Differential Effects of Ruminant and Industrial 18-Carbon trans-Monounsaturated Fatty Acids (trans Vaccenic and Elaidic) on the Inflammatory Responses of an Endothelial Cell Line

Endothelial dysfunction and inflammation are recognised factors in the development of atherosclerosis. Evidence suggests that intake of industrial trans fatty acids (TFAs) promotes endothelial dysfunction, while ruminant TFAs may have the opposite effect. The aim of this study was to compare the effects of elaidic acid (EA (18:1n-9t); an industrially produced TFA) and trans vaccenic acid (TVA (18:1n-7t); a natural TFA found in ruminant milk and meat) on inflammatory responses of endothelial cells (ECs). ECs (EA.hy926 cells) were cultured under standard conditions and exposed to TFAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments. TFAs were incorporated into ECs in a dose-dependent manner. Preincubation with EA (50 µM) increased production of MCP-1, RANTES, and IL-8 in response to TNF-α, while preincubation with TVA (1 µM) decreased production of ICAM-1 and RANTES in response to TNF-α. Preincubation with EA (50 µM) upregulated toll-like receptor 4 and cyclooxygenase 2 gene expression in response to TNF-α. In contrast, preincubation with TVA (1 µM) downregulated TNF-α induced nuclear factor kappa B subunit 1 gene expression. Preincubation of ECs with EA (50 µM) increased THP-1 monocyte adhesion. In contrast, preincubation of ECs with TVA (1 µM) reduced THP-1 monocyte adhesion, while preincubation of ECs with TVA (50 µM) decreased the level of surface expression of ICAM-1 seen following TNF-α stimulation. The results suggest that TVA has some anti-inflammatory properties, while EA enhances the response to an inflammatory stimulus. These findings suggest differential effects induced by the TFAs tested, fitting with the idea that industrial TFAs and ruminant TFAs can have different and perhaps opposing biological actions in an inflammatory context.     

Green synthesis of selenium based N-heterocyclic carbene compounds; structural,in-vitro anticancer and molecular docking studies

Benzimidazolium salts (3−6) were synthesized as stable N-Heterocyclic Carbene (NHC) precursors and their selenium-NHC compounds/Selenones (7−10) were prepared using water as a solvent. Characterization of each of the synthesized compounds was carried out by various analytical and spectroscopic (FT-IR, ¹H-, ¹³C NMR) methods. X-ray crystallographic analyses of single crystals obtained for salts 3 and 5 were carried out. Synthesized salts and their Se-NHCs were tested in-vitro for their anticancer potential against Cervical Cancer Cell line from Henrietta Lacks (HeLa), Breast cancer cell line (MDA-MB-231), Adenocarcinoma cell line (A549) and human normal endothelial cell line (EA.hy926). MTT assay was used for analysis and compared with standard drug 5-flourouracil. Benzimidazolium salts (3−6) and their selenium counter parts (7−10) were found potent anticancer agents. Salt 3–5 were found to be potent anticancer against HeLa with IC₅₀ values 0.072, 0.017 and 0.241 μM, respectively, which are less than standard drug (4.9 μM). The Se-NHCs (7–10) had also shown significant anticancer potential against HeLa with IC₅₀ values less than standard drug. Salts 3, 4 against EA.hy926, compounds 3,5,6, and 10 against MDA-MB-321, and compounds 4, 10 against A-549 cell line were found more potent anticancer agents with IC₅₀ values less than standard drug. Molecular docking for (7−10) showed their good anti-angiogenic potential having low binding energy and significant inhibition constant values with VEGFA (vascular endothelial growth factor), EGF (human epidermal growth factor), COX1 (cyclooxygenase-1) and HIF (hypoxia inducible factor).     

The Effect of Charge on Cellular Uptake and Phototoxicity of Polylysine Chlorine6Conjugates

Abstract— The effect of charge on the cellular uptake, localization and phototoxicity of conjugates between chlorin_e6 ( c _e6) and poly-L-lysine was studied in vitro. These conjugates (average MW35–55 kDa) were synthesized to have polycationic, polyanionic or neutral charges. Two human cell lines (A431 epidermoid carcinoma cells and EA.hy926 hybrid endothelial cells) were studied and the cellular uptake of c _e6 delivered by the conjugates of varying charge and free c _e6 was measured at conjugate c _e6 equivalent concentrations up to 0.4 μM. Uptake was time and concentration dependent and temperature dependent in the case of neutral and anionic conjugates. Relative uptake at 6 h for A431 cells was 73:15:4:1 and for EA. hy926 cells was 63:11:3:1 for cationic, anionic, neutral and free c _e6, respectively, but EA. hy926 cells took up 1.5-2 times as much c _e6 from all the conjugates as A431 cells. Localization as studied by fluorescence microscopy indicated that the cationic conjugate was in aggregates bound to the plasma membrane, while the other forms were internalized in organelles and membranes. Phototoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetra-zolium bromide (MTT) assay after irradiation with5–20 J cm⁻²of 666 nm light. In contrast to the uptake, the order of phototoxicity for both cell types per mole of c _e6 uptake per cell was neutral ≫ anionic > cationic > free c _e6. Polymeric c _e6 conjugates bearing positive, negative and neutral charges may have different tissue-localizing properties and could play a role in photodynamic therapy.     

Unsymmetrically substituted benzimidazolium based Silver(I)-N-heterocyclic carbene complexes: Synthesis,characterization and in vitro anticancer study against human breast cancer and colon cancer

The promising biomedical applications of silver complexes stimulated the researchers to test these compounds against cancer. The present research work was designed to achieve this goal. In this work, a series of 5-methyl benzimidazole based N-Heterocyclic carbene ligands and respective silver(I) complexes were synthesized and tested on cancer cell lines to assess their anticancer activity. Unsymmetrically substituted benzimidazole was found unique in its reactivity and generation of a single product during NHC ligand formation was only possible after two successive alkylations with same alkyl halide. The corresponding Ag(I)-NHC adducts were obtained by in situ deprotonation of the NHC ligands. Synthesized compounds were characterized by various physcio-chemical and spectroscopic methods. Single crystal X-ray diffraction study of complex 7 revealed its mononuclear structure. Preliminary in vitro anticancer study of azolium salts and respective Ag(I)-NHC complexes against human breast cancer (MDA-MB-231), colon cancer (HCT-116) and normal endothelial cells (EA.hy926) cells revealed that all the compounds are more cytotoxic to cancer cells than normal cells and the complexes are relatively more potent compared to the corresponding NHC ligands. It was found that increased chain length and presence of methyl substituent on benzimidazole ring enhance the biopotency of Ag(I)-NHC complexes. The synthesized compounds were further studied for pro-apoptotic mechanism of action via Rhodamine 123 test. The tested compounds were found to induce apoptosis via extrinsic mitochondrial pathway.     

5,2′-Dibromo-2,4′,5′-trihydroxydiphenylmethanone Inhibits LPS-Induced Vascular Inflammation by Targeting the Cav1 Protein

Vascular inflammation is directly responsible for atherosclerosis. 5,2′-Dibromo-2,4′,5′-trihydroxydiphenylmethanone (TDD), a synthetic bromophenol derivative, exhibits anti-atherosclerosis and anti-inflammatory effects. However, the underlying pathways are not yet clear. In this study, we first examined the effects of TDD on toll-like receptor-4 (TLR4) activity, the signaling receptor for lipopolysaccharide (LPS), and found that TDD does not inhibit LPS-induced TLR4 expression in EA.hy926 cells and the vascular wall in vivo. Next, we investigated the global protein alterations and the mechanisms underlying the action of TDD in LPS-treated EA.hy926 cells using an isobaric tag for the relative and absolute quantification technique. Western blot analysis revealed that TDD inhibited NF-κB activation by regulating the phosphorylation and subsequent degradation IκBα. Among the differentially expressed proteins, TDD concentration-dependently inhibited Caveolin 1(Cav1) expression. The interaction between Cav1 and TDD was determined by using biolayer interference assay, UV-vis absorption spectra, fluorescence spectrum, and molecular docking. We found that TDD can directly bind to Cav1 through hydrogen bonds and van der Waals forces. In conclusion, our results showed that TDD inhibited LPS-induced vascular inflammation and the NF-κB signaling pathway by specifically targeting the Cav1 protein. TDD may be a novel anti-inflammatory compound, especially for the treatment of atherosclerosis.     

Quantification of intracellular homocysteine by stable isotope dilution liquid chromatography/tandem mass spectrometry

A precise and accurate stable isotope dilution liquid chromatography/tandem mass spectrometry method for the analysis of intracellular homocysteine has been developed. An internal standard, [(2)H(8)]-homocystine, was added to cell pellets from EA.hy 926 cells grown in culture under low and high folate concentrations. D,L-dithiothreitol was used to reduce cellular homocystine to homocysteine. Cellular proteins were precipitated by the addition of formic acid in acetonitrile. After centrifugation, a portion of the supernatant was analyzed by liquid chromatography/tandem mass spectrometry. Using a Supelcosil cyano column with an Applied Biosystems API 4000 triple quadrupole mass spectrometer, the SRM transitions for homocysteine (m/z 136 to m/z 90) and [(2)H(4)]-homocysteine (m/z 140 to m/z 94) were monitored. The method was validated by conducting five replicate analyses on three different days at four different concentrations (concentrations at the lower limit of quantitation and expected lower quartile, mid-range and upper quartile). The limit of detection was 2 ng/10(6) EA.hy 926 cells. Using this method, the intracellular homocysteine concentration in EA.hy 926 cells ranged from 10 to 36 ng/10(6) cells.     

Enzymatic inhibitory activity of Ficus deltoidea leaf extract on matrix metalloproteinase-2, 8 and 9

Dysregulation of matrix metalloproteinases (MMPs) activity is known in many pathological conditions with which most of the conditions are related to elevate MMPs activities. Ficus deltoidea (FD) is a plant known for its therapeutic properties. In order to evaluate the therapeutic potential of FD leaf extract, we study the enzymatic inhibition properties of FD leaf extract and its major bioactive compounds (vitexin and isovitexin) on a panel of MMPs (MMP-2, MMP-8 and MMP-9) using experimental and computational approaches. FD leaf extract and its major bioactive compounds showed pronounced inhibition activity towards the MMPs tested. Computational docking analysis revealed that vitexin and isovitexin bind to the active site of the three tested MMPs. We also evaluated the cytotoxicity and cell migration inhibition activity of FD leaf extract in the endothelial EA.hy 926 cell line. Conclusively, this study provided additional information on the potential of FD leaf extract for therapeutical application.     

Fabrication of biodegradable poly(trimethylene carbonate) networks for potential tissue engineering scaffold applications

Biodegradable poly(trimethylene carbonate) (PTMC) networks were prepared by photopolymerization of linear (L)‐ and star (S)‐shaped PTMC macromonomers for potential tissue engineering scaffold applications. The L‐ (M_n, 6400) and S‐shaped (M_n, 5880) PTMC macromonomers were synthesized using 1,4‐butane diol and 2‐ethyl‐ 2‐hydroxyl‐propane‐1,3‐diol co‐initiated ring‐opening polymerization of trimethylene carbonate (TMC) in the presence of stannous octoate and subsequent acrylation with acryloyl chloride. Chemical structures of the PTMC macromonomers and their corresponding networks were characterized by ¹H NMR and ¹³C NMR spectroscopy. The human endothelial cell line, EA.hy926 was used to test the biocompatibility, cell adhesion, and proliferation behavior of both PTMC networks. The PTMC networks made from the S‐shaped macromonomers exhibited superior cell adhesion and proliferation behavior than those made of the linear macromonomers. Copyright © 2008 John Wiley & Sons, Ltd.