Damaging effects of advanced glycation end product in the murine macrophages cell line J774A.1.

The interaction of reducing sugars, such as aldose, with proteins and the subsequent molecular rearrangements, produces irreversible advanced glycation end-products (AGEs), a heterogeneous class of non-enzymatic glycated proteins or lipids. AGEs form cross-links, trap macromolecules and release reactive oxygen intermediates. AGEs are linked to aging, and increase in several related diseases. The aim of this study was to assess, in a murine macrophage cell line, J774A.1, the effects of 48 h of exposure to glycated serum containing a known amount of pentosidine, a well-known AGE found in the plasma and tissues of diabetic and uremic subjects. Fetal bovine serum was incubated with ribose (50 mm) for 7 days at 37 °C to obtain about 10 nmol/ml of pentosidine. The cytotoxic parameters studied were cell morphology and viability by neutral red uptake, lactate dehydrogenase release and tetrazolium salt test. In the medium and in the intracellular compartment, bound and free pentosidine were evaluated by HPLC, as sensitive and specific glycative markers, and thiobarbituric acid reactive substances (TBARs), as index of the extent of lipid peroxidation. Our results confirm that macrophages are able to take up pentosidine. It is conceivable that bound pentosidine is degraded and free pentosidine is released inside the cell and then into the medium. The AGE increase in the medium was combined with an increase in TBARs, meaning that an oxidative stress occurred; marked cytotoxic effects were observed, and were followed by the release of free pentosidine and TBARs into the culture medium.

Antioxidant status in J774A.1 macrophage cell line during chronic exposure to glycated serum

Advanced glycation end-products (AGEs) are linked to aging and correlated diseases. The aim of present study was to evaluate oxidative stress related parameters in J774A.1 murine macrophage cells during chronic exposure to a subtoxic concentration of AGE (5% ribose-glycated serum (GS)) and subsequently for 48 h to a higher dose (10% GS). No effects on cell viability were evident in either experimental condition. During chronic treatment, glycative markers (free and bound pentosidine) increased significantly in intra- and extracellular environments, but the production and release of thiobarbituric acid reactive substances (TBARs), as an index of lipid peroxidation, underwent a time-dependent decrease. Exposure to 10% GS evidenced that glycative markers rose further, while TBARs elicited a cellular defence against oxidative stress. Nonadapted cultures showed an accumulation of AGEs, a marked oxidative stress, and a loss of viability. During 10% GS exposure, reduced glutathione levels in adapted cultures remained constant, as did the oxidized glutathione to reduced glutathione ratio, while nonadapted cells showed a markedly increased redox ratio. A constant increase of heat shock protein 70 (HSP70) mRNA was observed in all experimental conditions. On the contrary, HSP70 expression became undetectable for a longer exposure time; this could be due to the direct involvement of HSP70 in the refolding of damaged proteins. Our findings suggest an adaptive response of macrophages to subtoxic doses of AGE, which could constitute an important factor in the spread of damage to other cellular types during aging.Key words: in vitro cytotoxicity, AGE, pentosidine, glycoxidation, oxidative stress, TBARs.

The effect of melanocortin peptides on Interleukin 1-beta induced chondrocyte cell death and inflammation

ntroduction: Osteoarthritis (OA) is a degenerative joint disease affecting more than 8.5 million people in the UK. Disruption in the catabolic and anabolic balance, with the catabolic cytokine Interleukin 1 beta (IL-1β) being involved in the initiation and progression of OA (1). Melanocortin peptides (α-MSH and D[Trp8]-γ-MSH) exert their anti-inflammatory effects via activation of melanocortin receptors (MC), with both MC1 and MC3 being identified as promising candidates as novel targets for OA (2). This study aims to assess the chondroprotective and anti-inflammatory effects of the pan melanocortin receptor agonist α-MSH and MC3 agonist D[Trp8]-γ-MSH following IL-1β chondrocyte stimulation. Methods: RT-PCR/ Western Blot: Human C-20/A4 chondrocytic cell-line were cultured in 6 well plates (1x106 cells/well) and harvested to determine MC and IL-1β expression by RT-PCR, and Western Blot. Cell-Culture: Cells were cultured in 96 well plates (1x106 cells/well) and stimulated with H2O2 (0.3%), TNF-α (60 pg/ml) or IL-1β (0-5000pg/ml) for 0-72h and cell viability determined. Drug Treatment: In separate experiments cells were pre-treated with 3 μg/ml α-MSH (Sigma-Aldrich Inc. Poole, UK), or D[Trp8]-γ-MSH (Phoenix Pharmaceuticals, Karlsrhue, Germany) (all dissolved in PBS) for 30 minutes prior to IL-1β (5000pg/ml) stimulation for 6-24h. Analysis: Cell viability was determined by using the three cell viability assays; Alamar Blue, MTT and the Neutral Red (NR) assay. Cell-free supernatants were collected and analysed for Interleukin -6 (IL-6) and IL-8 release by ELISA. Data expressed as Mean ± SD of n=4-8 determination in quadruplicate. *p≤ 0.05 vs. control. Results: Both RT-PCR, and Western Blot showed MC1 and MC3 expression on C-20/A4 cells. Cell viability analysis: IL-1β stimulation led to a maximal cell death of 35% at 6h (Alamar Blue), and 40% and 75% with MTT and Neutral Red respectively at 24h compared to control. The three cell viability assays have different cellular uptake pathways, which accounts for the variations observed in cell viability in response to the concentration of IL-1β, and time. Cytokine analysis by ELISA: IL-1β (5000pg/ml) stimulation for 6 and 24h showed maximal IL-6 production 292.3 ±3.8 and 275.5 ±5.0 respectively, and IL-8 production 353.3 ±2.6 and 598.3 ±8.6 respectively. Pre-treatment of cells with α-MSH and D[Trp8]-γ-MSH caused significant reductions in both IL-6 and IL-8 respectively following IL-1β stimulation at 6h. Conclusion: MC1/3 are expressed on C-20/A4 cells, activation by melanocortin peptides led to an inhibition of IL-1β induced cell death and pro-inflammatory cytokine release.

Effect of Short-Chain Fatty Acid Acetate on Colon Cancer

Acetate is a short chain fatty acid produced as a result of fermentation of ingested fibers by the gut microbiota. While it has been shown to reduce cell proliferation in some cancer cell lines1,2, more recent studies on liver3 and brain4 tumours suggest that acetate may actually promote tumour growth. Acetate in the cell is normally converted into acetyl-coA by two enzymes and metabolized; mitochondrial (ACSS1) and cytosolic (ACSS2) acetyl-coA synthetase. In the mitochondria acetyl-coA is utilized in the TCA cycle. In the cytosol it is utilized in lipid synthesis. In this study, the effect of acetate treatment on the growth of HT29 colon cancer cell line and its mechanism of action was assessed. HT29 human colorectal adenocarcinoma cells were treated with 10mM NaAc and cell viability, cellular bioenergetics and gene expression were investigated. Cell viability was assessed 24 hours after treatment using an MTT assay (Sigma, UK, n=8). Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured by XFe Analyzer (Seahorse Bioscience, USA). After a baseline reading cells were treated and OCR and ECAR measurements were observed for 18 hours (n=4). Total mRNA was isolated 24 hours after treatment using RNeasy kit (Qiagen, USA). Quantitative PCR reactions were performed using Taqman gene expression assays and Taqman Universal PCR Master Mix (ThermoFisher Scientific, UK) on Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, USA) and analysed using ΔΔCt method (n=3). Acetate treatment led to a significant reduction in cell viability (15.9%, Figure 1). OCR, an indicator of oxidative phosphorylation, was significantly increased (p<0.0001) while ECAR, an indicator of glycolysis, was significantly reduced (p<0.0001, Figure 2). Gene expression of ACSS1 was increased by 1.7 fold of control (p=0.07) and ACSS2 expression was reduced to 0.6 fold of control (p=0.06, Figure 3). In conclusion, in colon cancer cells acetate supplementation induces cell death and increases oxidative capacity. These changes together with the trending decrease in ACSS2 expression suggest suppression of lipid synthesis pathways. We hypothesize that the reduced tumor growth by acetate is a consequence of the suppression of ACSS2 and lipid synthesis, both effects reported previously to reduce tumor growth3–5. These effects clearly warrant further investigation.