Protein levels of nitric oxide synthase (NOS2) were also inhibited in cells treated with the GTA+ve fraction (particularly 20 and 40 ug/ml), but not in cells treated with the GTA-ve fraction (Figure 5). Figure 5 Western analysis of NFκB, IκBα and NOS2 in SW620 cells treated with three concentrations of GTA+ve and GTA-ve extracts and doxorubicin (DOX). Representative

Western blots showing protein levels of NFκB, IκBα and NOS2 in SW620 cells treated with GTA+ve and GTA-ve extracts (see ABT-888 mouse methods). To explore further the effect of GTAs on modulating inflammation, we employed the RAW264.7 mouse macrophage line in which a pro-inflammatory state can be induced by treatment with lipopolysaccharide (LPS). RAW264.7 cells were treated for 4 hours with GTA+ve and GTA-ve fractions prior to the addition of LPS, and the effects on various proinflammatory markers evaluated. We observed no affect on RAW264.7 cell growth or proliferation rates during the 20 hours post-GTA treatment. RAW264.7 THZ1 cost cells treated with GTA+ve fractions prior to LPS stimulation showed a significant dose-dependent reduction (p < 0.05) in the generation of nitric oxide as assessed through the production of nitrite using the Griess reagent system (Figure 6A), which was mirrored by low levels of NOS2 mRNA Epigenetics inhibitor transcripts (Figure 6B) and protein levels (Figure 6C). For comparison (and as controls), cells were also

treated with various combinations of free fatty acids including EPA, DHA and equimolar mixtures of 18:1, 18:2 and 18:3 (FA mix), of which only 100 uM DHA showed any protective effect on NOS2 protein induction (Figure 6C). Figure 6 Determination of nitric oxide status in RAW264.7 cells treated with GTA+ve and GTA-ve extracts. RAW264.7 cells were pre-treated for 4 hours with GTA+ve or GTA-ve extracts followed by the addition of LPS (1 ug/ml) for 20 hours. (A) Nitric oxide levels in cells were determined using Griess reagent, (B) NOS2 mRNA transcript levels were determined by real-time rtPCR, and (C) NOS protein (treatment with

80 ug/ml) assessed by Western blot (NS, non-specific). Asterisks indicate p < 0.05 relative to LPS treatment alone, and FA mix in (C) represents a 100 uM equal mixture of 18:1, 18:2 and 18:3 fatty acids. Data are expressed as the average of three duplicate experiments ± 1S.D. Similar effects were observed with TNFα upon treatment with 17-DMAG (Alvespimycin) HCl GTA+ve extract, which showed significantly reduced mRNA transcript levels (p < 0.05, Figure 7A) as well as protein levels in cell lysates and conditioned media (Figures 7B and 7C, respectively). Consistent with the above findings, transcript levels of COX2 and IL-1β (Figures 8A and 8B), as well as IL-1β protein levels (Figure 8C), were also significantly reduced (p < 0.05) with GTA+ve treatment. The results indicate that human blood extracts containing GTAs have anti-proliferative and anti-inflammatory properties that GTA-ve extracts lack. Figure 7 TNFα response in RAW264.7 cells treated with GTA+ve and GTA-ve extracts.