J Cachexia Sarcopenia Muscle (2011) 2:181–188 Table 2 Gene expression in skeletal muscle of mice with cancer and cancer cachexia Gene expression (AU) NOX2 p22phox p40phox p47phox p67phox NOX4 Rac1 SOD1 SOD2 SOD3 Gpx Catalase Cancer 11.8±2.3 88.7±2.9 21.4±2.8 5.7±1.2 367±92.7 4.3±0.6 3.6±1.1 350±36.5 122±8.0 2.6±0.3 23.5±2.3 49.3±7.3 Cachexia 5.0±1.1* 75.5±10.5 7.9±1.6* 7.3±1.4 15.6±3.3* 8.5±2.7 7.5±1.6 120±37.8* 59.4±5.4* 1.8±0.3 12.8±1.2* 43.2±5.6 The values represent the mean±SEM *P<0.003, statistically significant differences cancer alone (p < 0.001, Table 2). However, the antioxidant enzymes SOD3 and catalase had similar mRNA levels in both groups (Table 2). 3.4 Protein levels of NOX2 enzyme subunits in skeletal muscle of cancer and cancer cachectic mice Changes in the mRNA levels of the key NOX2 enzyme subunits—NOX2, p40phox and p67phox—lead to further analysis of protein expression. The protein expression of the regulatory and catalytic NOX2 enzyme subunits NOX2 and p40phox were lower in cachectic mice when compared to mice with cancer alone (p = 0.021; p < 0.012, Fig. 2). However, the protein expression of p67phox was similar in skeletal muscle from mice in both groups (Fig. 2). 3.5 Antioxidant enzyme activity and hydrogen peroxide levels in skeletal muscle of cancer and cancer cachectic mice To validate changes in mRNA and protein levels, SOD1, SOD2, catalase and GPx activity levels were measured in skeletal muscle of mice with cancer and cancer cachexia. SOD1 and GPx activities were significantly lower in skeletal muscle from cachectic mice (SOD1 9.9±0.9 U/mg protein, GPx 0.74±0.22 mmol/min/mg protein) when com- pared to mice with cancer alone (SOD1 11.2±0.8 U/mg protein, GPx 1.48±0.08 mmol/min/mg protein) (p<0.003, Table 3). However, levels of SOD2 and catalase activity were similar in both groups of (Table 3). H2O2 levels were similar (A) 15 n i treepo isseno10 itlaev repx 5 R 0 (B) 15 n i treepo isseon10 tilaev rexp 5 R 0 (C) 15 n i treepo isseon10 itlaev repx 5 R 0 Cancer Cachexia Cancer Cachexia * * * 185 NOX2 GAPDH p40 phox GAPDH p67 phox GAPDH Cancer Cachexia Fig. 2 Protein expression levels of NOX2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia. The values represent the mean±SEM. Statistically significant differences *P<0.05. a NOX2, b p40phox, c p67pho in skeletal muscle from cachectic mice and mice with cancer alone (Table 3).