Important Inhibitory Effects Research Articles

Determination of the effect of milk fat on the inactivation of Listeria monocytogenes by ohmic heating

Research in recent years has focused on innovative technologies that provide pathogen inactivation without damaging the structural properties of foods. Ohmic heating (OH) is an innovative technology, that provides an effective microbial inactivation with massive and rapid heating. This study aims to determine the effects of milk fat on the inactivation of Listeria monocytogenes by OH with a low voltage gradient. L. monocytogenes (ATCC 13932) inoculated 3.1%, 1.5%, and 0.1% fat-milk samples were heated up to 62°C by OH and conventional heating (CH) process. OH treatment lead to the inactivation of L. monocytogenes in both 1.5% and 0.1% groups and led to approximately 5.30 log decrease, however, there was a 3.10 log decrease in the 3.1% group at 6 min. CH lead to a few reduction as 0.21, 0.29 and 0.39 log in 3.1%, 1.5% and 0.1% fat-milk respectively. In OH, the sublethal injury ratio was higher than CH in all milk groups. However, OH did not statistically change color and pH values at the 6th min of the process, had a significant effect on hydroxymethylfurfural value only in 3.1% fat-milk. In conclusion, the increased fat content may have important inhibitory effects on pathogen inactivation in OH. Thus, the OH conditions should be chosen carefully to sufficient inactivation of pathogens in milk with high-fat content.

A Triazaspirane Derivative Inhibits Migration and Invasion in PC3 Prostate Cancer Cells.

Cancer is a serious health problem due to the complexity of establishing an effective treatment. The purpose of this work was to evaluate the activity of a triazaspirane as a migration and invasion inhibitor in PC3 prostatic tumor cells through a possible negative regulation of the FAK/Src signal transduction pathway and decreased secretion of metalloproteinases 2 and 9. Molecular docking analysis was performed using Moe 2008.10 software. Migration (wound-healing assay) and invasion (Boyden chamber assay) assays were performed. In addition, the Western blot technique was used to quantify protein expression, and the zymography technique was used to observe the secretion of metalloproteinases. Molecular docking showed interactions in regions of interest of the FAK and Src proteins. Moreover, the biological activity assays demonstrated an inhibitory effect on cell migration and invasion, an important suppression of metalloproteinase secretion, and a decrease in the expression of p-FAK and p-Src proteins in treated PC3 cells. Triazaspirane-type molecules have important inhibitory effects on the mechanisms associated with metastasis in PC3 tumor cells.

Allelopathic Activity and Chemical Constituents of Extracts from Leaves and Stems of Kherchoun (Hertia cheirifolia)

Allelopathy is a common biological phenomenon by which one organism produces biochemicals that influence the growth, survival, development, and reproduction of other organisms. The aim of this study was to investigate the phytochemical screening of the various extracts of Kherchoun (Hertia cheirifolia) and the evaluation of antioxidant capacities, by the DPPH test (2, 2-diphenyl-1-picrylhydrazyl), and of their allelopathic effects, vis-à-vis the two species: Vetch (Vicia sativa L.) and triticale (Triticosecale wittm L.). Biochemical analysis results showed that the highest total lipid content was observed at the leaf level (9%). The aqueous extracts of the leaves are rich in polyphenols, with a rate of 268.67mg EAG/g MS, compared to the stems (242.67mg EAG/g MS). The levels of flavonoids are higher in the leaves (38.67mg EAG/g MS) but lower in the stems (24.75mg EAG/g MS). On the other hand, the highest total tannin content recorded at leaf level is 608.33 mg EAG/g MS and the lowest is recorded in the stems (505 mg EAG/g MS). In contrast, the aqueous extract of the stems has the highest condensed tannin content (0.049 mg EAG/g MS), compared to that of the leaves (0.036 mg EAG/g MS). The two extract .Both aqueous extracts of Hertia cheirifolia are endowed with good antioxidant abilities. CI50 =139.43 2.99a μg/mL at leaf level and CI50 =111.38 22.2a μg/mL at stem level.Concerning the allelopathic study, the results obtained showed that the two extracts (Leaves and stems) of Hertia cheirifolia exerted different effects on the germination parameters of weed seeds of vetch (Vicia sativa L.) and triticale (Triticosecale wittm L.). Indeed, the two extracts exerted very important inhibitory effects, vis-à-vis the two seeds tested, on the germination and development of the seedlings (length of the root and length of the aerial part), in a significant and dose-dependent way.

Chemical Analysis and Investigation of Biological Effects of Salvia officinalis Essential Oils at Three Phenological Stages.

Salvia officinalis is a medicinal plant used to treat some diseases, including microbial infections and diabetes. Different studies showed the biological and pharmacological properties of this species. The aim of this study was the determination of the chemical compounds of S. officinalis essential oils and the investigation of their antimicrobial, antioxidant, antidiabetic, and anti-inflammatory properties. The chemical compounds of S. officinalis were determined by GC-MS analysis. The antioxidant activity was assessed by DPPH, ABTS, H2O2, and FRAP assays. The in vitro antidiabetic effect was evaluated by the inhibition of α-amylase, α-glucosidase, and lipase activities, and the anti-inflammatory effect was evaluated using the 5-lipoxygenase assay. Moreover, antibacterial activity was assessed against six bacterial strains using agar well diffusion assay and microdilution method. The main compounds in essential oils of S. officinalis at three phenological stages were naphthalenone, camphor, 1.8-cineole, and α-thujone. The full flowering stage essential oil showed the best antioxidant activity with different IC50 values according to the used tests. This oil also exhibited important inhibitory effects at the full flowering stage against α-amylase (IC50 = 69.23 ± 0.1 μg/mL), α-glucosidase (IC50 = 22.24 ± 0.07 μg/mL), and lipase (IC50 = 37.3 ± 0.03 μg/mL). The 5-lipoxygenase inhibitory effect was the best at the full flowering stage (IC50 = 9.24 ± 0.03 μg/mL). The results of the antibacterial evaluation revealed that, at three seasonal periods, S. officinalis essential oil demonstrated strong antibacterial activity. Although the full flowering stage had the best antibacterial activity, there were no significant differences between the three stages. Additionally, the essential oils showed bactericidal effects on Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, Proteus mirabilis, Escherichia coli, and Salmonella typhimurium, respectively. The findings of this work showed remarkably that S. officinalis synthesizes essential oils according to different developmental stages. Moreover, it has exhibited interesting biological and pharmacological properties justifying its medicinal effects and suggesting it as a very important source of natural drugs.

Determination of Volatile Compounds of Mentha piperita and Lavandula multifida and Investigation of Their Antibacterial, Antioxidant, and Antidiabetic Properties.

Mentha piperita and Lavandula multifida are widely used in Moroccan traditional medicine for the treatment of diabetes and infectious diseases. The aims of this work were the determination of the chemical composition of Mentha piperita (MPEO) and Lavandula multifida (LMEO) essential oils and the evaluation of their antibacterial, antioxidant, and antidiabetic activities. The chemical composition was determined by GC-MS analysis. The antibacterial effects were evaluated against several bacterial strains using disc diffusion, MIC, and MBC methods. The antioxidant activity was evaluated in vitro using DPPH, H2O2, and xanthine oxidase, and the antidiabetic activity was estimated by the inhibitory effects of α-amylase, α-glucosidase, and lipase activities. GC-MS results showed that the main compounds of MPEO were menthone (29.24%), levomenthol (38.73%), and eucalyptol (6.75%). However, eucalyptol (28.11%), 2-bornanone (11.57%), endo-borneol (7.82%), and linalyl acetate (5.22%) are the major compounds of LMEO. The results exhibited important inhibitory effects against some bacterial strains with MIC = MBC = 0.39 mg/mL for MPEO against Staphylococcus aureus ATCC. However, LMEO exhibited remarkable antioxidant and antidiabetic activities compared to MPEO. Indeed, LMEO inhibited DPPH, H2O2, and xanthine oxidase with concentrations of 15.23, 21.52, and 8.89 µg/mL, respectively. Moreover, LMEO exhibited α-amylase and α-glucosidase at IC50 = 85.34 and IC50 = 59.36 µg/mL, respectively. The findings showed that both MPEO and LMEO exhibit promising biological properties. However, the application of these species or their main bioactive compounds requires further investigation.

Chemical characterization, comprehensive antioxidant capacity, and enzyme inhibitory potential of leaves from Pistacia terebinthus L. (Anacardiaceae)

Pistacia terebinthus, Anacardiaceae, is used in traditional medicine and its fruits are consumed as coffee in Turkey. However, the research on the chemical and biological activity of P. terebinthus leaves is almost lack. In the present study, we aimed to evaluate the comprehensive antioxidant capacity (DPPH, ABTS, reducing power and metal chelating activity) and enzyme inhibitory effects (against AChE, BChE, α-amylase, α-glucosidase, and tyrosinase) as well as phytochemical content of the leaves extracts from P. terebinthus. The phytochemical composition in extracts were analysed using UHPLC-MS. Total phenolics and flavonoids contents of the extracts were 65.51–210.54 mg GAE/g and 54.94–169.89 mg RE/g, respectively. The highest level of total phenolic content was found in the methanol extract while the maximum total flavonoid content observed in ethyl acetate extract. The antioxidant results showed that methanol extract (ABTS: 3.29 mmol TE/g, DPPH: 2.06 mmol TE/g, CUPRAC: 2.67 mmol TE/g, FRAP: 1.62 mmol TE/g, Metal chelating activity: 65.65 mg EDTA/g) exhibited promising antioxidant capacity. The methanol extract also demonstrated important enzyme inhibitory effects. The results suggest that the extracts of P. terebinthus leaves has the potential to be used as good candidate of natural antioxidants and enzyme inhibitors.

Novel anti-invasive properties of a Fascin1 inhibitor on colorectal cancer cells.

Tumor invasion and metastasis involve processes in which actin cytoskeleton rearrangement induced by Fascin1 plays a crucial role. Indeed, Fascin1 has been found overexpressed in tumors with worse prognosis. Migrastatin and its analogues target Fascin1 and inhibit its activity. However, there is need for novel and smaller Fascin1 inhibitors. The aim of this study was to assess the effect of compound G2 in colorectal cancer cell lines and compare it to migrastatin in in vitro and in vivo assays. Molecular modeling, actin-bundling, cell viability, inmunofluorescence, migration, and invasion assays were carried out in order to test anti-migratory and anti-invasive properties of compound G2. In addition, the in vivo effect of compound G2 was evaluated in a zebrafish model of invasion. HCT-116 cells exhibited the highest Fascin1 expression from eight tested colorectal cancer cell lines. Compound G2 showed important inhibitory effects on actin bundling, filopodia formation, migration, and invasion in different cell lines. Moreover, compound G2 treatment resulted in significant reduction of invasion of DLD-1 overexpressing Fascin1 and HCT-116 in zebrafish larvae xenografts; this effect being less evident in Fascin1 known-down HCT-116 cells. This study proves, for the first time, the in vitro and in vivo anti-tumoral activity of compound G2 on colorectal cancer cells and guides to design improved compound G2-based Fascin1 inhibitors. KEY MESSAGES: • Fascin is crucial for tumor invasion and metastasis and is overexpressed in bad prognostic tumors. • Several adverse tumors overexpress Fascin1 and lack targeted therapy. • Anti-fascin G2 is for the first time evaluated in colorectal carcinoma and compared with migrastatin. • Filopodia formation, migration activity, and invasion in vitro and in vivo assays were performed. • G2 blocks actin structures, migration, and invasion of colorectal cancer cells as fascin-dependent.

Essential oils of Mentha viridis rich phenolic compounds show important antioxidant, antidiabetic, dermatoprotective, antidermatophyte and antibacterial properties

Abstract The aim of this study was the investigation of the in vitro antioxidant, antidiabetic, dermatoprotective, antidermatophyte, and antibacterial properties of Mentha viridis essential oil (MVEO). Chemical composition of MVEO was determined using GC-MS analysis. Antioxidant activity was estimated by three complementary methods: DPPH, FRAP and ABTS. The in vitro antidiabetic effect was evaluated by the inhibition of α-Amylase and α-Glucosidase. Inhibitory effects of tyrosinase and elastase were used to reveal the dermatoprotective property of MVEO. Antibacterial activity was tested against five strains using agar well diffusion assay and microtitration method. The chemical composition determination of MVEO revealed the presence of carvone (37.26), 1.8-Cineole (11.82), and Terpinen-4-ol (08.72%) as the main compounds. MVEO presented remarkable antioxidant effects by IC50 values of 80.45 ± 1.86 μg/mL, 101.78 ± 3.14 μg/mL, and 139.59 ± 3.12 μg/mL obtained by DPPH, FRAP and ABTS tests respectively. In vitro inhibition of enzymes implicated in hydrocarbures degradation showed that MVEO exhibited important inhibitory effects against α-Amylase (IC50 = 101.72 ± 1.86 μg/mL) and α-Glucosidase (IC50 = 86.93 ± 2.43 μg/mL). Moreover, MVEO demonstrated remarkable inhibition of tyrosinase (IC50 = 55.13 ± 1.01 μg/mL) and elastase (IC50 = 114.24 ± 1.22 μg/mL). The antifungal activity showed important inhibitions of three dermatophyte species (T. mentagrophytes, T. tonsurans and T. violaceum). The antibacterial activity showed an important bactericidal action against tested strains, especially, Staphylococcus aureus and Listeria monocytogenes (MIC = MBC = 0.25%). The findings of this work showed that volatile components of MVEO could constitute a new source for antioxidant, antidiabetic, dermatoprotective, antidermatophyte and antibacterial properties. However, further investigations regarding the isolation of MVEO main compounds and investigations of their biological properties are required.

CdpR Inhibits CRISPR-Cas Adaptive Immunity to Lower Anti-viral Defense while Avoiding Self-Reactivity

SummaryCRISPR-Cas systems as adaptive immunity in bacteria and archaea battle against bacteriophages. However, little is known how CRISPR-Cas systems are precisely regulated to effectively eliminate intruders while not inducing self-reactivity. Here, we identify intrinsic negative modulator of CRISPR-Cas that influences interference and adaptation functions. LasI/RhlI-derived autoinducers activate cas operon by enhancing the binding of virulence factor regulator (Vfr) cis-response elements to cas1 promoter, whereas CdpR represses this intracellular signaling and blocks transcription of cas operon. Importantly, inhibition of Vfr reduces cas1 expression and impairs immunization and immune memory mediated by CRISPR-Cas, leading to more severe phage infection but lower self-targeting activities. In addition, CdpR-mediated LasI/RhlI/Vfr intracellular signaling represses cleavage of bacterial endogenous sequences by impeding Cas3 RNA cleavage activity. Thus, CdpR renders important inhibitory effects on CRISPR-Cas systems to avoid possible self-reactivity but potentially heightening infection risk. Our study provides insight into fine regulation of CRISPR-Cas systems for maintaining homeostasis.

Antioksidan ve antimiktobiyal özellikleri ile birlikte Craterellus cornucopioides (L.) Pers. türünün hücre büyümesi baskılama potansiyeli

Craterellus cornucopioides (L.) Pers which is also known as trumpet of death or horn of plenty, is a wild edible macrofungus. This study was conducted to elucidate the potential health beneficial properties of C. cornucopioides. Bioactive ingredients (phenolics, flavonoids, β-carotene and lycopene) and DPPH radical scavenging activities were determined. Additionally, cell growth inhibitory effects on HepG2 cells together with some bacteria were evaluated. Accordingly, water and methanol extracts contains 37.71±1.42 μg/mg and 13.78±1.60 μg/mg phenolic contents, respectively. Similarly, methanolic extracts have higher β-caroten and lycopene content as compared to aqueous extracts. In parallel with these antioxidants, methanolic extracts have also higher DPPH scavenging activity (IC50: 5.26±0.67 mg/ml). Besides, water extracts have higher flavonoid contents (2.13±0.06 μg/mg) then the methanolic extracts. C. cornucopioides has also an important cell growth inhibitory effects on HepG2 cell (IC50: 18.41±1.10 mg/ml for aqueous extracts and IC50: 3.14±1.07 mg/ml for methanolic extracts). Moreover, both extracts were effective on six different bacteria tested. As a result, this study indicates that C. cornucopioides could reduce the cellular oxidative stress because of its high antioxidant ingredients, inhibit the growth of pathogen microrganisms and have some degree of cell growth inhibitory potential at least to the HepG2 cells.Pers which is (L.) Craterellus cornucopioides

Physicochemical and Biological Properties of Eel By-Products Protein Hydrolysates: Potential Application to Meat Product Preservation

European eel by-products have been hydrolysed in order to produce novel bioactive peptides with antibacterial and antioxidant activity. Protein hydrolysates from Anguilla by-products obtained by treatment with Savinase® (PHAB-s) have high protein content than that produced with Protamex® (PHAB-p). Furthemore, both protein hydrolysates had a high percentage of essential amino acids. In addition, results indicated that PHAB-s and PHAB-p exhibited different degrees of antioxidant activities, evaluated by several antioxidant assays. Thus, PHAB-s displayed high antioxidant activity, in terms of DPPH-radical scavenging effect (IC50 = 5.2 mg/ml), β-carotene bleaching inhibitory effect (CI50 = 4.4 mg/ml) and metal chelating activity. At 10 mg/ml, PHAB-p showed the highest reducing power. Antibacterial activities of PHAB-s and PHAB-p were also evaluated. PHAB-s showed the most important inhibitory effects against seven bacterial species. The application of PHAB-s and PHAB-p on minced meat as preservative reduced the lipid oxidation and inhibited the microbial growth during chilled storage for 11 days.

Synthetic chalcones as potential tool for acute- and chronic-pain control

The purpose of this study was to validate the potential anti-hypersensitive activity of two chalcones, (2E)-1-(4-aminophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (ANCh) and N-{4-[(2E)-3-(4-nitrophenyl)prop-2-enoil]phenyl}acetamide (AcANCh), by different models of acute and persistent pain in mice, besides in silico analysis. Molecules computational investigation for prediction of Lipinki’s and Veber's rules to determine solubility, % absorption, drug likeness and toxicity liabilities was performed. Male and female C57BL/6 mice (20–30 g, n = 6) were used. Firstly, mice were pre-treated with the compounds ANCh or AcANCh and then submitted to the models of acute hypersensitivity by the intraplantar injection of different phlogistic agents. The mechanical sensitivity was assessed using von Frey hairs (0.6 g). The obtained data shows that both compounds presented important inhibitory effects on mechanical hypersensitivity induced by carrageenan (with oral bioavailability). The anti-hypersensitive effect was also accompanied by the interference in leukocyte migration, interleukin-1β (IL-1β) and tumour necrosis factor (TNF) levels reduction and by the absence of unspecific effects. Added to the in vivo results, the in silico analysis presented none violation in Lipinski’s or Veber’s rules, good probability to cell membrane permeability and oral bioavailability, positive values of drug likeness and few risk of computational toxicity. ANCh partially reduced the hypersensitivity induced by IL-1β and TNF, epinephrine and prostaglandin E2 (PGE2). AcANCh had similar effect, except for the absent of inhibition in PGE2-injected mice. Both compounds were capable of reducing the mechanical hypersensitivity presented in all persistent models of hypersensitivity (inflammatory pain, chronic nerve constriction and cancer pain), with emphasis for ANCh. These results suggest that both chalcones could represent good strategies for the control of acute and chronic pain, without important side effects. ANCh seems to involve cell migration and cytokines production as the main mechanism, together with interference in PGE2 neuronal sensitization pathway. In vivo and in silico analyses reinforce the potential characteristics of the compounds to become future drugs.

Metformin reverses bFGF-induced epithelial-mesenchymal transition in HCC cells.

Metformin had exerted important inhibitory effects in multiple cancers. However, the correlation between metformin and hepatocellular carcinoma (HCC) metastasis, and the relevant mechanisms are still unclear. By quantitative proteomics analysis technique, we found metformin could suppress FGF signalling significantly. In FGF signalling basic fibroblast growth factor (bFGF) is a crucial member, it initially binds to its receptors, the complex of bFGF and receptors activate FGF signallings, and promote many cancers progressions. When treating HCC cell lines HepG2 and Huh7 with bFGF, we observed the cells exhibited epithelial mesenchymal transition (EMT) and these cells metastasis potential was enhanced dramaticlly. However, when treating with metformin and bFGF together, EMT and metastasis induced by bFGF could be inhibited in these cells. Furthermore, bFGF could activate AKT/GSK-3β signalling, sequentially decrease the interaction between GSK-3β and Twist1 and decrease ubiquitination of Twist1 leading to Twist1 degradation reducing. While metformin could repress the bFGF-mediated activation in AKT/GSK-3β signalling, inhibition on interaction between GSK-3β and Twist1, enhancement of Twist1 stability. Taken together, our findings suggested that metformin had prominent negative effects on bFGF-induced EMT and metastasis in HCC cells.

Extraction, characterization and antimicrobial activity of sulfated polysaccharides from fish skins

Sulfated polysaccharides were extracted from gray triggerfish (GTSP) and smooth hound (SHSP) skins. Their chemical and physical characteristics were determined using X-ray diffraction and Infrared spectroscopic analysis. The antibacterial activities of GTSP and SHSP against Listeria monocytogenes (ATCC 43251), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Salmonella enterica (ATCC 43972) and Enterobacter sp were evaluated by determining clear growth inhibition zone diameters and the minimum inhibitory concentration (MIC) values and by essays in liquid media. GTSP and SHSP were fractionated by a Diethylaminoethyl-cellulose chromatography. Fraction FGII, from GTSP, and fraction FSII, from SHSP, showed the most important inhibitory effects against the tested bacterial species. The sulfated polysaccharides from fish skins did not show hemolytic activity towards bovine erythrocytes. Overall, the results suggested that those polysaccharides could offer promising sources of polysaccharides for future application as dietary ingredients in the nutraceutical industry.

Vitronectin Inhibits Efferocytosis through Interactions with Apoptotic Cells as well as with Macrophages

Effective removal of apoptotic cells, particularly apoptotic neutrophils, is essential for the successful resolution of acute inflammatory conditions. In these experiments, we found that whereas interaction between vitronectin and integrins diminished the ability of macrophages to ingest apoptotic cells, interaction between vitronectin with urokinase-type plasminogen activator receptor (uPAR) on the surface of apoptotic cells also had equally important inhibitory effects on efferocytosis. Preincubation of vitronectin with plasminogen activator inhibitor-1 eliminated its ability to inhibit phagocytosis of apoptotic cells. Similarly, incubation of apoptotic cells with soluble uPAR or Abs to uPAR significantly diminished efferocytosis. In the setting of LPS-induced ALI, enhanced efferocytosis and decreased numbers of neutrophils were found in bronchoalveolar lavage obtained from vitronectin-deficient (vtn(-/-)) mice compared with wild type (vtn(+/+)) mice. Furthermore, there was increased clearance of apoptotic vtn(-/-) as compared with vtn(+/+) neutrophils after introduction into the lungs of vtn(-/-) mice. Incubation of apoptotic vtn(-/-) neutrophils with purified vitronectin before intratracheal instillation decreased efferocytosis in vivo. These findings demonstrate that the inhibitory effects of vitronectin on efferocytosis involve interactions with both the engulfing phagocyte and the apoptotic target cell.

Systemic inhibition of myeloid dendritic cells by circulating HLA class I molecules in HIV-1 infection

BackgroundHIV-1 infection is associated with profound dysfunction of myeloid dendritic cells, for reasons that remain ill-defined. Soluble HLA class I molecules can have important inhibitory effects on T cells and NK cells, but may also contribute to reduced functional properties of professional antigen-presenting cells. Here, we investigated the expression of soluble HLA class I isoforms during HIV-1 infection and assessed their functional impact on antigen-presenting characteristics of dendritic cells.ResultsSoluble HLA class I molecules were highly upregulated in progressive HIV-1 infection as determined by quantitative Western blots. This was associated with strong increases of intracellular expression of HLA class I isoforms in dendritic cells and monocytes. Using mixed lymphocyte reactions, we found that soluble HLA class I molecules effectively inhibited the antigen-presenting properties of dendritic cells, however, there was no significant influence of HLA class I molecules on the cytokine-secretion properties of these cells. The immunomodulatory effects of soluble HLA class I molecules were mediated by interactions with inhibitory myelomonocytic MHC class I receptors from the Leukocyte Immunoglobulin Like Receptor (LILR) family.ConclusionsDuring progressive HIV-1 infection, soluble HLA class I molecules can contribute to systemic immune dysfunction by inhibiting the antigen-presenting properties of myeloid dendritic cells through interactions with inhibitory myelomonocytic HLA class I receptors.

Fluorescent cyclin-dependent kinase inhibitors block the proliferation of human breast cancer cells

Inhibitors of cyclin-dependent kinases (CDKs) are an emerging class of drugs for the treatment of cancers. CDK inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combination with radiation therapy and chemotherapies. Drugs that target CDKs could have important inhibitory effects on cancer cell cycle progression, an extremely important mechanism in the control of cancer cell growth. Using rational drug design, we designed and synthesized fluorescent CDK inhibitors ( VMY-1-101 and VMY-1-103) based on a purvalanol B scaffold. The new agents demonstrated more potent CDK inhibitory activity, enhanced induction of G2/M arrest and modest apoptosis as compared to purvalanol B. Intracellular imaging of the CDK inhibitor distribution was performed to reveal drug retention in the cytoplasm of treated breast cancer cells. In human breast cancer tissue, the compounds demonstrated increased binding as compared to the fluorophore. The new fluorescent CDK inhibitors showed undiminished activity in multidrug resistance (MDR) positive breast cancer cells, indicating that they are not a substrate for p-glycoprotein. Fluorescent CDK inhibitors offer potential as novel theranostic agents, combining therapeutic and diagnostic properties in the same molecule.

Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of cytoskeletal regulators of VSMC motility

Vascular smooth muscle cell (VSMC) migration is an important cellular event in multiple vascular diseases, including atherosclerosis, restenosis, and transplant vasculopathy. Little is known regarding the effects of anti-inflammatory interleukins on VSMC migration. This study tested the hypothesis that an anti-inflammatory Th2 interleukin, interleukin-19 (IL-19), could decrease VSMC motility. IL-19 significantly decreased platelet-derived growth factor (PDGF)-stimulated VSMC chemotaxis in Boyden chambers and migration in scratch wound assays. IL-19 significantly decreased VSMC spreading in response to PDGF. To determine the molecular mechanism(s) for these cellular effects, we examined the effect of IL-19 on activation of proteins that regulate VSMC cytoskeletal dynamics and locomotion. IL-19 decreased PDGF-driven activation of several cytoskeletal regulatory proteins that play an important role in smooth muscle cell motility, including heat shock protein-27 (HSP27), myosin light chain (MLC), and cofilin. IL-19 decreased PDGF activation of the Rac1 and RhoA GTPases, important integrators of migratory signals. IL-19 was unable to inhibit VSMC migration nor was able to inhibit activation of cytoskeletal regulatory proteins in VSMC transduced with a constitutively active Rac1 mutant (RacV14), suggesting that IL-19 inhibits events proximal to Rac1 activation. Together, these data are the first to indicate that IL-19 can have important inhibitory effects on VSMC motility and activation of cytoskeletal regulatory proteins. This has important implications for the use of anti-inflammatory cytokines in the treatment of vascular occlusive disease.

Inhibition of Phosphodiesterase 9A (PDE9A) Significantly Reduces Cytokine-Stimulated Adhesion of Neutrophils From Sickle Cell Disease Individuals, in Vitro, but Not Red Cell Adhesion.

Abstract 1520Poster Board I-543The adhesion of both red and white cells to the vessel walls of the microcirculation initiates vaso-occlusion in sickle cell disease (SCD). The chronic inflammatory nature of SCD leads to elevation of circulating cytokines in patients, which may contribute significantly to the activation of blood cells and their consequent adhesion. Nitric oxide (NO) has important inhibitory effects on cellular adhesive properties and drugs that enhance NO bioavailability or NO-cGMP-dependent signaling may hold potential for treatment of various aspects of SCD. This study aimed to observe the effect of cytokines, often found augmented in the plasma of SCD individuals, on the in vitro adhesive properties of neutrophils (neu) and red blood cells (RBC) from healthy control (CON) and steady-state SCD (SCD) individuals. Furthermore, the effects of BAY 73-6691, an inhibitor of the cGMP-hydrolyzing enzyme, phosphodiesterase 9A (PDE9A) and BAY 41-2271, a guanylate cylase activator, on non-stimulated and cytokine-stimulated cell adhesion were determined. Neus and RBCs were isolated from peripheral blood of CON and SCD individuals. Cell adhesion (5×106neu/ml in RPMI or 2×108RBC/ml in HBSS) to immobilized fibronectin (FN;20μg/ml) was assessed using static adhesion assays (30min, 37°C, 5%CO2) in the presence or absence of cytokines IL-8 (100-500ng/ml), TNF-alpha (0.1-1μg/ml) and GM-CSF (1-100ng/ml) and/or in the presence/absence of BAY 73-6691 (10-60μM), BAY 41-2271 (60-150nM) or DMSO vehicle (0.02%v/v). As previously demonstrated, SCDneu have a greater capacity to adhere to FN than CONneu (see Table). Stimulation of cells in vitro with all three cytokines significantly augmented both CONneu adhesion to FN and further increased SCDneu adhesion (Table). Co-incubation of both CONneu and SCDneu with BAY 73-6691, but not BAY 41-2271, significantly reduced their adhesions to FN (Table). Furthermore, BAY 73-6691, but essentially not BAY 41-2271, significantly inhibited CONneu and SCDneu adhesion stimulated by IL-8, TNF-alpha and GM-CSF (Table). DMSO vehicle had no significant effect upon neu adhesion (data not shown). As previously reported, SCD RBC have a greater capacity to adhere to FN, in vitro, compared to CON RBC (12.8±1.7% comp. 7.8±1.0%, n=7, p<0.05). However, in contrast to neutrophils, cytokines IL-8 (10-500ng/ml) and TNF-alpha (0.1-1μg/ml) did not alter the capacities of neither CON RBC nor SCD RBC to adhere to FN (200ng/ml IL-8: CON RBC, 6.8±0.8%; SCD RBC, 13.5±1.8%, n≥6, p>0.05) (1μg/ml TNF-alpha: CON RBC, 7.0±1.0%; SCD RBC, 11.4±1.9%, n=6, p>0.05). Furthermore, BAY 73-6691 and BAY 41-2271 did not affect either basal CON RBC (data not shown) or SCD RBC adhesion (SCD RBC basal; 14.8±1.0%; 60μM BAY 73-6691, 16.4±1.3%; 150nM BAY 41-2271, 15.7±1.2%, n=6). Key SCD inflammatory mediators were found, at physiologically relevant concentrations, to augment the adhesive properties of neutrophils, but not RBC, from control and SCD individuals. Circulating inflammatory cytokines may play a role in the induction of leukocyte adhesive properties in SCD; in contrast factors other than inflammatory stimuli may be more important for induction of SCD RBC adhesion. Data suggest that elevation of intracellular cGMP may be an important approach for reducing SCD leukocyte, but not SCD RBC, adhesive properties, even in an inflammatory environment. PDE9A is highly expressed in hematopoietic cells and inhibition of this enzyme, with consequent augmentation of cGMP, may represent a tissue/cell-specific therapeutic drug target worthy of further in vitro and in vivo studies as a therapy for SCD.TableEffect of cytokine stimulation and cGMP-elevating agents upon adhesive properties of neutrophils from healthy control and steady-state SCD individuals% Neutrophil adhesion to Fibronectin (mean±SEM)ControlsSCDBasal N=18IL-8 (200ng/ml) N≥7TNF-alpha (500ng/ml) N≥7GM-CSF (100ng/ml) N≥7Basal N=18IL-8 (200ng/ml) N≥7TNF-alpha (500ng/ml) N≥7GM-CSF (100ng/ml) N≥7Basal16.1±1.526.6±2.827.4±5.521.4±1.621.4±1.932.5±3.529.2±3.025.2±2.2* p<0.05* p<0.05* p<0.05&p<0.05* p<0.05* p<0.05* p<0.05BAY13.5±2.214.5±2.917.4±4.69.42±2.315.5±1.210.5±3.523.2±3.115.4±1.9736691 (60μM)# p<0.01# p<0.001# p<0.01# p<0.01# p<0.001# p<0.001# p<0.05# p<0.05BAY 412271 (60 nM)17.5±1.923.6±3.825.8±5.018.2±2.119.2±1.220.4±3.5 # p<0.0527.4±2.720.0±1.4&, comp. to control neutrophils (Mann-Whitney); *, comp. to non-cytokine stimulated adhesion; #, comp. to basal adhesion without co-incubation with BAY-736691/412271(Repeated measures ANOVA, Dunn post test) DisclosuresNo relevant conflicts of interest to declare.

Protective effect of 3,5,3′-triiodothyroacetic and 3,5,3′,5′-tetraiodothyroacetic acids on serum albumin fibrillation

Inhibition or reversion of protein self-aggregation has been suggested as a possible preventive mechanism against amyloid diseases, and many efforts are underway to found out molecules capable to restrain the protein aggregation process. In this paper, the inhibitory effects of thyroid hormone analogues on heat-induced fibrillation process of serum albumin are reported. Among the analogues tested, 3,5,3',5'-tetraiodothyroacetic and 3,5,3'-triiodothyroacetic acid showed the most important inhibitory effects on amyloid formation. Thyroxine exhibits a lesser protective effect, while 3,5,3'-triiodothyronine showed no significant inhibition. The gaining of a negative charge together with a size reduction of the hormone molecule could play an essential role in the inhibition of fibrils formation. According to infrared spectroscopy results, the thyroid hormones analogues protective effects proceed via the stabilization of the protein native structure. The current work demonstrates the effectiveness of naturally occurring molecules in the inhibition of albumin fibril formation.