoterol, concentrations increased cytomix-induced CXCL10 release and at 0.01 nM enhanced NF-κB activity. Salmeterol 0.1 nM together with fluticasone 0.1 and 10 nM still increased CXCL10 release. The thiazolidinediones ciglitazone and rosiglitazone (at 25 and 100 μM) inhibited cytomix-induced CXCL10 release but these inhibitory effects were not prevented by the PPAR-g antagonist GW9662. Ciglitazone did not affect early NF-κB activity and CXCL10 mRNA production. Thus the thiazolidinediones inhibited asthmatic ASMC CXCL10 release under conditions when common asthma therapies were ineffective or enhanced it. They may provide an alternative strategy to reduce mast cell-ASM interactions and restore normal airway physiology in asthma.

Clotrimazole and econazole inhibit Streptococcus mutans biofilm and virulence in vitro.

PubMed

Qiu, Wei; Ren, Biao; Dai, Huanqin; Zhang, Lixin; Zhang, Qiong; Zhou, Xuedong; Li, Yuqing

2017-01-01

The aim of this study was to determine the inhibitory effect of eight antifungal drugs on S. mutans growth, biofilm formation and virulence factors. The actions of antifungal drugs on S. mutans were determined by recovery plates and survival kinetic curves. Biofilms were observed by scanning electron microscopy and the viable cells were recovered on BHI plates, meanwhile biofilms were stained by BacLight live/dead kit to investigate the biofilm viability. Bacteria/extracellular polysaccharides staining assays were performed to determine the EPS production of S. mutans biofilms. Acidogenicity and acidurity of S. mutans were determined using pH drop and acid tolerance assays, and the expression of ldh gene was evaluated using qPCR. We found that clotrimazole (CTR) and econazole (ECO) showed antibacterial activities on S. mutans UA159 and S. mutans clinical isolates at 12.5 and 25mg/L, respectively. CTR and ECO could also inhibit S. mutans biofilm formation and reduce the viability of preformed biofilm. CTR and ECO affected the live/dead ratio and the EPS/bacteria ratio of S. mutans biofilms. CTR and ECO also inhibited the pH drop, lactate acid production, and acid tolerance. The abilities of CTR and ECO to inhibit S. mutans ldh expression were also confirmed. We found that two antifungal azoles, CTR and ECO, had the abilities to inhibit the growth and biofilm formation of S. mutans and more importantly, they could also inhibit the virulence factors of S. mutans. Copyright © 2016 Elsevier Ltd. All rights reserved.

[INFLUENCE OF INHIBITION OF ACTIN POLYMERIZATION ON ADIPOGENIC DIFFERENTIATION OF RAT Achilles-DERIVED TENDON STEM CELLS IN VITRO].

PubMed

Chen, Bo; Tang, Kanglai; Zhang, Jiqiang; Guo, Yupeng; Liu, Xiangzhou; Shi, Youxin

2015-02-01

To investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/ soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor y (PPARγ), lipoprotein lipase (LPL), and fatty acid binding protein (aP2). The final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P < 0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary

Inhibition of polo-like kinase 1 leads to the suppression of osteosarcoma cell growth in vitro and in vivo.

PubMed

Liu, Xianzhe; Choy, Edwin; Harmon, David; Yang, Shuhua; Yang, Cao; Mankin, Henry; Hornicek, Francis J; Duan, Zhenfeng

2011-06-01

Osteosarcoma is the most common type of primary bone cancer in children and adolescents. Treatment options for osteosarcoma may include surgery, chemotherapy, and radiotherapy. Unfortunately, many patients eventually relapse, resulting in an unsatisfactory outcome. The serine/threonine-specific polo-like kinase 1 (PLK1) is a kinase that plays an important role in mitosis and the maintenance of genomic stability. PLK1 has been found to be highly expressed in the malignant cells of osteosarcoma. Here, we describe the in-vitro and in-vivo effects of BI 2536, a small-molecule inhibitor of PLK1, which through inhibiting PLK1 enzymatic activity, causes mitotic arrest and eventually induces cancer cell apoptosis. In this study, we show that the PLK1 inhibitor, BI 2536, inhibits proliferation and induces apoptosis in two-dimensional and three-dimensional cultures of osteosarcoma cell lines, KHOS and U-2OS. A proliferation assay performed both in two-dimensional and three-dimensional culture showed that the growth of both cell lines was inhibited by BI 2536. Cell cycle analysis showed that the cells treated with BI 2536 were mainly arrested in the G2/M phase. Immunofluorescence and western blotting analysis confirmed that the administration of BI 2536 led to significant decrease of PLK1 and Mcl-1 protein expression levels in dose-dependent and time-dependent manners. Furthermore, BI 2536-induced apoptosis in the osteosarcoma cell lines was shown by poly (ADP-ribose) polymerase cleavage and caspase assay. Finally, in mouse osteosarcoma xenografts, BI 2536-treated mice had significantly smaller tumors compared with the control mice. These findings offer evidence of the potential role for targeting PLK1 in osteosarcoma therapy.

[Effects of cornel iridoid glycoside on activity of cholinesterases in vitro].

PubMed

Chu, Si-Juan; Zhang, Lan; Liu, Gang; Zhou, Wen-Xia; Li, Lin

2013-05-01

The purpose of the present study was to investigate the effects of cornel iridoid glycoside (CIG) on the activity of cholinesterases in vitro, and to investigate the mechanism of CIG's treating Alzheimer's disease (AD). The sources of cholinesterases were prepared from human blood cells, rat brain homogenate and human blood plasma, respectively. The biochemical methods were used to detect the activity of acetylcholine esterase (AChE) and butyryl cholinesterase (BuChE) to investigate the influence of CIG on cholinesterases. The results showed that CIG inhibited the activity of AChE of human blood cells and rat brain homogenate, with the 50% inhibition rate (IC50) of 1.6 g . L-1 and 3.3 g . L-1, respectively; and the inhibition of AChE of CIG is reversible. CIG also inhibited the activity of BuChE of human blood plasma, with the IC50 of 2.9 g . L-1. In conclusion, CIG can inhibit the activity of AChE and BuChE in vitro, which may be one of the mechanisms of CIG to treat AD.

Atorvastatin inhibits insulin synthesis by inhibiting the Ras/Raf/ERK/CREB pathway in INS-1 cells

PubMed Central

Sun, Hongxi; Li, Yu; Sun, Bei; Hou, Ningning; Yang, Juhong; Zheng, Miaoyan; Xu, Jie; Wang, Jingyu; Zhang, Yi; Zeng, Xianwei; Shan, Chunyan; Chang, Bai; Chen, Liming; Chang, Baocheng

2016-01-01

pathway were inhibited by 50 μM atorvastatin in INS-1 cells in vitro. Moreover, 50 μM atorvastatin reduced the binding of p-CREB with deoxyribonucleic acid (DNA) in INS-1 cells in vitro. Conclusion: Atorvastatin inhibits insulin synthesis in beta cells by inhibiting the activation of the Ras complex pathway. PMID:27684825

Glycine inhibits melanogenesis in vitro and causes hypopigmentation in vivo.

PubMed

Ishikawa, Masago; Kawase, Ichiro; Ishii, Fumio

2007-11-01

The simplest amino acid, glycine, is important in protein composition and plays a significant role in numerous physiological events in mammals. Despite the inhibitory effect of glycine on spontaneous melanogenesis in B16F0 melanoma cells, the details of the underlying mechanisms remain unknown. The present study was conducted to investigate the further effects and the mechanisms of inhibitory effect of glycine on melanogenesis using B16F0 melanoma cells and hair follicle melanogenesis in C57BL/6J mice. Treatment with glycine (1-16 mM) for 72 h inhibited alpha-melanocyte stimulating hormone (alpha-MSH)-induced melanogenesis in a concentration-dependent manner without any effects on cell proliferation in B16F0 melanoma cells. Treatment with kojic acid (2.5 mM) for 72 h also inhibited alpha-MSH-induced melanogenesis in B16F0 melanoma cells. The highest dose of glycine inhibited the alpha-MSH-induced increment of tyrosinase protein levels in B16F0 melanoma cells. In hair follicle melanogenesis in C57BL/6J mice, treatment with glycine (1250 or 2500 mg/kg, i.p.) for 5 d prevented the decrement of L* and C* values and inhibited the increment of tyrosinase protein levels and melanin content within the skin. Treatment with hydroquinone (100 mg/kg, i.p.) for 5 d had a similar hypopigmenting effect to that of high dose glycine. These results suggest that glycine has an inhibitory effect on melanogenesis that is mediated by down-regulation of tyrosinase protein levels, leading to a hypopigmenting effect in C57BL/6J mice.

Caspase Inhibition Prevents Tumor Necrosis Factor-α-Induced Apoptosis and Promotes Necrotic Cell Death in Mouse Hepatocytes in Vivo and in Vitro.

PubMed

Ni, Hong-Min; McGill, Mitchell R; Chao, Xiaojuan; Woolbright, Benjamin L; Jaeschke, Hartmut; Ding, Wen-Xing

2016-10-01

How different cell death modes and cell survival pathways cross talk remains elusive. We determined the interrelation of apoptosis, necrosis, and autophagy in tumor necrosis factor (TNF)-α/actinomycin D (ActD) and lipopolysaccharide/D-galactosamine (GalN)-induced hepatotoxicity in vitro and in vivo. We found that TNF-α/ActD-induced apoptosis was completely blocked by a general caspase inhibitor ZVAD-fmk at 24 hours but hepatocytes still died by necrosis at 48 hours. Inhibition of caspases also protected mice against lipopolysaccharide/GalN-induced apoptosis and liver injury at the early time point, but this protection was diminished after prolonged treatment by switching apoptosis to necrosis. Inhibition of receptor-interacting protein kinase (RIP)1 by necrostatin 1 partially inhibited TNF-α/ZVAD-induced necrosis in primary hepatocytes. Pharmacologic inhibition of autophagy or genetic deletion of Atg5 in hepatocytes did not protect against TNF-α/ActD/ZVAD-induced necrosis. Moreover, pharmacologic inhibition of RIP1 or genetic deletion of RIP3 failed to protect and even exacerbated liver injury after mice were treated with lipopolysaccharide/GalN and a pan-caspase inhibitor. In conclusion, our results suggest that different cell death mode and cell survival pathways are closely integrated during TNF-α-induced liver injury when both caspases and NF-κB are blocked. Moreover, results from our study also raised concerns about the safety of currently ongoing clinical trials that use caspase inhibitors. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

In vitro kinetic interactions of DEET, pyridostigmine and organophosphorus pesticides with human cholinesterases.

PubMed

Wille, Timo; Thiermann, Horst; Worek, Franz

2011-04-25

The simultaneous use of the repellent DEET, pyridostigmine, and organophosphorus pesticides has been assumed as a potential cause for the Gulf War Illness and combinations have been tested in different animal models. However, human in vitro data on interactions of DEET with other compounds are scarce and provoked the present in vitro study scrutinizing the interactions of DEET, pyridostigmine and pesticides with human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE). DEET showed to be a weak and reversible inhibitor of hAChE and hBChE. The IC(50) of DEET was calculated to be 21.7mM DEET for hAChE and 3.2mM DEET for hBChE. The determination of the inhibition kinetics of pyridostigmine, malaoxon and chlorpyrifos oxon with hAChE in the presence of 5mM DEET resulted in a moderate reduction of the inhibition rate constant k(i). The decarbamoylation velocity of pyridostigmine-inhibited hAChE was not affected by DEET. In conclusion, the in vitro investigation of interactions between human cholinesterases, DEET, pyridostigmine, malaoxon and chlorpyrifos oxon showed a weak inhibition of hAChE and hBChE by DEET. The inhibitory potency of the tested cholinesterase inhibitors was not enhanced by DEET and it did not affect the regeneration velocity of pyridostigmine-inhibited AChE. Hence, this in vitro study does not give any evidence of a synergistic effect of the tested compounds on human cholinesterases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

In vitro modification of Candida albicans invasiveness.

PubMed

Fontenla de Petrino, S E; de Jorrat, M E; Sirena, A; Valdez, J C; Mesón, O

1986-05-01

Candida albicans produces germ-tubes (GT) when it is incubated in animal or human serum. This dimorphism is responsible for its invasive ability. The purpose of the present paper is (1) to evaluate the ability of rat peritoneal macrophages to inhibit GT production of ingested Candida albicans, obtained from immunized rats and then activated in vitro with Candida-induced lymphokines; (2) to determinate any possible alteration of phagocytic and candidacidal activities. The phagocytes were obtained from rats immunized with viable C. albicans. Some of them were exposed to Candida-induced lymphokines in order to activate the macrophages in vitro. The monolayers of activated, immune and normal macrophages were infected with a C. albicans suspension during 4 hr. Activated macrophages presented not only the highest phagocytic and candidacidal activities but a noticeable inhibition of GT formation and incremented candidacidal activity.

Lipid-Lowering Pharmaceutical Clofibrate Inhibits Human Sweet Taste

PubMed Central

Kochem, Matthew

2017-01-01

T1R2-T1R3 is a heteromeric receptor that binds sugars, high potency sweeteners, and sweet taste blockers. In rodents, T1R2-T1R3 is largely responsible for transducing sweet taste perception. T1R2-T1R3 is also expressed in non-taste tissues, and a growing body of evidence suggests that it helps regulate glucose and lipid metabolism. It was previously shown that clofibric acid, a blood lipid-lowering drug, binds T1R2-T1R3 and inhibits its activity in vitro. The purpose of this study was to determine whether clofibric acid inhibits sweetness perception in humans and is, therefore, a T1R2-T1R3 antagonist in vivo. Fourteen participants rated the sweetness intensity of 4 sweeteners (sucrose, sucralose, Na cyclamate, acesulfame K) across a broad range of concentrations. Each sweetener was prepared in solution neat and in mixture with either clofibric acid or lactisole. Clofibric acid inhibited sweetness of every sweetener. Consistent with competitive binding, inhibition by clofibric acid was diminished with increasing sweetener concentration. This study provides in vivo evidence that the lipid-lowering drug clofibric acid inhibits sweetness perception and is, therefore, a T1R carbohydrate receptor inhibitor. Our results are consistent with previous in vitro findings. Given that T1R2-T1R3 may in part regulate glucose and lipid metabolism, future studies should investigate the metabolic effects of T1R inhibition. PMID:27742692

Red Pepper (Capsicum baccatum) Extracts Present Anti-Inflammatory Effects In Vivo and Inhibit the Production of TNF-α and NO In Vitro.

PubMed

Allemand, Alexandra; Leonardi, Bianca Franco; Zimmer, Aline Rigon; Moreno, Susana; Romão, Pedro Roosevelt Torres; Gosmann, Grace

2016-08-01

Capsicum baccatum is the most consumed red pepper species in Brazil. Our previous studies demonstrated the anti-inflammatory properties of its crude extract, whose activity is yet to be fully characterized. Herein, we examined the anti-inflammatory in vivo effects of enriched extracts obtained through bioguided fractionation as dichloromethane (DCM), butanol (BUT), and residual aqueous (RAq) extracts and its influence on inflammatory mediators produced by macrophages in vitro. We demonstrated that all C. baccatum extracts presented anti-inflammatory activity in vivo. In addition, we showed that BUT and RAq were more effective in inhibiting the neutrophil migration induced by carrageenan (Cg) to peritoneal cavity and both extracts inhibited paw edema induced by Cg, prostaglandin E2, and histamine in mice. Furthermore, the pretreatment with C. baccatum extracts significantly reduced the levels of myeloperoxidase (MPO) in the paw tissues of mice compared with the carrageenan group. Once again, RAq and BUT caused the greatest reduction in MPO levels. Moreover, it was demonstrated for the first time that C. baccatum inhibited the nitric oxide and tumor necrosis factor-alpha production by lipopolysaccharide/interferon gamma (IFN-γ)-stimulated macrophages. These anti-inflammatory effects seem to be at least, in part, independent of capsaicin. Hence, red pepper has bioactive compounds and might be used to develop food-derived extracts to treat related inflammatory diseases.

Inhibition of thrombin by functionalized C60 nanoparticles revealed via in vitro assays and in silico studies.

PubMed

Liu, Yanyan; Fu, Jianjie; Pan, Wenxiao; Xue, Qiao; Liu, Xian; Zhang, Aiqian

2018-01-01

The studies on the human toxicity of nanoparticles (NPs) are far behind the rapid development of engineered functionalized NPs. Fullerene has been widely used as drug carrier skeleton due to its reported low risk. However, different from other kinds of NPs, fullerene-based NPs (C 60 NPs) have been found to have an anticoagulation effect, although the potential target is still unknown. In the study, both experimental and computational methods were adopted to gain mechanistic insight into the modulation of thrombin activity by nine kinds of C 60 NPs with diverse surface chemistry properties. In vitro enzyme activity assays showed that all tested surface-modified C 60 NPs exhibited thrombin inhibition ability. Kinetic studies coupled with competitive testing using 3 known inhibitors indicated that six of the C 60 NPs, of greater hydrophobicity and hydrogen bond (HB) donor acidity or acceptor basicity, acted as competitive inhibitors of thrombin by directly interacting with the active site of thrombin. A simple quantitative nanostructure-activity relationship model relating the surface substituent properties to the inhibition potential was then established for the six competitive inhibitors. Molecular docking analysis revealed that the intermolecular HB interactions were important for the specific binding of C 60 NPs to the active site canyon, while the additional stability provided by the surface groups through van der Waals interaction also play a key role in the thrombin binding affinity of the NPs. Our results suggest that thrombin is a possible target of the surface-functionalized C 60 NPs relevant to their anticoagulation effect. Copyright © 2017. Published by Elsevier B.V.

MicroRNA-494 inhibits cell proliferation and invasion of chondrosarcoma cells in vivo and in vitro by directly targeting SOX9.

PubMed

Li, Jingyuan; Wang, Lijuan; Liu, Zongzhi; Zu, Chao; Xing, Fanfan; Yang, Pei; Yang, Yongkang; Dang, Xiaoqian; Wang, Kunzheng

2015-09-22

Accumulating evidence indicates that dysregulation of miRNAs could contribute to tumor growth and metastasis of chondrosarcoma by infuencing cell proliferation and invasion. In the current study, we are interested to examine the role of miRNAs in the carcinogenesis and progression of chondrosarcoma. Here, using comparative miRNA profiling of tissues and cells of chondrosarcoma and cartilage, we identified miR-494 as a commonly downregulated miRNA in the tissues of patients with chondrosarcoma and chondrosarcoma cancer cell line, and upregulation of miR-494 could inhibit proliferation and invasion of chondrosarcoma cancer cells in vivo and in vitro. Moreover, our data demonstrated that SOX9, the essential regulator of the process of cartilage differentiation, was the direct target and functional mediator of miR-494 in chondrosarcoma cells. And downregulation of SOX9 could also inhibit migration and invasion of chondrosarcoma cells. In the last, we identified low expression of miR-494 was significantly correlated with poor overall survival and prognosis of chondrosarcoma patients. Thus, miR-494 may be a new common therapeutic target and prognosis biomarker for chondrosarcoma.

MicroRNA-494 inhibits cell proliferation and invasion of chondrosarcoma cells in vivo and in vitro by directly targeting SOX9

PubMed Central

Liu, Zongzhi; Zu, Chao; Xing, Fanfan; Yang, Pei; Yang, Yongkang; Dang, Xiaoqian; Wang, Kunzheng

2015-01-01

Accumulating evidence indicates that dysregulation of miRNAs could contribute to tumor growth and metastasis of chondrosarcoma by infuencing cell proliferation and invasion. In the current study, we are interested to examine the role of miRNAs in the carcinogenesis and progression of chondrosarcoma. Here, using comparative miRNA profiling of tissues and cells of chondrosarcoma and cartilage, we identified miR-494 as a commonly downregulated miRNA in the tissues of patients with chondrosarcoma and chondrosarcoma cancer cell line, and upregulation of miR-494 could inhibit proliferation and invasion of chondrosarcoma cancer cells in vivo and in vitro. Moreover, our data demonstrated that SOX9, the essential regulator of the process of cartilage differentiation, was the direct target and functional mediator of miR-494 in chondrosarcoma cells. And downregulation of SOX9 could also inhibit migration and invasion of chondrosarcoma cells. In the last, we identified low expression of miR-494 was significantly correlated with poor overall survival and prognosis of chondrosarcoma patients. Thus, miR-494 may be a new common therapeutic target and prognosis biomarker for chondrosarcoma. PMID:26317788

Inhibition of angiogenesis in vitro by Arg-Gly-Asp-containing synthetic peptide.

PubMed Central

Nicosia, R. F.; Bonanno, E.

1991-01-01

This study was designed to evaluate the effect of the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) on angiogenesis in serum-free collagen gel culture of rat aorta. The GRGDS peptide contains the amino acid sequence Arg-Gly-Asp (RGD), which has been implicated as a recognition site in interactions between extracellular matrix (ECM) molecules and cell membrane receptors. RGD-containing synthetic peptides are known to inhibit attachment of endothelial cells to substrates, but their effect on angiogenesis has not been fully characterized. Aortic explants embedded in collagen gel in the absence of GRGDS generated branching microvessels through a process of endothelial migration and proliferation. Addition of GRGDS to the culture medium caused a marked inhibition of angiogenesis. In contrast, GRGES, a control peptide lacking the RGD sequence, failed to inhibit angiogenesis. The inhibitory effect of GRGDS was nontoxic and reversible. The angiogenic activity of aortic explants previously inhibited with GRGDS could be restored by incubating the cultures in GRGDS-free medium. These findings suggest that angiogenesis is an anchorage-dependent process that can be inhibited by interfering with the attachment of endothelial cells to the ECM. It also indicates that synthetic peptides can be used as probes to study the mechanisms by which the ECM regulates angiogenesis. Images Figure 1 PMID:1707235

Stable knockdown of LRG1 by RNA interference inhibits growth and promotes apoptosis of glioblastoma cells in vitro and in vivo.

PubMed

Zhong, Di; Zhao, Siren; He, Guangxu; Li, Jinku; Lang, Yanbin; Ye, Wei; Li, Yongli; Jiang, Chuanlu; Li, Xianfeng

2015-06-01

Leucine-rich α2 glycoprotein 1 (LRG1) has been shown to be aberrantly expressed in multiple human malignancies. However, the biological functions of LRG1 in human glioblastoma remain unknown. Here, we report for the first time the role of LRG1 in glioblastoma development based on the preliminary in vitro and in vivo data. We first confirmed the expression of LRG1 in human glioblastoma cell lines. Next, to investigate the role of LRG1 in the tumorigenesis and development of glioblastoma, a short hairpin RNA (shRNA) construct targeting LRG1 mRNA was transfected into U251 glioblastoma cells to generate a cell line with stably silenced LRG1 expression. The results showed that silencing of LRG1 significantly inhibited cell proliferation, induced cell cycle arrest at G0/G1 phase, and enhanced apoptosis in U251 cells in vitro. Consistently, LRG1 silencing resulted in the downregulation of key cell cycle factors including cyclin D1, B, and E and apoptotic gene Bcl-2 while elevated the levels of pro-apoptotic Bax and cleaved caspase-3, as determined by Western blot analysis. We further demonstrate that the silencing of LRG1 expression effectively reduced the tumorigenicity of U251 cells, delayed tumor formation, and promoted apoptosis in a xenograft tumor model in vivo. In conclusion, silencing the expression of LRG1 suppresses the growth of glioblastoma U251 cells in vitro and in vivo, suggesting that LRG1 may play a critical role in glioblastoma development, and it may have potential clinical implications in glioblastoma therapy.

Identification of a Phosphodiesterase-Inhibiting Fraction from Roasted Coffee (Coffea arabica) through Activity-Guided Fractionation.

PubMed

Röhrig, Teresa; Liesenfeld, David; Richling, Elke

2017-05-17

Recent reports that coffee can significantly inhibit cAMP phosphodiesterases (PDEs) in vitro, as well as in vivo, have described another beneficial effect of coffee consumption. However, the PDE-inhibiting substances remain mostly unknown. We chose activity-guided fractionation and an in vitro test system to identify the coffee components that are responsible for PDE inhibition. This approach indicated that a fraction of melanoidins reveals strong PDE-inhibiting potential (IC 50 = 130 ± 42 μg/mL). These melanoidins were characterized as water-soluble, low-molecular weight melanoidins (<3 kDa) with a nitrogen content of 4.2% and a carbohydrate content lower than those of other melanoidins. Fractions containing known PDE inhibitors such as chlorogenic acids, alkylpyrazines, or trigonelline as well as N-caffeoyl-tryptophan and N-p-coumaroyl-tryptophan did not exert PDE-inhibiting activity. We also observed that the known PDE inhibitor caffeine does not contribute to the PDE-inhibiting effects of coffee.

In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herraez, Elisa; Macias, Rocio I.R.; Vazquez-Tato, Jose

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 TCAmore » 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.« less

Recent advances in evaluation of oxime efficacy in nerve agent poisoning by in vitro analysis.

PubMed

Worek, F; Eyer, P; Aurbek, N; Szinicz, L; Thiermann, H

2007-03-01

The availability of highly toxic organophosphorus (OP) warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE). Reactivators (oximes) of inhibited AChE are a mainstay of treatment, however, the commercially available compounds, obidoxime and pralidoxime, are considered to be rather ineffective against various nerve agents, e.g. soman and cyclosarin. This led to the synthesis and investigation of numerous oximes in the past decades. Reactivation of OP-inhibited AChE is considered to be the most important reaction of oximes. Clinical data from studies with pesticide-poisoned patients support the assumption that the various reactions between AChE, OP and oxime, i.e. inhibition, reactivation and aging, can be investigated in vitro with human AChE. In contrast to animal experiments such in vitro studies with human tissue enable the evaluation of oxime efficacy without being affected by species differences. In the past few years numerous in vitro studies were performed by different groups with a large number of oximes and methods were developed for extrapolating in vitro data to different scenarios of human nerve agent poisoning. The present status in the evaluation of new oximes as antidotes against nerve agent poisoning will be discussed.

Bacterial inhibition potential of 3D rapid-prototyped magnesium-based porous composite scaffolds–an in vitro efficacy study

PubMed Central

Ma, Rui; Lai, Yu-xiao; Li, Long; Tan, Hong-lue; Wang, Jia-li; Li, Ye; Tang, Ting-ting; Qin, Ling

2015-01-01

Bone infections are common in trauma-induced open fractures with bone defects. Therefore, developing anti-infection scaffolds for repairing bone defects is desirable. This study develoepd novel Mg-based porous composite scaffolds with a basal matrix composed of poly(lactic-co-glycolicacid) (PLGA) and tricalcium phosphate (TCP). A unique low-temperature rapid prototyping technology was used to fabricate the scaffolds, including PLGA/TCP (PT), PLGA/TCP/5%Mg (PT5M), PLGA/TCP/10%Mg (PT10M), and PLGA/TCP/15%Mg (PT15M). The bacterial adhesion and biofilm formation of Staphylococcus aureus were evaluated. The results indicated that the Mg-based scaffolds significantly inhibited bacterial adhesion and biofilm formation compared to PT, and the PT10M and PT15M exhibited significantly stronger anti-biofilm ability than PT5M. In vitro degratation tests revealed that the degradation of the Mg-based scaffolds caused an increase of pH, Mg2+ concentration and osmolality, and the increased pH may be one of the major contributing factors to the antibacterial function of the Mg-based scaffolds. Additionally, the PT15M exhibited an inhibitory effect on cell adhesion and proliferation of MC3T3-E1 cells. In conclusion, the PLGA/TCP/Mg scaffolds could inhibit bacterial adhesion and biofilm formation, and the PT10M scaffold was considered to be an effective composition with considerable antibacterial ability and good cytocompatibility. PMID:26346217

A preliminary in vitro study into the use of IL-1Ra gene therapy for the inhibition of intervertebral disc degeneration

PubMed Central

Le Maitre, Christine L; Freemont, Anthony J; Hoyland, Judith A

2006-01-01

Conventional therapies for low back pain (LBP) are purely symptomatic and do not target the cause of LBP, which in approximately 40% of cases is caused by degeneration of the intervertebral disc (DIVD). Targeting therapies to inhibit the process of degeneration would be a potentially valuable treatment for LBP. There is increasing evidence for a role for IL-1 in DIVD. A natural inhibitor of IL-1 exists, IL-1Ra, which would be an ideal molecular target for inhibiting IL-1-mediated effects involved in DIVD and LBP. In this study, the feasibility of ex vivo gene transfer of IL-1Ra to the IVD was investigated. Monolayer and alginate cultures of normal and degenerate human intervertebral disc (IVD) cells were infected with an adenoviral vector carrying the IL-1Ra gene (Ad-IL-1Ra) and protein production measured using an enzyme-linked immunosorbent assay. The ability of these infected cells to inhibit the effects of IL-1 was also investigated. In addition, normal and degenerate IVD cells infected with Ad-IL-1Ra were injected into degenerate disc tissue explants and IL-1Ra production in these discs was assessed. This demonstrated that both nucleus pulposus and annulus fibrosus cells infected with Ad-IL-1Ra produced elevated levels of IL-1Ra for prolonged time periods, and these infected cells were resistant to IL-1. When the infected cells were injected into disc explants, IL-1Ra protein expression was increased which was maintained for 2 weeks of investigation. This in vitro study has shown that the use of ex vivo gene transfer to degenerate disc tissue is a feasible therapy for the inhibition of IL-1-mediated events during disc degeneration. PMID:16436110

6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan, Barden, E-mail: cchan@bidmc.harvard.edu; VanderLaan, Paul A.; Sukhatme, Vikas P.

2013-09-20

Highlights: •Expression of 6PGD positively correlates with advancing stage of lung carcinoma. •Knockdown of 6PGD by shRNA potently inhibits c-Met tyrosine phosphorylation. •Exogenous HGF fails to restore c-Met phosphorylation in cells with 6PGD knocked down. •6PGD knockdown results in inhibition of cell migration in vitro. •Constitutively active TPR-cMet significantly restores migration of cells without 6PGD. -- Abstract: 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functionalmore » role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.« less

Ex-vivo in-vitro inhibition of lipopolysaccharide stimulated tumor necrosis factor-alpha and interleukin-1 beta secretion in human whole blood by extractum urticae dioicae foliorum.

PubMed

Obertreis, B; Ruttkowski, T; Teucher, T; Behnke, B; Schmitz, H

1996-04-01

An extract of Urtica dioica folium (IDS 23, Rheuma-Hek), monographed positively for adjuvant therapy of rheumatic diseases and with known effects in partial inhibition of prostaglandin and leukotriene synthesis in vitro, was investigated with respect to effects of the extract on the lipopolysaccharide (LPS) stimulated secretion of proinflammatory cytokines in human whole blood of healthy volunteers. In the assay system used, LPS stimulated human whole blood showed a straight increase of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion reaching maximum concentrations within 24 h following a plateau and slight decrease up to 65 h, respectively. The concentrations of these cytokines was strongly positively correlated with the number of monocytes/macrophages of each volunteer. TNF-alpha and IL-1 beta concentration after LPS stimulation was significantly reduced by simultaneously given IDS 23 in a strictly dose dependent manner. At time 24 h these cytokine concentrations were reduced by 50.8% and 99.7%, respectively, using the highest test IDS 23 assay concentration of 5 mg/ml (p < 0.001). After 65 h the corresponding inhibition was 38.9% and 99.9%, respectively (p < 0.001). On the other hand IDS 23 showed no inhibition but stimulated IL-6 secretion in absence of LPS alone. Simultaneously given LPS and IDS 23 resulted in no further increase. In contrast to described effects on arachidonic acid cascade in vitro, tested Urtica dioica phenol carbon acid derivates and flavonoides such as caffeic malic acid, caffeic acid, chlorogenic acid, quercetin and rutin did not influence LPS stimulated TNF-alpha, IL-1 beta and IL-6 secretion in tested concentrations up to 5 x 10(-5) mol/l. These further findings on the pharmacological mechanism of action of Urticae dioica folia may explain the positive effects of this extract in the treatment of rheumatic diseases.

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Franco, Gilson C.N.; Department of Pharmacology, FOP/UNICAMP, Piracicaba, SP; Kajiya, Mikihito

2011-06-10

Tetracycline antibiotics, including doxycycli/e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography andmore » Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.« less

Graviola: A Novel Promising Natural-Derived Drug That Inhibits Tumorigenicity and Metastasis of Pancreatic Cancer Cells In Vitro and In Vivo Through Altering Cell Metabolism

PubMed Central

Torres, María P.; Rachagani, Satyanarayana; Purohit, Vinee; Pandey, Poomy; Joshi, Suhasini; Moore, Erik D.; Johansson, Sonny L.; Singh, Pankaj K.; Ganti, Apar K.; Batra, Surinder K.

2012-01-01

Pancreatic tumors are resistant to conventional chemotherapies. The present study was aimed at evaluating the potential of a novel plant-derived product as a therapeutic agent for pancreatic cancer (PC). The effects of an extract from the tropical tree Annona Muricata, commonly known as Graviola, was evaluated for cytotoxicity, cell metabolism, cancer-associated protein/gene expression, tumorigenicity, and metastatic properties of PC cells. Our experiments revealed that Graviola induced necrosis of PC cells by inhibiting cellular metabolism. The expression of molecules related to hypoxia and glycolysis in PC cells (i.e. HIF-1α, NF-κB, GLUT1, GLUT4, HKII, and LDHA) were downregulated in the presence of the extract. In vitro functional assays further confirmed the inhibition of tumorigenic properties of PC cells. Overall, the compounds that are naturally present in a Graviola extract inhibited multiple signaling pathways that regulate metabolism, cell cycle, survival, and metastatic properties in PC cells. Collectively, alterations in these parameters led to a decrease in tumorigenicity and metastasis of orthotopically implanted pancreatic tumors, indicating promising characteristics of the natural product against this lethal disease. PMID:22475682

Structure–inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Zhong-Ze; Joint Center for Translational Medicine, Dalian Institute of Chemical Physics Chinese Academy of Sciences and The first Affiliated Hospital of Liaoning Medical University, No.457, Zhongshan Road, Dalian 116023; Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892

The wide utilization of ginseng provides the high risk of herb–drug interaction (HDI) with many clinical drugs. The inhibition of ginsenosides towards drug-metabolizing enzymes (DMEs) has been regarded as an important reason for herb–drug interaction (HDI). Compared with the deep studies on the ginsenosides' inhibition towards cytochrome P450 (CYP), the inhibition of ginsenosides towards the important phase II enzymes UDP-glucuronosyltransferases (UGTs) remains to be unclear. The present study aims to evaluate the inhibition behavior of ginsenosides towards important UGT isoforms located in the liver and intestine using in vitro methods. The recombinant UGT isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employedmore » as in vitro probe reaction. The results showed that structure-dependent inhibition existed for the inhibition of ginsenosides towards UGT isoforms. To clarify the possibility of in vivo herb–drug interaction induced by this kind of inhibition, the ginsenoside Rg{sub 3} was selected as an example, and the inhibition kinetic type and parameters (K{sub i}) were determined. Rg{sub 3} competitively inhibited UGT1A7, 2B7 and 2B15-catalyzed 4-MU glucuronidation reaction, and exerted noncompetitive inhibition towards UGT1A8-catalyzed 4-MU glucuronidation. The inhibition parameters (K{sub i} values) were calculated to be 22.6, 7.9, 1.9, and 2.0 μM for UGT1A7, 1A8, 2B7 and 2B15. Using human maximum plasma concentration of Rg{sub 3} (400 ng/ml (0.5 μM)) after intramuscular injection of 60 mg Rg{sub 3}, the area under the plasma concentration-time curve (AUC) was extrapolated to increase by 2.2%, 6.3%, 26.3%, and 25% for the co-administered drugs completely undergoing the metabolism catalyzed by UGT1A7, 1A8, 2B7 and 2B15, respectively. All these results indicated that the ginsenosides' inhibition towards UGT isoforms might be an important reason for ginseng–drug interaction. - Highlights: ► Structure-dependent inhibition

Amelioration of bleomycin-induced pulmonary fibrosis by chlorogenic acid through endoplasmic reticulum stress inhibition.

PubMed

Wang, Yi-Chun; Dong, Jing; Nie, Jing; Zhu, Ji-Xiang; Wang, Hui; Chen, Qiong; Chen, Jun-Yi; Xia, Jia-Mei; Shuai, Wei

2017-09-01

To investigate the inhibitory effects of chlorogenic acid on pulmonary fibrosis and the internal mechanisms in vivo and in vitro. 30 male BALB/C mice were randomized into 5 groups: control group, pulmonary fibrosis model group, low, middle and high dose of chlorogenic acid groups. Mice in pulmonary fibrosis model group were administered 5.0 mg/kg bleomycin with intracheal instillation and mice in 3 chlorogenic acid groups were treated with chlorogenic acid every day for 28 days after bleomycin administration. Lung tissue histology was observed using HE staining. Primary pulmonary fibroblasts were isolated and cultured. The expressions of fibrosis related factors (α-SMA and collagen I), as well as ER stress markers (CHOP and GRP78) were determined by both real-time PCR assay and Western blotting, while the expressions of other ER stress signaling pathway factors PERK, IRE-1, ATF-6 and protein levels of caspase-12, caspase-9, caspase-3, PARP were determined by Western blotting. RLE-6TN cell line induced by TGF-β1 was also used to verify the amelioration effects in vitro study. In both in vivo and in vitro studies, TUNEL staining was used to evaluate cell apoptosis. Expressions of collagen I, α-SMA, GRP78, and CHOP were significantly inhibited by chlorogenic acid in dose-dependent manner. Similarly, decreasing levels of cleaved caspase-12, caspase-9, caspase-3 and increasing level of uncleaved PARP were observed in chlorogenic acid groups compared with those in the fibrosis group both in vivo and in vitro. Chlorogenic acid could also significantly down-regulate the level of phosphorylation of PERK and cleaved ATF-6 in vivo study. Moreover, MTT assay demonstrated chlorogenic acid could enhance proliferation of RLE-6TN cells induced by TGFβ1 in vitro. And the apoptosis assays indicated that chlorogenic acid could significantly inhibit cell apoptosis both in vivo and in vitro studies. Chlorogenic acid could inhibit the pulmonary fibrosis through endoplasmic

Comparative in vitro inhibition of urinary tract pathogens by single- and multi-strain probiotics.

PubMed

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

2013-09-01

Multi-species probiotic preparations have been suggested as having a wide spectrum of application, although few studies have compared their efficacy with that of individual component strains at equal concentrations. We therefore tested the ability of 4 single probiotics and 4 probiotic mixtures to inhibit the urinary tract pathogens Escherichia coli NCTC 9001 and Enterococcus faecalis NCTC 00775. We used an agar spot test to test the ability of viable cells to inhibit pathogens, while a broth inhibition assay was used to assess inhibition by cell-free probiotic supernatants in both pH-neutralised and non-neutralised forms. In the agar spot test, all probiotic treatments showed inhibition, L. acidophilus was the most inhibitory single strain against E. faecalis, L. fermentum the most inhibitory against E. coli. A commercially available mixture of 14 strains (Bio-Kult(®)) was the most effective mixture, against E. faecalis, the 3-lactobacillus mixture the most inhibitory against E. coli. Mixtures were not significantly more inhibitory than single strains. In the broth inhibition assays, all probiotic supernatants inhibited both pathogens when pH was not controlled, with only 2 treatments causing inhibition at a neutral pH. Both viable cells of probiotics and supernatants of probiotic cultures were able to inhibit growth of two urinary tract pathogens. Probiotic mixtures prevented the growth of urinary tract pathogens but were not significantly more inhibitory than single strains. Probiotics appear to produce metabolites that are inhibitory towards urinary tract pathogens. Probiotics display potential to reduce the incidence of urinary tract infections via inhibition of colonisation.

Kaempferol in red and pinto bean seed (Phaseolus vulgaris L.) coats inhibits iron bioavailability using an in vitro digestion/human Caco-2 cell model.

PubMed

Hu, Ying; Cheng, Zhiqiang; Heller, Larry I; Krasnoff, Stuart B; Glahn, Raymond P; Welch, Ross M

2006-11-29

Four different colored beans (white, red, pinto, and black beans) were investigated for factors affecting iron bioavailability using an in vitro digestion/human Caco-2 cell model. Iron bioavailability from whole beans, dehulled beans, and their hulls was determined. The results show that white beans contained higher levels of bioavailable iron compared to red, pinto, and black beans. These differences in bioavailable iron were not due to bean-iron and bean-phytate concentrations. Flavonoids in the colored bean hulls were found to be contributing to the low bioavailability of iron in the non-white colored beans. White bean hulls contained no detectable flavonoids but did contain an unknown factor that may promote iron bioavailability. The flavonoids, kaempferol and astragalin (kaempferol-3-O-glucoside), were identified in red and pinto bean hulls via HPLC and MS. Some unidentified anthocyanins were also detected in the black bean hulls but not in the other colored bean hulls. Kaempferol, but not astragalin, was shown to inhibit iron bioavailability. Treating in vitro bean digests with 40, 100, 200, 300, 400, 500, and 1000 microM kaempferol significantly inhibited iron bioavailability (e.g., 15.5% at 40 microM and 62.8% at 1000 microM) in a concentration-dependent fashion. Thus, seed coat kaempferol was identified as a potent inhibitory factor affecting iron bioavailability in the red and pinto beans studied. Results comparing the inhibitory effects of kaempferol, quercitrin, and astragalin on iron bioavailability suggest that the 3',4'-dihydroxy group on the B-ring in flavonoids contributes to the lower iron bioavailability.

Nonsteroidal anti-inflammatory drugs inhibit gastric peroxidase activity.

PubMed

Banerjee, R K

1990-06-20

The peroxidase activity of the mitochondrial fraction of rat gastric mucosa was inhibited with various nonsteroidal anti-inflammatory drugs (NSAIDs) in vitro. Indomethacin was found to be more effective than phenylbutazone (PB) or acetylsalicylic acid (ASA). Mouse gastric peroxidase was also very sensitive to indomethacin inhibition. Indomethacin has no significant effect on submaxillary gland peroxidase activity of either of the species studied. Purified rat gastric peroxidase activity was inhibited 75% with 0.15 mM indomethacin showing half-maximal inhibition at 0.04 mM. The inhibition could be withdrawn by increasing the concentration of iodide but not by H2O2. NSAIDs inhibit gastric peroxidase activity more effectively at acid pH (pH 5.2) than at neutral pH. Spectral studies showed a bathochromic shift of the Soret band of the enzyme with indomethacin indicating its interaction at or near the heme part of the enzyme.

In vivo prediction of CYP-mediated metabolic interaction potential of formononetin and biochanin A using in vitro human and rat CYP450 inhibition data.

PubMed

Arora, Sumit; Taneja, Isha; Challagundla, Muralikrishna; Raju, Kanumuri Siva Rama; Singh, Sheelendra Pratap; Wahajuddin, Muhammad

2015-11-19

Formononetin (FMN) and Biochanin A (BCA) are the principal isoflavones present in commercially available extracts of red clover that are widely been consumed for various health benefits. We investigated the in vitro effects of FMN and BCA on catalytic activity of human/rat cytochrome P450 enzymes to assess the drug interaction potential of red clover. IC50 and Ki values of FMN and BCA for CYPs were determined in human/rat liver microsomes. FMN and BCA showed concentration-dependent inhibition of CYP1A2 activity with IC50 values of 13.42 and 24.98μM in human liver microsomes and 38.57 and 11.86μM in rat liver microsomes, respectively. The mode of inhibition of human CYP1A2 by FMN was found to be competitive with apparent Ki value of 10.13±1.96μM. FMN also inhibited human CYP2D6. BCA exerted moderately inhibitory effects on human CYP2C9. The predicted in vivo inhibition for CYP1A2 was insignificant (R value <1.1) at hepatic level while at intestinal level, it was significant (R value >11). The inhibitory effects on other CYPs were found to be minimal. Red clover may be considered safe to be consumed along with co-prescribed medications; however, precaution must be taken while co-administering it with CYP1A2 substrates. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Isorhapontigenin (ISO) Inhibits Invasive Bladder Cancer Formation In Vivo and Human Bladder Cancer Invasion In Vitro by Targeting STAT1/FOXO1 Axis.

PubMed

Jiang, Guosong; Wu, Amy D; Huang, Chao; Gu, Jiayan; Zhang, Liping; Huang, Haishan; Liao, Xin; Li, Jingxia; Zhang, Dongyun; Zeng, Xingruo; Jin, Honglei; Huang, Haojie; Huang, Chuanshu

2016-07-01

Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancer's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1-FOXO1-MMP-2 axis. Cancer Prev Res; 9(7); 567-80. ©2016 AACR. ©2016 American

Inhibition of breast cancer cell growth by methyl pyropheophenylchlorin photodynamic therapy is mediated though endoplasmic reticulum stress-induced autophagy in vitro and vivo.

PubMed

Zhu, Jiang; Tian, Si; Li, Kai-Ting; Chen, Qing; Jiang, Yuan; Lin, Hai-Dan; Yu, Le-Hua; Bai, Ding-Qun

2018-05-01

Autophagy and ER stress participated in the inhibition of MPPa-PDT on tumor growth, but the molecular links between them remain undefined. We just explore the molecular mechanism between them in vitro and vivo. CCK-8 assay and flow cytometer were used to detect the cytotoxicity and mode of cell death after MPPa-PDT. Furthermore, the role of autophagy was verified in MPPa-PDT. Confocal microscopy was used to show the intracellular distribution of MPPa. ER stress markers and PERK signaling pathway were detected by western blot. While in vivo, tumor histology and immunohistochemistry were performed to show the effect of MPPa-PDT in mice. After MPPa-PDT, cells viability decreased in dose-dependent manner. Besides, the cell apoptosis increased along with the increasing of Beclin-1and LC3B II but declining of P62. When pretreated with 3-MA, LC3B II formation and the cytotoxicity declined. MPPa-PDT caused increasing of ER stress markers (GRP78, CHOP) as MPPa accumulated in ER. However, pretreatment with ER stress inhibitor 4PBA, the expression of GRP78 and LC3B II was blocked but the PERK signaling pathway activated and the expression of P62 increased. In vivo, the tumor growth was significantly inhibited by MPPa-PDT. Besides, the appearance of ER stress and autophagy was further demonstrated by immunohistochemistry. Our findings demonstrate that autophagy mediated by MPPa-PDT was regulated by ER stress, via PERK signaling pathway, to kill MDA-MB-231 cells in vitro and vivo. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Evaluation of new antimicrobial agents on Bacillus spp. strains: docking affinity and in vitro inhibition of glutamate-racemase.

PubMed

Tamay-Cach, Feliciano; Correa-Basurto, José; Villa-Tanaca, Lourdes; Mancilla-Percino, Teresa; Juárez-Montiel, Margarita; Trujillo-Ferrara, José G

2013-10-01

Three glutamic acid derivatives, two boron-containing and one imide-containing compound, were synthesized and tested for antimicrobial activity targeting glutamate-racemase. Antimicrobial effect was evaluated over Bacillus spp. Docking analysis shown that the test compounds bind near the active site of racemase isoforms, suggesting an allosteric effect. The boron derivatives had greater affinity than the imide derivative. In vitro assays shown good antimicrobial activity for the boron-containing compounds, and no effectiveness for the imide-containing compounds. The minimum inhibitory concentration of tetracycline, used as standard, was lower than that of the boron-containing derivatives. However, it seems that the boron-containing derivatives are more selective for bacteria. Experimental evidence suggests that the boron-containing derivatives act by inhibiting the racemase enzyme. Therefore, these test compounds probably impede the formation of the bacterial cell wall. Thus, the boron-containing glutamic acid derivatives should certainly be of interest for future studies as antimicrobial agents for Bacillus spp.

Sodium selenite induces apoptosis and inhibits autophagy in human synovial sarcoma cell line SW982 in vitro.

PubMed

Yang, Le; Cai, Yong-Song; Xu, Ke; Zhu, Jia-Lin; Li, Yuan-Bo; Wu, Xiao-Qing; Sun, Jian; Lu, She-Min; Xu, Peng

2018-05-01

The present study aimed to examine the effects of sodium selenite on the SW982 human synovial sarcoma cell line in relation to cell viability, apoptosis and autophagy. The results indicated that sodium selenite reduced cell viability and induced apoptosis by activating caspase‑3 and members of the poly (ADP‑ribose) polymerase and Bcl‑2 protein families in SW982 cells. Furthermore, autophagy was also suppressed by sodium selenite treatment in SW982 cells, and apoptosis was upregulated in cells co‑treated with sodium selenite and the autophagy inhibitor 3‑methyladenine. By contrast, apoptosis was downregulated when sodium selenite was combined with rapamycin, an inducer of autophagy. The results indicated that autophagy may protect cells from the cytotoxicity of sodium selenite. The present study results demonstrated that sodium selenite induced apoptosis and inhibited autophagy and autophagy‑protected cells from death by antagonizing sodium selenite‑induced apoptosis in SW982 cells in vitro.

Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system.

PubMed

Fukaya, Yoshitaka; Kuroda, Masayuki; Aoyagi, Yasuyuki; Asada, Sakiyo; Kubota, Yoshitaka; Okamoto, Yoshitaka; Nakayama, Toshinori; Saito, Yasushi; Satoh, Kaneshige; Bujo, Hideaki

2012-05-31

Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-α/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti- apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.

Inhibition of trypsin by condensed tannins and wine.

PubMed

Gonçalves, Rui; Soares, Susana; Mateus, Nuno; de Freitas, Victor

2007-09-05

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.

Inhibition of T cell-mediated functions by MVM(i), a parvovirus closely related to minute virus of mice.

PubMed

Engers, H D; Louis, J A; Zubler, R H; Hirt, B

1981-12-01

A purified preparation of MVM(i), a murine parvovirus closely related to minute virus of mice (MVM), was found to inhibit various functions mediated by murine T cells in vitro. Addition of MVM(i) virus to secondary allogeneic mixed leukocyte cultures resulted in the inhibition of both lymphocyte proliferation (3H-thymidine incorporation) and the generation of cytolytic T lymphocyte activity but not interferon production. MVM(i) virus also inhibited the growth and cytolytic activity of several cloned, long-term Lyt-2+ cytolytic T cell lines. Furthermore, the antigen-induced proliferative responses of parasite- (Leishmania) specific Lyt-1+ T cells in vitro was abrogated by the addition of MVM(i) virus to the culture. Finally, the suppression of an in vitro antibody response to SRBC by MVM(i) virus was the result of the inhibition of T helper cells required for the B cell response. These suppressive effects were specific for MVM(i); parallel studies in which the prototype MVM parvovirus was used showed no significant inhibition in the various systems tested.

Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

PubMed Central

Fazal, Fabeha; Gu, Lianzhi; Ihnatovych, Ivanna; Han, YooJeong; Hu, WenYang; Antic, Nenad; Carreira, Fernando; Blomquist, James F.; Hope, Thomas J.; Ucker, David S.; de Lanerolle, Primal

2005-01-01

Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis. PMID:15988034

CO2 laser and fluoride on the inhibition of root caries—an in vitro microbial model

NASA Astrophysics Data System (ADS)

Steiner-Oliveira, C.; Rodrigues, L. K. A.; Parisotto, T. M.; Sousa E Silva, C. M.; Hara, A. T.; Nobre-Dos-Santos, M.

2010-09-01

An increase in the dental caries prevalence on root surfaces has been observed mainly in elderly. This research assessed, in vitro, the effectiveness of a pulsed CO2 (λ = 10.6 μm) laser associated or not with fluoride, in reducing human root dentine demineralization in conditions that mimic an oral high cariogenic challenge. After sterilization, root dentine specimens were randomly assigned into 6 groups ( n = 30), in triplicate. The groups were Control (C), Streptococcus mutans (SM), Fluoride (F), Laser (L), Fluoride + laser (FL), and Laser + fluoride (LF). Except for the control group, all the specimens were inoculated with SM and immersed 3 times a day in a 40% sucrose bath. After a 7-day cariogenic challenge, the mineral loss and lesion depth were evaluated by transverse microradiography and fluoride in the biofilm was determined using an ion-selective electrode. Results were statistically analyzed by analysis of variance, at 5% of significance level. For groups C, SM, F, L, FL and LF, the means (standard-deviation) of mineral loss were 816.3 (552.5)a, 3291.5 (1476.2)c, 2508.5 (1240.5)bc, 2916.2 (1323.7)c, 1839.7 (815.2)b and 1955.0 (1001.4)b, respectively; while lesion depths were 39.6 (22.8)a, 103.1 (38.9)c, 90.3 (44.6)bc, 91.7 (27.0)bc, 73.3 (26.6)b, 75.1 (35.2)b, respectively (different superscript letters indicate significant differences among groups). In conclusion, irradiation of root dentine with a pulsed CO2 laser at fluency of 12.0 J/cm2 was able to inhibit root surface demineralization only when associated with fluoride. No synergy effect on the inhibition of root dentine mineral loss was provided by the combination of fluoride application and laser irradiation.

Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway In Vitro and In Vivo

PubMed Central

He, Haiyang; Wu, Yuzhang

2013-01-01

Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted. PMID:23990993

Sanguiin H-6, a constituent of Rubus parvifolius L., inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-α-induced osteoclast formation in vivo.

PubMed

Sakai, Eiko; Aoki, Yuri; Yoshimatsu, Masako; Nishishita, Kazuhisa; Iwatake, Mayumi; Fukuma, Yutaka; Okamoto, Kuniaki; Tanaka, Takashi; Tsukuba, Takayuki

2016-07-15

Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated. To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption. Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-α (TNF-α)-mediated osteoclastogenesis was examined in vivo. R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-κB alpha (IκBα) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-κB in vitro, as well as TNF-α-mediated osteoclastogenesis in vivo. Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction. Copyright © 2016 Elsevier GmbH. All rights reserved.

A novel imidazopyridine derivative, X22, attenuates sepsis-induced lung and liver injury by inhibiting the inflammatory response in vitro and in vivo

PubMed Central

Ge, Xiangting; Feng, Zhiguo; Xu, Tingting; Wu, Beibei; Chen, Hongjin; Xu, Fengli; Fu, Lili; Shan, Xiaoou; Dai, Yuanrong; Zhang, Yali; Liang, Guang

2016-01-01

Sepsis remains a leading cause of death worldwide. Despite years of extensive research, effective drugs to treat sepsis in the clinic are lacking. In this study, we found a novel imidazopyridine derivative, X22, which has powerful anti-inflammatory activity. X22 dose-dependently inhibited lipopolysaccharide (LPS)-induced proinflammatory cytokine production in mouse primary peritoneal macrophages and RAW 264.7 macrophages. X22 also downregulated the LPS-induced proinflammatory gene expression in vitro. In vivo, X22 exhibited a significant protection against LPS-induced death. Pretreatment or treatment with X22 attenuated the sepsis-induced lung and liver injury by inhibiting the inflammatory response. In addition, X22 showed protection against LPS-induced acute lung injury. We additionally found that pretreatment with X22 reduced the inflammatory pain in the acetic acid and formalin models and reduced the dimethylbenzene-induced ear swelling and acetic acid-increased vascular permeability. Together, these data confirmed that X22 has multiple anti-inflammatory effects and may be a potential therapeutic option in the treatment of inflammatory diseases. PMID:27390516

A novel imidazopyridine derivative, X22, attenuates sepsis-induced lung and liver injury by inhibiting the inflammatory response in vitro and in vivo.

PubMed

Ge, Xiangting; Feng, Zhiguo; Xu, Tingting; Wu, Beibei; Chen, Hongjin; Xu, Fengli; Fu, Lili; Shan, Xiaoou; Dai, Yuanrong; Zhang, Yali; Liang, Guang

2016-01-01

Sepsis remains a leading cause of death worldwide. Despite years of extensive research, effective drugs to treat sepsis in the clinic are lacking. In this study, we found a novel imidazopyridine derivative, X22, which has powerful anti-inflammatory activity. X22 dose-dependently inhibited lipopolysaccharide (LPS)-induced proinflammatory cytokine production in mouse primary peritoneal macrophages and RAW 264.7 macrophages. X22 also downregulated the LPS-induced proinflammatory gene expression in vitro. In vivo, X22 exhibited a significant protection against LPS-induced death. Pretreatment or treatment with X22 attenuated the sepsis-induced lung and liver injury by inhibiting the inflammatory response. In addition, X22 showed protection against LPS-induced acute lung injury. We additionally found that pretreatment with X22 reduced the inflammatory pain in the acetic acid and formalin models and reduced the dimethylbenzene-induced ear swelling and acetic acid-increased vascular permeability. Together, these data confirmed that X22 has multiple anti-inflammatory effects and may be a potential therapeutic option in the treatment of inflammatory diseases.

Gemcitabine-loaded albumin nanospheres (GEM-ANPs) inhibit PANC-1 cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Li, Ji; Di, Yang; Jin, Chen; Fu, Deliang; Yang, Feng; Jiang, Yongjian; Yao, Lie; Hao, Sijie; Wang, Xiaoyi; Subedi, Sabin; Ni, Quanxing

2013-04-01

With the development of nanotechnology, special attention has been given to the nanomaterial application in tumor treatment. Here, a modified desolvation-cross-linking method was successfully applied to fabricate gemcitabine-loaded albumin nanospheres (GEM-ANPs), with 110 and 406 nm of mean diameter, respectively. The aim of this study was to assess the drug distribution, side effects, and antitumor activity of GEM-ANPs in vivo. The metabolic viability and flow cytometry analysis revealed that both GEM-ANPs, especially 406-nm GEM-ANPs, could effectively inhibit the metabolism and proliferation and promote the apoptosis of human pancreatic carcinoma (PANC-1) in vitro. Intravenous injection of 406-nm GEM-ANPs exhibited a significant increase of gemcitabine in the pancreas, liver, and spleen of Sprague-Dawley rats ( p < 0.05). Moreover, no signs of toxic side effects analyzed by blood parameter changes were observed after 3 weeks of administration although a high dose (200 mg/kg) of GEM-ANPs were used. Additionally, in PANC-1-induced tumor mice, intravenous injection of 406-nm GEM-ANPs also could effectively reduce the tumor volume by comparison with free gemcitabine. With these findings, albumin nanosphere-loading approach might be efficacious to improve the antitumor activity of gemcitabine, and the efficacy is associated with the size of GEM-ANPs.

Ebselen exhibits glycation-inhibiting properties and protects against osmotic fragility of human erythrocytes in vitro.

PubMed

Soares, Julio C M; Folmer, Vanderlei; Da Rocha, João B T; Nogueira, Cristina W

2014-05-01

Diabetic status is associated with an increase on oxidative stress markers in humans and animal models. We have investigated the in vitro effects of high concentrations of glucose on the profile of oxidative stress and osmotic fragility of blood from control and diabetic patients; we considered whether its antioxidant properties could afford some protection against glucose-induced osmotic fragility, and whether ebselen could act as an inhibitor of hemoglobin glycation. Raising blood glucose to 5-100 mmol/L resulted in a concentration-dependent increase of glycated hemoglobin (HbA1c; P < 0.001) and thiobarbituric acid reactive species (TBA-RS) content (P < 0.004). Non-protein SH groups (NPSH) also increased significantly as the concentration of glucose increased up to 30 mmol/L (P < 0.001). The osmotic fragility was more pronounced in blood of uncontrolled diabetic patients than in these non-diabetic subjects. Ebselen significantly reduced the glucose-induced increase in osmotic fragility and inhibited HbA1c formation (P < 0.0001). These results indicate that blood from patients with uncontrolled diabetes are more sensitive to osmotic shock than from patients with controlled diabetes and control subjects in relation to increased production of free radicals in vivo. © 2014 International Federation for Cell Biology.

Sodium Phenylbutyrate Inhibits Tumor Growth and the Epithelial-Mesenchymal Transition of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

PubMed

Qian, Kun; Sun, Laiyu; Zhou, Guoqing; Ge, Haixia; Meng, Yue; Li, Jingfen; Li, Xiao; Fang, Xinqiang

2018-05-01

Sodium phenylbutyrate (SPB) as a salt of 4-phenylbutyric acid (4-PBA) has been reported to be an ammonia scavenger, histone deacetylase inhibitor, and an endoplasmic reticulum stress inhibitor in various diseases, including neurological diseases, inflammatory disorders, and carcinogenesis. Although phenylbutyrate showed effective antitumor properties in many cancers, its role in oral squamous cell carcinoma (OSCC) remains further characterized. Thus, the OSCC cell lines CAL27, HSC3, and SCC4 were treated with a series of doses of SPB for different times. The IC 50 of three cell lines for SPB was determined to be 4.0, 3.7, and 3.0 mM. The CCK-8 assay indicated that the treatment of SPB induced continuous inhibition of cell vitality of three cell lines. Apoptosis was assessed by Hoechst assay that showed that SPB could significantly promote cell apoptosis. Moreover, the apoptosis-related pathway was analyzed, and the results showed that the expression of antiapoptosis factor BCL-2 was downregulated by SPB but the cleavage of caspase-3 was increased. Meanwhile, it was found that SPB also impaired the migration and invasion of OSCC cells in vitro. Mechanistically, the transforming growth factor-β (TGFB) related epithelial-mesenchymal transition (EMT) was inhibited by SPB with decreased mesenchymal marker N-cadherin and increased epithelial marker E-cadherin. Furthermore, the antitumor effect of SPB in vivo was also demonstrated. The administration of SPB induced remarkably tumor regression with decreased tumor volume, and the TGFB level and EMT phenotype in vivo were also inhibited. These data demonstrated that the treatment of SPB could function as antitumor therapeutics for OSCC.

Digestion products of the PH20 hyaluronidase inhibit remyelination.

PubMed

Preston, Marnie; Gong, Xi; Su, Weiping; Matsumoto, Steven G; Banine, Fatima; Winkler, Clayton; Foster, Scott; Xing, Rubing; Struve, Jaime; Dean, Justin; Baggenstoss, Bruce; Weigel, Paul H; Montine, Thomas J; Back, Stephen A; Sherman, Larry S

2013-02-01

Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases, including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions. Copyright © 2012 American Neurological Association.

Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma

PubMed Central

Zeng, Ling; Beggs, Reena R.; Cooper, Tiffiny S.; N.Weaver, Alice; S.Yang, Eddy

2017-01-01

EGFR inhibition and radiotherapy are potent inducers of DNA damage. Checkpoint kinases 1 and 2 (Chk1/2) are critical regulators of the DNA-damage response, controlling cell-cycle checkpoints that may permit recovery from therapy-associated genomic stress. We hypothesized that Chk1/2 inhibition (CHKi) with prexasertib may enhance cytotoxicity from EGFR inhibition plus radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study, we found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in human papillomavirus virus (HPV)-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed in vivo in both HPV-positive and HPV-negative cell line xenografts receiving triple combination therapy with CHKi, cetuximab, and radiotherapy without a concomitant increase in toxicity as assessed by mouse body weight. Taken together, the combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects in HNSCCs both in vitro and in vivo, suggesting that this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644). PMID:28138028

Intermittent hydrostatic pressure inhibits shear stress-induced nitric oxide release in human osteoarthritic chondrocytes in vitro.

PubMed

Lee, Mel S; Trindade, Michael C D; Ikenoue, Takashi; Schurman, David J; Goodman, Stuart B; Smith, R Lane

2003-02-01

To test the effects of intermittent hydrostatic pressure (IHP) on nitric oxide (NO) release induced by shear stress and matrix macromolecule gene expression in human osteoarthritic chondrocytes in vitro. Chondrocytes isolated from cartilage samples from 9 patients with osteoarthritis were cultured and exposed to either shear stress or an NO donor. Nitrite concentration was measured using the Griess reaction. Matrix macromolecule mRNA signal levels were determined using reverse-transcriptase polymerase chain reaction and quantified by imaging analysis software. Exposure to shear stress upregulated NO release in a dose and time-dependent manner. Application of IHP inhibited shear stress induced NO release but did not alter NO release from chondrocytes not exposed to shear stress. Shear stress induced NO or addition of an NO donor (sodium nitroprusside) was associated with decreased mRNA signal levels for the cartilage matrix proteins, aggrecan, and type II collagen. Intermittent hydrostatic pressure blocked the inhibitory effects of sodium nitroprusside but did not alter the inhibitory effects of shear stress on cartilage macromolecule gene expression. Our data show that shear stress and IHP differentially alter chondrocyte metabolism and suggest that a balance of effects between different loading forces preserve cartilage extracellular matrix in vivo.

Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry.

PubMed

Yang, Shu; Xu, Miao; Lee, Emily M; Gorshkov, Kirill; Shiryaev, Sergey A; He, Shihua; Sun, Wei; Cheng, Yu-Shan; Hu, Xin; Tharappel, Anil Mathew; Lu, Billy; Pinto, Antonella; Farhy, Chen; Huang, Chun-Teng; Zhang, Zirui; Zhu, Wenjun; Wu, Yuying; Zhou, Yi; Song, Guang; Zhu, Heng; Shamim, Khalida; Martínez-Romero, Carles; García-Sastre, Adolfo; Preston, Richard A; Jayaweera, Dushyantha T; Huang, Ruili; Huang, Wenwei; Xia, Menghang; Simeonov, Anton; Ming, Guoli; Qiu, Xiangguo; Terskikh, Alexey V; Tang, Hengli; Song, Hongjun; Zheng, Wei

2018-01-01

The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC 50 ) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.

OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine both in vitro and in vivo.

PubMed

Zhi, Xiao; Chen, Wei; Xue, Fei; Liang, Chao; Chen, Bryan Wei; Zhou, Yue; Wen, Liang; Hu, Liqiang; Shen, Jian; Bai, Xueli; Liang, Tingbo

2015-09-22

Despite its relative rarity, pancreatic ductal adenocarcinoma (PDAC) accounts for a large percentage of cancer deaths. In this study, we investigated the in vitro efficacy of OSI-027, a selective inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, to treat PDAC cell lines alone, and in combination with gemcitabine (GEM). Similarly, we tested the efficacy of these two compounds in a xenograft mouse model of PDAC. OSI-027 significantly arrested cell cycle in G0/G1 phase, inhibited the proliferation of Panc-1, BxPC-3, and CFPAC-1 cells, and downregulated mTORC1, mTORC2, phospho-Akt, phospho-p70S6K, phospho-4E-BP1, cyclin D1, and cyclin-dependent kinase 4 (CDK4) in these cells. Moreover, OSI-027 also downregulated multidrug resistance (MDR)-1, which has been implicated in chemotherapy resistance in PDAC cells and enhanced apoptosis induced by GEM in the three PDAC cell lines. When combined, OSI-027 with GEM showed synergistic cytotoxic effects both in vitro and in vivo. This is the first evidence of the efficacy of OSI-027 in PDAC and may provide the groundwork for a new clinical PDAC therapy.

OSI-027 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine both in vitro and in vivo

PubMed Central

Xue, Fei; Liang, Chao; Chen, Bryan Wei; Zhou, Yue; Wen, Liang; Hu, Liqiang; Shen, Jian; Bai, Xueli; Liang, Tingbo

2015-01-01

Despite its relative rarity, pancreatic ductal adenocarcinoma (PDAC) accounts for a large percentage of cancer deaths. In this study, we investigated the in vitro efficacy of OSI-027, a selective inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, to treat PDAC cell lines alone, and in combination with gemcitabine (GEM). Similarly, we tested the efficacy of these two compounds in a xenograft mouse model of PDAC. OSI-027 significantly arrested cell cycle in G0/G1 phase, inhibited the proliferation of Panc-1, BxPC-3, and CFPAC-1 cells, and downregulated mTORC1, mTORC2, phospho-Akt, phospho-p70S6K, phospho-4E-BP1, cyclin D1, and cyclin-dependent kinase 4 (CDK4) in these cells. Moreover, OSI-027 also downregulated multidrug resistance (MDR)-1, which has been implicated in chemotherapy resistance in PDAC cells and enhanced apoptosis induced by GEM in the three PDAC cell lines. When combined, OSI-027 with GEM showed synergistic cytotoxic effects both in vitro and in vivo. This is the first evidence of the efficacy of OSI-027 in PDAC and may provide the groundwork for a new clinical PDAC therapy. PMID:26213847

Differential in vitro inhibition of M3G and M6G formation from morphine by (R)- and (S)-methadone and structurally related opioids

PubMed Central

Morrish, Glynn A; Foster, David J R; Somogyi, Andrew A

2006-01-01

Aims To determine the in vitro kinetics of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation and the inhibition potential by methadone enantiomers and structurally related opioids. Methods M3G and M6G formation kinetics from morphine were determined using microsomes from five human livers. Inhibition of glucuronide formation was investigated with eight inhibitors (100 µm) and the mechanism of inhibition determined for (R)- and (S)-methadone (70–500 µm) using three microsomal samples. Results Glucuronide formation displayed single enzyme kinetics. The M3G Vmax (mean ± SD) was 4.8-fold greater than M6G Vmax (555 ± 110 vs. 115 ± 19 nmol mg−1 protein h−1; P = 0.006, mean of difference 439; 95% confidence interval 313, 565 nmol mg−1 protein h−1). Km values for M3G and M6G formation were not significantly different (1.12 ± 0.37 vs. 1.11 ± 0.31 mm; P = 0.89, 0.02; −0.29, 0.32 mm). M3G and M6G formation was inhibited (P < 0.01) with a significant increase in the M3G/M6G ratio (P < 0.01) for all compounds tested. Detailed analysis with (R)- and (S)-methadone revealed noncompetitive inhibition with (R)-methadone Ki of 320 ± 42 µm and 192 ± 12 µm for M3G and M6G, respectively, and (S)-methadone Ki of 226 ± 30 µm and 152 ± 20 µm for M3G and M6G, respectively. Ki values for M3G inhibition were significantly greater than for M6G for (R)-methadone (P = 0.017, 128; 55, 202 µm) and (S)-methadone (P = 0.026, 75; 22, 128 µm). Conclusions Both methadone enantiomers noncompetitively inhibited the formation of morphine's primary metabolites, with greater inhibition of M6G formation compared with M3G. These findings indicate a mechanism for reduced morphine clearance in methadone-maintained patients and reduced relative formation of the opioid active M6G compared with M3G. PMID:16487227

Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo.

PubMed

Jia, Xiao-Qin; Cheng, Hai-Qing; Li, Hong; Zhu, Yan; Li, Yu-Hua; Feng, Zhen-Qing; Zhang, Jian-Ping

2011-11-01

We have previously found that connective tissue growth factor (CTGF) is highly expressed in a rat model of liver cancer. Here, we examined expression of CTGF in human hepatocellular carcinoma (HCC) cells and its effect on cell growth. Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2, SMMC-7721, MHCC-97H and LO2. siRNA for the CTGF gene was designed, synthesized and cloned into a Plk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF. CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to evaluate the growth effect, and a colony formation assay was used for observing clonogenic growth. In vivo, tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation. Statistical significance was determined by t test for comparison between two groups, or analysis of variance (ANOVA) for multiple groups. Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples (87.5%). CTGF was overexpressed 5-fold in 20 HCC tissues, compared with surrounding non-tumor liver tissue. CTGF mRNA level was 5 - 8-fold higher in HepG2, SMMC-7721 and MHCC-97H than in LO2 cells. This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression (P < 0.05). Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells (P < 0.05). The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells (P < 0.05). CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo. Knockdown of CTGF may be a potential therapeutic strategy for treatment of HCC.

A novel taspine analog, HMQ1611, inhibits growth of non-small cell lung cancer by inhibiting angiogenesis

PubMed Central

LU, WEN; DAI, BINGLING; MA, WEINA; ZHANG, YANMIN

2012-01-01

In the present study, we investigated the antitumor activity of HMQ1611, a novel synthetic taspine derivative, in vivo and evaluated associated potential antiangiogenesis mechanisms. The proliferation of A549 cells was examined by WST-1 assay in vitro. Tube formation and lung tissue vessel models were used to observe the antiangiogenic activity of HMQ1611. In addition, vascular enodthelial growth factor (VEGF) secretion and KDR kinase activities were measured by ELISA and the HTRF®KinEASE™-TK assay. In vivo, the antitumor activity was assessed by implantation of A549 cells in athymic mice. The results showed that HMQ1611 inhibited A549 cell proliferation and VEGF secretion, while it significantly inhibited tube formation and tissue vascularization. Furthermore, HMQ1611 inhibited A549 xenograft tumor growth. In conclusion, the results of our study suggest that HMQ1611 has latent properties for the inhibition of angiogenesis which are involved in its antitumor activity. PMID:23162661

A novel taspine analog, HMQ1611, inhibits growth of non-small cell lung cancer by inhibiting angiogenesis.

PubMed

Lu, Wen; Dai, Bingling; Ma, Weina; Zhang, Yanmin

2012-11-01

In the present study, we investigated the antitumor activity of HMQ1611, a novel synthetic taspine derivative, in vivo and evaluated associated potential antiangiogenesis mechanisms. The proliferation of A549 cells was examined by WST-1 assay in vitro. Tube formation and lung tissue vessel models were used to observe the antiangiogenic activity of HMQ1611. In addition, vascular enodthelial growth factor (VEGF) secretion and KDR kinase activities were measured by ELISA and the HTRF(®)KinEASE(™)-TK assay. In vivo, the antitumor activity was assessed by implantation of A549 cells in athymic mice. The results showed that HMQ1611 inhibited A549 cell proliferation and VEGF secretion, while it significantly inhibited tube formation and tissue vascularization. Furthermore, HMQ1611 inhibited A549 xenograft tumor growth. In conclusion, the results of our study suggest that HMQ1611 has latent properties for the inhibition of angiogenesis which are involved in its antitumor activity.

SPARC Overexpression Inhibits Cell Proliferation in Neuroblastoma and Is Partly Mediated by Tumor Suppressor Protein PTEN and AKT

PubMed Central

Bhoopathi, Praveen; Gorantla, Bharathi; Sailaja, G. S.; Gondi, Christopher S.; Gujrati, Meena; Klopfenstein, Jeffrey D.; Rao, Jasti S.

2012-01-01

Secreted protein acidic and rich in cysteine (SPARC) is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell–cell and cell–matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo. PMID:22567126

Synthesis, Antiplasmodial Activity, and β-Hematin Inhibition of Hydroxypyridone–Chloroquine Hybrids

PubMed Central

2013-01-01

A series of noncytotoxic 4-aminoquinoline-3-hydroxypyridin-4-one hybrids were synthesized on the basis of a synergistic in vitro combination of a precursor N-alkyl-3-hydroxypyridin-4-one with chloroquine (CQ) and tested in vitro against CQ resistant (K1 and W2) and sensitive (3D7) strains of Plasmodium falciparum. In vitro antiplasmodial activity of the precursors was negated by blocking the chelator moiety via complexation with gallium(III) or benzyl protection. None of the precursors inhibited β-hematin formation. Most hybrids were more potent inhibitors of β-hematin formation than CQ, and a correlation between antiplasmodial activity and inhibition of β-hematin formation was observed. Potent hybrids against K1, 3D7, and W2, respectively, were 8c (0.13, 0.004, and 0.1 μM); 8d (0.08, 0.01, and 0.02 μM); and 7g (0.07, 0.03, and 0.08 μM). PMID:24900724

Iminosugars Inhibit Dengue Virus Production via Inhibition of ER Alpha-Glucosidases--Not Glycolipid Processing Enzymes.

PubMed

Sayce, Andrew C; Alonzi, Dominic S; Killingbeck, Sarah S; Tyrrell, Beatrice E; Hill, Michelle L; Caputo, Alessandro T; Iwaki, Ren; Kinami, Kyoko; Ide, Daisuke; Kiappes, J L; Beatty, P Robert; Kato, Atsushi; Harris, Eva; Dwek, Raymond A; Miller, Joanna L; Zitzmann, Nicole

2016-03-01

It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages in vitro, in which it inhibits DENV secretion with an EC50 of 5 μM. We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that

Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.

2006-09-10

Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis.more » Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.« less

In vitro and in vivo inhibition of aldose reductase and advanced glycation end products by phloretin, epigallocatechin 3-gallate and [6]-gingerol.

PubMed

Sampath, Chethan; Sang, Shengmin; Ahmedna, Mohamed

2016-12-01

Hyperglycemic stress activates polyol pathway and aldose reductase (AR) key enzyme responsible for generating secondary complications during diabetes. In this study the therapeutic potential of phloretin, epigallocatechin 3-gallate (EGCG) and [6]-gingerol were evaluated for anti-glycating and AR inhibitory activity in vitro and in vivo systems. Human retinal pigment epithelial (HRPE) cells were induced with high glucose supplemented with the phloretin, EGCG and [6]-gingerol. Aldose reductase activity, total advanced glycation end products (AGEs) and enzyme inhibitor kinetics were assessed. Male C57BL/6J mice were randomly assigned to one of the different treatments (bioactive compounds at 2 concentrations each) with either a low fat diet or high fat diet (HFD). After sixteen weeks, AGE accumulation and AR activity was determined in heart, eyes and kidney. High glucose induced toxicity decreased cell viability compared to the untreated cells and AR activity increased to 2-5 folds from 24 to 96h. Pre-treatment of cells with phloretin, EGCG and [6]-gingerol improved cell viability and inhibited AR activity. The enzyme inhibition kinetics followed a non-competitive mode of inhibition for phloretin and EGCG whereas [6]-gingerol indicated uncompetitive type of inhibition against AR. Data from the animal studies showed high plasma glucose levels in HFD group over time, compared to the low fat diet. HFD group developed cataract and AR activity increased to 4 folds compared to the group with low fat diet. Administration of EGCG, phloretin and [6]-gingerol significantly reduced blood sugar levels, AGEs accumulation, and AR activity. These findings could provide a basis to consider using the selected dietary components alone or in combination with other therapeutic approaches to prevent diabetes-related complications in humans. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Ferulic acid destabilizes preformed {beta}-amyloid fibrils in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ono, Kenjiro; Hirohata, Mie; Yamada, Masahito

2005-10-21

Inhibition of the formation of {beta}-amyloid fibrils (fA{beta}), as well as the destabilization of preformed fA{beta} in the CNS, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that curcumin (Cur) inhibits fA{beta} formation from A{beta} and destabilizes preformed fA{beta} in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of ferulic acid (FA) on the formation, extension, and destabilization of fA{beta} at pH 7.5 at 37 deg C in vitro. We next compared the anti-amyloidogenic activities of FA with Cur, rifampicin, and tetracycline. Ferulic acid dose-dependentlymore » inhibited fA{beta} formation from amyloid {beta}-peptide, as well as their extension. Moreover, it destabilized preformed fA{beta}s. The overall activity of the molecules examined was in the order of: Cur > FA > rifampicin = tetracycline. FA could be a key molecule for the development of therapeutics for AD.« less

Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro.

PubMed

Ademiluyi, Adedayo O; Oboh, Ganiyu

2013-03-01

This study sought to assess the inhibitory activities of phenolic-rich extracts from soybean on α-amylase, α-glucosidase and angiotensin I converting enzyme (ACE) activities in vitro. The free phenolic extract of the soybean was obtained by extraction with 80% acetone, while that of the bound phenolic extract was done by extracting the alkaline and acid hydrolyzed residue with ethyl acetate. The inhibitory action of these extracts on the enzymes activity as well as their antioxidant properties was assessed. Both phenolic-rich extracts inhibited α-amylase, α-glucosidase and ACE enzyme activities in a dose dependent pattern. However, the bound phenolic extract exhibited significantly (P < 0.05) higher α-amylase and ACE inhibition while the free phenolic extract had significantly (P < 0.05) higher α-glucosidase inhibitory activity. Nevertheless, the free phenolic extract had higher α-glucosidase inhibitory activity when compared to that of α-amylase; this property confer an advantage on soybean phenolic-rich extracts over commercial antidiabetic drugs with little or no side effect. And inhibition of ACE suggests the antihypertension potential of soybean phenolic-rich extracts. Furthermore, the enzyme inhibitory activities of the phenolic-rich extracts were not associated with their phenolic content. Therefore, phenolic-rich extracts of soybean could inhibit key-enzyme linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (ACE) and thus could explain in part the mechanism by which soybean renders these health promoting effect. Copyright © 2011 Elsevier GmbH. All rights reserved.

Targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin squamous cell carcinoma cell growth in vitro and in vivo.

PubMed

Zou, Ying; Ge, Minggai; Wang, Xuemin

2017-08-19

Abnormal activation of PI3K-AKT-mTOR signaling is detected in human skin squamous cell carcinoma (SCC). LY3023414 is a novel, potent, and orally bio-available PI3K-AKT-mTOR inhibitor. Its activity against human skin SCC cells was tested. We demonstrated that LY3023414 was cytotoxic when added to established (A431 line) and primary (patient-derived) human skin SCC cells. LY3023414 induced G0/1-S arrest and inhibited proliferation of skin SCC cells. Moreover, LY3023414 induced activation of caspase-3/-9 and apoptosis in skin SCC cells. Intriguingly, LY3023414 was yet non-cytotoxic nor pro-apoptotic to normal human skin cells (melanocytes, keratinocytes and fibroblasts). At the molecular level, LY3023414 blocked PI3K-AKT-mTOR activation in skin SCC cells, as it dephosphorylated PI3K-AKT-mTOR substrates: P85, AKT and S6K1. In vivo studies showed that oral administration of LY3023414 at well-tolerated doses inhibited A431 xenograft tumor growth in severe combined immunodeficiency (SCID) mice. AKT-mTOR activation in LY3023414-treated tumors was also largely inhibited. Together, these results suggest that targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin SCC cell growth in vitro and in vivo, establishing the rationale for further clinical testing. Copyright © 2017 Elsevier Inc. All rights reserved.

Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus.

PubMed

Cacciatore, Luis Claudio; Kristoff, Gisela; Verrengia Guerrero, Noemí R; Cochón, Adriana C

2012-07-01

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC(50) values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary

Lipopolysaccharide-induced inhibition of transcription of tlr4 in vitro is reversed by dexamethasone and correlates with presence of conserved NFκB binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonin, Camila P., E-mail: mila_bonin@yahoo.com.br; Baccarin, Raquel Y.A., E-mail: baccarin@usp.br; Nostell, Katarina, E-mail: katarina.nostell@slu.se

2013-03-08

Highlights: ► Chimpanzees, horses and humans have regions of similarity on TLR4 and MD2 promoters. ► Rodents have few regions of similarity on TLR4 promoter when compared to primates. ► Conserved NFkB binding sites were found in the promoters of TLR4 and MD2. ► LPS-induced inhibition of TLR4 transcription is reversed by dexamethasone. ► LPS-induced transcription of MD2 is inhibited by dexamethasone. -- Abstract: Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) is a master trigger of the deleterious effects of septic shock. Horses and humans are considered the most sensitive species to septic shock, but the mechanisms explainingmore » these phenomena remain elusive. Analysis of tlr4 promoters revealed high similarity among LPS-sensitive species (human, chimpanzee, and horse) and low similarity with LPS-resistant species (mouse and rat). Four conserved nuclear factor kappa B (NFκB) binding sites were found in the tlr4 promoter and two in the md2 promoter sequences that are likely to be targets for dexamethasone regulation. In vitro treatment of equine peripheral blood mononuclear cells (eqPBMC) with LPS decreased transcripts of tlr4 and increased transcription of md2 (myeloid differentiation factor 2) and cd14 (cluster of differentiation 14). Treatment with dexamethasone rescued transcription of tlr4 after LPS inhibition. LPS-induced transcription of md2 was inhibited in the presence of dexamethasone. Dexamethasone alone did not affect transcription of tlr4 and md2.« less

A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro.

PubMed

Rathore, Kusum; Cekanova, Maria

2015-01-01

Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo.

Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

PubMed

Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

2013-02-28

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

Role of inhibition of osteogenesis function by Sema4D/Plexin-B1 signaling pathway in skeletal fluorosis in vitro.

PubMed

Liu, Xiao-li; Song, Jing; Liu, Ke-jian; Wang, Wen-peng; Xu, Chang; Zhang, Yu-zeng; Liu, Yun

2015-10-01

Skeletal fluorosis is a chronically metabolic bone disease with extensive hyperostosis osteosclerosis caused by long time exposure to fluoride. Skeletal fluorosis brings about a series of abnormal changes of the extremity, such as joint pain, joint stiffness, bone deformity, etc. Differentiation and maturation of osteoblasts were regulated by osteoclasts via Sema4D/Plexin-B1 signaling pathway. Furthermore, the differentiation and maturation of osteoclasts are conducted by osteoblasts via RANKL/RANK/OPG pathway. Both of these processes form a feedback circuit which is a key link in skeletal fluorosis. In this study, an osteoblast-osteoclast co-culture model in vitro was developed to illustrate the mechanism of skeletal fluorosis. With the increase of fluoride concentration, the expression level of Sema4D was decreased and TGF-β1 was increased continuously. OPG/RANKL mRNA level, however, increased gradually. On the basis of that, the inhibition of Sema4D/Plexin-B1/RhoA/ROCK signaling pathway caused by fluoride promoted the level of TGF-β1 and activated the proliferation of osteoblasts. In addition, osteroprotegerin (OPG) secreted by osteoblasts was up-regulated by fluoride. The competitive combination of OPG and RANKL was strengthened and the combination of RANKL and RANK was hindered. And then the differentiation and maturation of osteoclasts were inhibited, and bone absorption was weakened, leading to skeletal fluorosis.

Inhibition of Wnt/beta-catenin signaling downregulates expression of aldehyde dehydrogenase isoform 3A1 (ALDH3A1) to reduce resistance against temozolomide in glioblastoma in vitro.

PubMed

Suwala, Abigail Kora; Koch, Katharina; Rios, Dayana Herrera; Aretz, Philippe; Uhlmann, Constanze; Ogorek, Isabella; Felsberg, Jörg; Reifenberger, Guido; Köhrer, Karl; Deenen, René; Steiger, Hans-Jakob; Kahlert, Ulf D; Maciaczyk, Jaroslaw

2018-04-27

Glioblastoma is the most aggressive type of glioma. The Wingless (Wnt) signaling pathway has been shown to promote stem cell properties and resistance to radio- and chemotherapy in glioblastoma. Here, we demonstrate that pharmacological Wnt pathway inhibition using the porcupine inhibitor LGK974 acts synergistically with temozolomide (TMZ), the chemotherapeutic drug currently used as standard treatment for glioblastoma, to suppress in vitro growth of glioma cells. Synergistic growth inhibition was independent of the O 6 -alkylguanine DNA alkyltransferase ( MGMT ) promoter methylation status. Transcriptomic analysis revealed that expression of aldehyde dehydrogenase 3A1 ( ALDH3A1 ) was significantly down-regulated when cells were treated with LGK974 and TMZ. Suppressing ALDH3A1 expression increased the efficacy of TMZ and reduced clonogenic potential accompanied by decreased expression of stem cell markers CD133, Nestin and Sox2. Taken together, our study suggests that previous observations concerning Wnt signaling blockade to reduce chemoresistance in glioblastoma is at least in part mediated by inhibition of ALDH3A1.

Enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7.

PubMed

Fang, Zhong-Ze; Wang, Haina; Cao, Yun-Feng; Sun, Dong-Xue; Wang, Li-Xuan; Hong, Mo; Huang, Ting; Chen, Jian-Xing; Zeng, Jia

2015-03-01

UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0 μM and 31.1 μM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7 μM and 3.1 μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism. © 2014 Wiley Periodicals, Inc.

Annexin A11 knockdown inhibits in vitro proliferation and enhances survival of Hca-F cell via Akt2/FoxO1 pathway and MMP-9 expression.

PubMed

Liu, Shuqing; Wang, Jiasheng; Guo, Chunmei; Qi, Houbao; Sun, Ming-Zhong

2015-03-01

Annexin A11 (Anxa11), a Ca(2+)-regulated phospholipid-binding protein, is involved in cell apoptosis, differentiation, vesicle trafficking, cancer progression and autoimmune diseases. Previous study from our group indicated that Anxa11 was associated with lymphatic metastatic potential of murine hepatocarcinoma cells. Herein, we investigated the effects and action mechanism of Anxa11 knockdown on in vitro cell proliferation and apoptosis of Hca-F, a murine hepatocarcinoma cell with∼75% lymph node metastatic potential. Real-time PCR and western blotting assays indicated that Anxa11 was significantly downregulated in monoclonal Anxa11-shRNA-transfected Hca-F cells. Anxa11 knockdown in Hca-F suppressed its in vitro proliferation and cell apoptosis capacities. Following Anxa11 knockdown in Hca-F cells, Bax/Bcl-2 expression level ratio, Akt2 and FoxO1 (pSer319) expression levels as well as MMP-9 mRNA and active MMP-9 protein levels were significantly elevated in Hca-F cells. In conclusion, Annexin A11 knockdown inhibits the in vitro proliferation and cell apoptosis of Hca-F cell via Akt2/FoxO1 and/or MMP-9 expression pathway. Anxa11 might play an important role in hepatocarcinoma cell invasion and metastasis and hepatocarcinoma malignancy. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Doxycycline Inhibits Inflammation-Induced Lymphangiogenesis in Mouse Cornea by Multiple Mechanisms

PubMed Central

Huang, Jingwen; Zhou, Jingwen; Qiu, Sujuan; Liang, Dan

2014-01-01

Lymphangiogenesis is significantly involved in the pathogenesis of diseases, including graft rejection, cancer metastasis and various inflammatory conditions. The inhibition of lymphangiogenesis has become a new therapeutic target for the treatment of these diseases. Here, we explored the anti-lymphangiogenic effects of doxycycline in inflammation-induced lymphangiogenesis (ILA) in the cornea and the underlying mechanisms. In the present study, mice with ILA of the cornea were treated with topical doxycycline (0.1%) or vehicle control. Lymphangiogenesis was quantified using corneal immunostaining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Human dermal lymphatic endothelial cells (HDLECs) and a murine macrophage cell line (RAW264.7) were used to further explore the underlying mechanisms of doxycycline-mediated anti-lymphangiogenesis in vitro. Our results showed that doxycycline treatment dramatically inhibited ILA in the mouse cornea (p<0.001), with a significant decrease in vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 signalling, macrophage infiltration and inflammatory cytokine expression. Doxycycline also significantly inhibited VEGF-C-induced HDLEC proliferation in vitro by modulating the PI3K/Akt/endothelial nitric oxide (NO) synthase (eNOS) pathway and significantly suppressed interleukin-1β (IL-1β), TNF-α and VEGF-C production in the RAW264.7 cell line by modulating the PI3K/Akt/nuclear factor-kappaB (NF-κB) pathway. Additionally, doxycycline treatment dramatically reduced the phosphorylation of NF-κBp65, Akt and eNOS in ILA and significantly inhibited matrix metalloproteinases (MMPs) activity in vitro and in ILA. In conclusion, doxycycline inhibited ILA, possibly through suppression of VEGF-C signalling, macrophage function and MMPs activity. This observation suggests that doxycycline is a potential therapeutic agent for lymphangiogenesis-related diseases. PMID:25268699

Potency and selectivity of carprofen enantiomers for inhibition of bovine cyclooxygenase in whole blood assays.

PubMed

Brentnall, Claire; Cheng, Zhangrui; McKellar, Quintin A; Lees, Peter

2012-12-01

Whole blood in vitro assays were used to determine the potency and selectivity of carprofen enantiomers for inhibition of the isoforms of cyclooxygenase (COX), COX-1 and COX-2, in the calf. S(+)-carprofen possessed preferential activity for COX-2 inhibition but, because the slopes of inhibition curves differed, the COX-1:COX-2 inhibition ratio decreased from 9.04:1 for inhibitory concentration (IC)10 to 1.84:1 for IC95. R(-) carprofen inhibited COX-2 preferentially only for low inhibition of the COX isoforms (IC10 COX-1:COX-2=6.63:1), whereas inhibition was preferential for COX-1 for a high level of inhibition (IC95 COX-1:COX-2=0.20:1). S(+) carprofen was the more potent inhibitor of COX isoforms; potency ratios S(+):R(-) carprofen were 11.6:1 for IC10 and 218:1 for IC90. Based on serum concentrations of carprofen enantiomers obtained after administration of a therapeutic dose of 1.4 mg/kg to calves subcutaneously, S(+)-carprofen concentrations exceeded the in vitro IC80 COX-2 value for 32 h and the IC20 for COX-1 for 33 h. The findings are discussed in relation to efficacy and safety of carprofen in calves. Copyright © 2012 Elsevier Ltd. All rights reserved.

Myelin-induced inhibition in a spiral ganglion organ culture - Approaching a natural environment in vitro.

PubMed

Kramer, Benedikt; Tropitzsch, Anke; Müller, Marcus; Löwenheim, Hubert

2017-08-15

The performance of a cochlear implant depends on the defined interaction between afferent neurons of the spiral ganglion and the inserted electrode. Neurite outgrowth can be induced by neurotrophins such as brain-derived neurotrophic factor (BDNF) via tropomyosin kinase receptor B (TrkB). However, neurotrophin signaling through the p75 neurotrophin receptor (p75) inhibits neurite outgrowth in the presence of myelin. Organotypic cultures derived from postnatal (P3-5) mice were used to study myelin-induced inhibition in the cochlear spiral ganglion. Neurite outgrowth was analyzed and quantified utilizing an adapted Sholl analysis. Stimulation of neurite outgrowth was quantified after application of BDNF, the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) and a selective inhibitor of the Rho-associated kinase (Y27632), which inhibits the p75 pathway. Myelin-induced inhibition was assessed by application of myelin-associated glycoprotein (MAG-Fc) to stimulate the inhibitory p75 pathway. Inhibition of neurite outgrowth was achieved by the selective TrkB inhibitor K252a. Stimulation of neurite outgrowth was observed after treatment with BDNF, 7,8 DHF and a combination of BDNF and Y27632. The 7,8-DHF-induced growth effects could be inhibited by K252a. Furthermore, inhibition of neurite outgrowth was observed after supplementation with MAG-Fc. Myelin-induced inhibition could be overcome by 7,8-DHF and the combination of BDNF and Y27632. In this study, myelin-induced inhibition of neurite outgrowth was established in a spiral ganglion model. We reveal that 7,8-DHF is a viable novel compound for the stimulation of neurite outgrowth in a myelin-induced inhibitory environment. The combination of TrkB stimulation and ROCK inhibition can be used to overcome myelin inhibition. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma.

PubMed

Zeng, Ling; Beggs, Reena R; Cooper, Tiffiny S; Weaver, Alice N; Yang, Eddy S

2017-04-01

EGFR inhibition and radiotherapy are potent inducers of DNA damage. Checkpoint kinases 1 and 2 (Chk1/2) are critical regulators of the DNA-damage response, controlling cell-cycle checkpoints that may permit recovery from therapy-associated genomic stress. We hypothesized that Chk1/2 inhibition (CHKi) with prexasertib may enhance cytotoxicity from EGFR inhibition plus radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study, we found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in human papillomavirus virus (HPV)-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed in vivo in both HPV-positive and HPV-negative cell line xenografts receiving triple combination therapy with CHKi, cetuximab, and radiotherapy without a concomitant increase in toxicity as assessed by mouse body weight. Taken together, the combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects in HNSCCs both in vitro and in vivo , suggesting that this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644). Mol Cancer Ther; 16(4); 591-600. ©2017 AACR . ©2017 American Association for Cancer Research.

Inhibition of glycogen synthase kinase 3[beta] activity with lithium in vitro attenuates sepsis-induced changes in muscle protein turnover.

PubMed

Bertsch, Stephen; Lang, Charles H; Vary, Thomas C

2011-03-01

Loss of lean body mass is a characteristic feature of the septic response, and the mechanisms responsible for this decrease and means of prevention have not been fully elucidated. The present study tested the hypothesis that in vitro treatment of skeletal muscle with lithium chloride (LiCl), a glycogen synthase kinase (GSK) 3 inhibitor, would reverse both the sepsis-induced increase in muscle protein degradation and inhibition of protein synthesis. Sepsis decreased GSK-3[beta] phosphorylation and increased GSK-3[beta] activity, under basal conditions. Sepsis increased muscle protein degradation, with a concomitant increase in atrogin 1 and MuRF1 mRNA and 26S proteosome activity. Incubation of septic muscle with LiCl completely reversed the increased GSK-3[beta] activity and decreased proteolysis to basal nonseptic values, but only partially reduced proteosome activity and did not diminish atrogene expression. Lithium chloride also did not ameliorate the sepsis-induced increase in LC3-II, a marker for activated autophagy. In contrast, LiCl increased protein synthesis only in nonseptic control muscle. The inability of septic muscle to respond to LiCl was independent of its ability to reverse the sepsis-induced increase in eukaryotic initiation factor (eIF) 2B[varepsilon] phosphorylation, decreased eIF2B activity, or the reduced phosphorylation of FOXO3, but instead was more closely associated with the continued suppression of mTOR (mammalian target of rapamycin) kinase activity (e.g., reduced phosphorylation of 4E-BP1 and S6). These data suggest that in vitro lithium treatment, which inhibited GSK-3[beta] activity, (a) effectively reversed the sepsis-induced increase in proteolysis, but only in part by a reduction in the ubiquitin-proteosome pathway and not by a reduction in autophagy; and (b) was ineffective at reversing the sepsis-induced decrease in muscle protein synthesis. This lithium-resistant state seems mediated at the level of mTOR and not eIF2/eIF2B. Hence

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.

In vitro inhibition of commercial douche products against vaginal microflora.

PubMed Central

Pavlova, S I; Tao, L

2000-01-01

Recently, vaginal douching has been associated with many health risks in women. The aim of this study was to analyze the effect of commercial douche products against various vaginal microorganisms, including lactobacilli. Seven commercial douches were tested against eight Lactobacillus clinical isolates and three type strains from the American Type Culture Collection. BV-associated bacteria included six strains of five genera: Gardnerella, Mobiluncus, Mycoplasma, Peptostreptococcus, and Ureaplasma. Two isolates of group B Streptococcus, and three species of Candida were also tested. The minimal inhibition concentrations and minimal contact times for these products against vaginal microorganisms were determined in broth cultures. Four antiseptic-containing douche products showed a strong inhibitory effect against all vaginal microorganisms tested with a short contact time (less than 1 min). Three vinegar-containing douche products selectively inhibited vaginal pathogens associated with bacterial vaginosis, group B streptococcal vaginitis, and candidiasis, but not lactobacilli. The antimicrobial effects of the commercial douche products varied among different brands and microbial species tested. PMID:10805365

Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujimura, Masatake, E-mail: fujimura@nimd.go.jp; Usuki, Fusako; Kawamura, Miwako

Methylmercury (MeHg) is an environmental neurotoxicant which induces neuropathological changes in both the central nervous and peripheral sensory nervous systems. Our recent study demonstrated that down-regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which is known to promote neuritic extension, preceded MeHg-induced damage in cultured cortical neurons, suggesting that MeHg-mediated axonal degeneration is due to the disturbance of neuritic extension. Therefore we hypothesized that MeHg-induced axonal degeneration might be caused by neuritic extension/retraction incoordination. This idea brought our attention to the Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) pathway because it has been known to be associatedmore » with the development of axon and apoptotic neuronal cell death. Here we show that inhibition of the Rho/ROCK pathway prevents MeHg-intoxication both in vitro and in vivo. A Rho inhibitor, C3 toxin, and 2 ROCK inhibitors, Fasudil and Y-27632, significantly protected against MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured cortical neuronal cells exposed to 100 nM MeHg for 3 days. Furthermore, Fasudil partially prevented the loss of large pale neurons in dorsal root ganglia, axonal degeneration in dorsal spinal root nerves, and vacuolar degeneration in the dorsal columns of the spinal cord in MeHg-intoxicated model rats (20 ppm MeHg in drinking water for 28 days). Hind limb crossing sign, a characteristic MeHg-intoxicated sign, was significantly suppressed in this model. The results suggest that inhibition of the Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction incoordination and is effective for the prevention of MeHg-induced axonal degeneration and apoptotic neuronal cell death.« less

Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro.

PubMed

Yi, Young-Joo; Manandhar, Gaurishankar; Sutovsky, Miriam; Jonáková, Vera; Park, Chang-Sik; Sutovsky, Peter

2010-03-01

The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization. Copyright 2009. Published by Elsevier Ireland Ltd.

Green synthesis of fluorescent carbon dots from spices for in vitro imaging and tumour cell growth inhibition

PubMed Central

Gallo, Juan; Cerqueira, María de Fátima; Menéndez-Miranda, Mario; Costa-Fernández, José Manuel; Diéguez, Lorena; Espiña, Begoña

2018-01-01

Carbon dots have demonstrated great potential as luminescent nanoparticles in bioapplications. Although such nanoparticles appear to exhibit low toxicity compared to other metal luminescent nanomaterials, today we know that the toxicity of carbon dots (C-dots) strongly depends on the protocol of fabrication. In this work, aqueous fluorescent C-dots have been synthesized from cinnamon, red chilli, turmeric and black pepper, by a one-pot green hydrothermal method. The synthesized C-dots were firstly characterized by means of UV–vis, fluorescence, Fourier transform infrared and Raman spectroscopy, dynamic light scattering and transmission electron microscopy. The optical performance showed an outstanding ability for imaging purposes, with quantum yields up to 43.6%. Thus, the cytotoxicity of the above mentioned spice-derived C-dots was evaluated in vitro in human glioblastoma cells (LN-229 cancer cell line) and in human kidney cells (HK-2 non-cancerous cell line). Bioimaging and viability studies were performed with different C-dot concentrations from 0.1 to 2 mg·mL−1, exhibiting a higher uptake of C-dots in the cancer cultures compared to the non-cancerous cells. Results showed that the spice-derived C-dots inhibited cell viability dose-dependently after a 24 h incubation period, displaying a higher toxicity in LN-229, than in HK-2 cells. As a control, C-dots synthesized from citric acid did not show any significant toxicity in either cancerous or non-cancerous cells, implying that the tumour cell growth inhibition properties observed in the spice-derived C-dots can be attributed to the starting material employed for their fabrication. These results evidence that functional groups in the surface of the C-dots might be responsible for the selective cytotoxicity, as suggested by the presence of piperine in the surface of black pepper C-dots analysed by ESI-QTOF-MS. PMID:29527430

Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

PubMed

Thupari, J N; Pinn, M L; Kuhajda, F P

2001-07-13

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy. Copyright 2001 Academic Press.

Growth inhibition of Saccharomyces cerevisiae by the immunosuppressant leflunomide is due to the inhibition of uracil uptake via Fur4p.

PubMed

Fujimura, H

1998-10-01

The immunosuppressant leflunomide inhibits cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of the eukaryotic microorganism Saccharomyces cerevisiae. To elucidate the molecular mechanism of action of the drug, two yeast genes which suppress the anti-proliferative effect when present in multiple copies were cloned and designated MLF1 and MLF2 for multicopy suppressor of leflunomide sensitivity. DNA sequencing analysis revealed that the MLF1 gene is identical to the FUR4 gene, which encodes a uracil permease and functions to import uracil efficiently. The MLF2 was found to be identical to the URA3 gene. Excess exogenous uracil also overcomes the anti-proliferative effect of leflunomide on yeast cells. Uracil prototrophy also conferred resistance to leflunomide. Uracil uptake was inhibited by leflunomide. Thus, the growth inhibition by leflunomide seen in a S. cerevisiae ura3 auxotroph is due to the inhibition of the entry of exogenous uracil via the Fur4 uracil permease.

Estimation of plasma tacrine concentrations using an in vitro cholinesterase inhibition assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moriearty, P.L.; Kenny, W.; Kumar, V.

THA (9-amino, 1,2,3,4-tetrahydroacridine; tacrine) is currently under study as a cholinesterase (ChE) inhibitor in Alzheimer disease. In this study, a sensitive radiometric assay for THA inhibition of human plasma ChE, suitable for detection of effects of orally administered drug, is described. The assay is sensitive in a range of 4-50 ng/ml plasma. Reversibility of the inhibition permits distinguishing of drug effects on ChE from changes in amount of enzyme synthesized during treatment.

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

Stanniocalcin-2 Inhibits Mammalian Growth by Proteolytic Inhibition of the Insulin-like Growth Factor Axis*

PubMed Central

Jepsen, Malene R.; Kløverpris, Søren; Mikkelsen, Jakob H.; Pedersen, Josefine H.; Füchtbauer, Ernst-Martin; Laursen, Lisbeth S.; Oxvig, Claus

2015-01-01

Mammalian stanniocalcin-2 (STC2) is a secreted polypeptide widely expressed in developing and adult tissues. However, although transgenic expression in mice is known to cause severe dwarfism, and targeted deletion of STC2 causes increased postnatal growth, its precise biological role is still unknown. We found that STC2 potently inhibits the proteolytic activity of the growth-promoting metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). Proteolytic inhibition requires covalent binding of STC2 to PAPP-A and is mediated by a disulfide bond, which involves Cys-120 of STC2. Binding of STC2 prevents PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF, required for normal growth. Concordantly, we show that STC2 efficiently inhibits PAPP-A-mediated IGF receptor signaling in vitro and that transgenic mice expressing a mutated variant of STC2, STC2(C120A), which is unable to inhibit PAPP-A, grow like wild-type mice. Our work identifies STC2 as a novel proteinase inhibitor and a previously unrecognized extracellular component of the IGF system. PMID:25533459

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.