Effects of fish oil on lymphocyte proliferation,cytokine production and intracellular signalling in weanling pigs

It has been widely documented that fish oil attenuates inflammatory responses partially via down-regulation of T-lymphocyte function. To determine the anti-inflammatory role of fish oil in weanling pigs, we investigated the effects of fish oil and its functional constituents on peripheral blood lymphocyte proliferation, cytokine production and subsequent intracellular signalling in inflammatory-challenged weanling pigs and in in vitro cultured lymphocytes. Fish oil (7%) or corn oil (7%) was supplemented to 72 crossbred pigs (7.6 ± 0.3 kg BW and 28 ± 3 days of age) in a 2 × 2 factorial experiment that included an Escherichia coli lipopolysaccharide (LPS) challenge (challenged or not challenged). On day 14 and 28 of the experiment, 200 μg/kg BW of LPS or an equivalent amount of sterile saline was administered to the pigs by intraperitoneal injection. Blood samples were collected on days 15 and 29 to determine peripheral blood lymphocyte proliferation, interleukin-1β (IL-1β) and interleukin-2 (IL-2) production. The results showed that inflammatory challenge decreased average daily gain (P < 0.05) and average daily feed intake (P < 0.05) during days 15 - 28. Fish oil supplementation had no effect on growth performance. Inflammatory challenge increased lymphocyte proliferative response to concanavalin A (Con A) (P < 0.05) following each challenge. Fish oil tended to suppress (P < 0.1) the proliferation following the first challenge. Similarly, fish oil tended to reduce IL-1β production (P < 0.1) following the second challenge and IL-2 (P < 0.1) production following the first challenge in both challenged and unchallenged pigs compared with corn oil. In parallel in vitro experiments, peripheral blood lymphocytes of weanling pigs were incubated with various concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or linoleic acid (LA) (0, 20, 40, 60, 80, 100 μg/ml). EPA, DHA and high levels of LA predominantly suppressed IL-1β (P < 0.05), IL-2 (P < 0.05) production and subsequent lymphocyte proliferation (P < 0.05). Low levels of LA increased (P < 0.05) IL-2 production. Compared with LA, EPA resulted in a stronger inhibition of lymphocyte proliferation (P < 0.05) and IL-2 (P < 0.01), and DHA resulted in a stronger inhibition of IL-1β (P < 0.05) and IL-2 (P < 0.01). To elucidate the mechanism(s) by which fish oil and its functional constituents suppressed lymphocyte function, the kinetics of intracellular [Ca^{2 +}]_i and protein kinase C activity were determined in in vitro experiments. EPA, DHA and LA exerted very similar dose-dependent stimulatory effects on intracellular Ca^{2 +}. EPA and DHA inhibited protein kinase C activity (P < 0.05), while LA had no significant effect (P > 0.05). These results suggest that fish oil and its functional constituents (EPA and DHA) exerted an anti-inflammatory effect by down-regulation of lymphocyte activation in weanling pigs, possibly by manipulation of intracellular signalling.     

Potential urinary and plasma biomarkers of peroxisome proliferation in the rat: identification of N-methylnicotinamide and N-methyl-4-pyridone-3-carboxamide by 1H nuclear magnetic resonance and high performance liquid chromatography

This study identified two potential novel biomarkers of peroxisome proliferation in the rat. Three peroxisome proliferator-activated receptor (PPAR) ligands, chosen for their high selectivity towards the PPARα, -δ and -γ subtypes, were given to rats twice daily for 7 days at doses known to cause a pharmacological effect or peroxisome proliferation. Fenofibrate was used as a positive control. Daily treatment with the PPARα and -δ agonists produced peroxisome proliferation and liver hypertrophy. ¹H nuclear magnetic resonance spectroscopy and multivariate statistical data analysis of urinary spectra from animals given the PPARα and -δ agonists identified two new potential biomarkers of peroxisome proliferation - N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY) - both endproducts of the tryptophan-nicotinamide adenine dinucleotide (NAD⁺) pathway. After 7 days, excretion of NMN and 4PY increased 24- and three-fold, respectively, following high doses of fenofibrate. The correlation between total NMN excretion over 7 days and the peroxisome count was r=0.87 (r²=0.76). Plasma NMN, measured using a sensitive high performance liquid chromatography method, was increased up to 61-fold after 7 days' treatment with high doses of fenofibrate. Hepatic gene expression of aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) was downregulated following treatment with the PPARα and -δ agonists. The decrease was up to 11-fold compared with controls in the groups treated with high doses of fenofibrate. This supports the link between increased NMN and 4PY excretion and regulation of the tryptophan-NAD⁺ pathway in the liver. In conclusion, NMN, and possibly other metabolites in the pathway, are potential non-invasive surrogate biomarkers of peroxisome proliferation in the rat.     

Difference in Ki67 and Thymidylate Synthase Expression in Primary Tumour Compared with Metastatic Nodes in Breast Cancer Patients

Breast cancer is a heterogeneous disease, so therapeutic predictive biological markers need to be identified. To date an accurate evaluation of predictive markers is mainly done at the primary site; however, the main goal of adjuvant therapy for breast cancer is the control of micrometastases. The aim of this study is to assess as therapeutic and/or prognostic marker, the proliferation status of primary tumors and involved nodes as measured by Ki67 and thymidylate synthase (TS) expression, in 30 breast cancer node positive patients. TS is the main target of 5-fluorouracil (5-FU) activity, and its overexpression is one of the mechanisms of 5-FU drug resistance; however, in some studies its absence is responsible for a worse response to 5-FU. Our results show that malignant cells of involved nodes were in a post mitotic phase of the cell cycle, and show a low proliferation index and TS expression, while the primary tumours and controls, were strongly positive. On these basis we can hypothesize that these cells could be less sensitive to 5-FU. Further studies are necessary to identify other mechanisms responsible for their metastasing capability and/or for their aggressiveness.     

乙酰胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchR1表达的影响

用CCK-8比色法和流式细胞术,检测乙酰胆碱（acetylcholine,Ach）及拮抗剂阿托品（atropine,Atro）对SK-N-SH细胞增殖活性和周期分布的调节作用;进一步用荧光定量PCR、免疫印迹和流式细胞间接免疫荧光技术,分析SK-N-SH细胞毒蕈碱受体亚型Ⅰ型（mAchR1）和c-fos的表达差异。结果表明,1mmol／L Ach对SK-N-SH细胞有明显促增殖作用,而1mmoL／L Atro阻滞细胞从S期向G2／M期移行;1mmol／L Ach与1mmol／L Atro均反馈调节mAchR1的蛋白水平。但mAchR1 mRNA的表达不受影响;1mmol／L Ach显著上调c-fos mRNA和Fos蛋白的表达,但这种作用可被Atro逆转。提示胆碱类受体参与配基对肿瘤细胞的促增殖作用。     

Effect of Molecular Characteristics on Cellular Uptake,Subcellular Localization,and Phototoxicity of Zn(II) N-Alkylpyridylporphyrins

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.     

MEKK2 Kinase Association with 14-3-3 Protein Regulates Activation of c-Jun N-terminal Kinase

MEKK2 (MAP/ERK kinase kinase-2) is a serine/threonine kinase that belongs to the MEKK/STE11 family of MAP kinase kinase kinases (MAP(3)Ks). MEKK2 integrates stress and mitogenic signals to the activation of NF-κB, JNK1/2, p38, and ERK5 pathways. We have found that MEKK2 is regulated through a phosphorylation-dependent association with 14-3-3, a group of adapters that modulate dimerization and association between proteins. We found that MEKK2 was phosphorylated at Thr-283, which resulted in decreased activation loop phosphorylation at Ser-519 and consequently reduced activity. Mechanistically, we found that MEKK2 associated with inactive MEKK2 in the absence of 14-3-3 binding, which led to trans-autophosphorylation of Ser-519. Enforced binding with 14-3-3 reduced Ser-519 trans-autophosphorylation. Expression of T283A MEKK2 within a MEKK2^−/− background enhanced stress-activated c-Jun N-terminal kinase activity while elevating IL-6 expression, but also reduced ERK activation with a corresponding reduced proliferation rate. These results indicate that Thr-283 phosphorylation is an important regulatory mechanism for MEKK2 activation.     

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na/Ca exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca²⁺ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca²⁺ concentration [Ca²⁺]_i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca²⁺-mediated [Ca²⁺]_i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca²⁺ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca²⁺ entry. KB-R7943, a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and/or replacement of Na⁺ with NMDG⁺ inhibited thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca²⁺ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca²⁺ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca²⁺ influx pathways requiring Ca²⁺ signals for its effect on fibroblast proliferation.     

The stimulatory effect of ROCK inhibitor on bovine corneal endothelial cells

Reagents which can promote the proliferation, adhesion and migration of cultured corneal endothelial cells (CECs) will be helpful for the treatment of reduced visual acuity due to CECs deficiency. The objectives of this study were to investigate the potential use of an inhibitor of Rho-associated protein kinase (ROCK), Y-27632, to cultured bovine corneal endothelial cells (B-CECs) and evaluated its effects on the proliferation, adhesion and migration of B-CECs. The proliferation of cultured B-CECs was moderately enhanced by 10 μM Y-27632. Y-27632 induced fibroblast-like morphological changes in the cultured B-CECs and normal cell morphology could recover after Y-27632 removal. In addition, Y-27632 was found to significantly enhance the adhesion and migration of B-CECs. Furthermore, the hanging drop aggregation assay showed that Y-27632 promoted B-CECs to form cellular networks and sheets, which proliferated along the liquid–air interface and migrated to the surface of the lid of dish. Our study demonstrated that Y-27632 is a potentially powerful reagent which can enhance the proliferation of cultured B-CECs. Y-27632 will be useful in CEC injection therapy and topical application for CEC deficiency.     

Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg²⁺ concentrations. Changes in Mg²⁺ concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca²⁺/Mg²⁺ ratio facilitated Ca²⁺ influx in both control and prostate cancer cells, a significantly higher Ca²⁺ influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca²⁺/Mg²⁺ ratio per se. Additionally, an increase in the extracellular Ca²⁺/Mg²⁺ ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca²⁺/Mg²⁺ ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca²⁺/Mg²⁺ ratio could be essential for the initiation/progression of prostate cancer.     

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.