184 2.6 Statistical analysis Statistical analysis was performed using SPSS statisti- cal package (version 15.0). Results are expressed as mean ± SEM. Differences were determined by one-way ANOVA with Tukey HSD as posthoc to determine significant differences between groups, and results were considered statistically significant if p values were equal to or <0.05. 3 Results 3.1 Establishment of murine model of cancer and cancer cachexia Female BALB/c nu/nu mice were implanted with the MAC13 cell line (n = 12) that had previously developed a tumour in the same mouse model at approximately 9– 12 days post implantation (n = 3) and where the animal did not lose weight. Another group of female BALB/c nu/nu mice were implanted with the MAC16 cell line (n = 16) that had previously developed a tumour in the same mouse model at approximately 9–12 days post implantation (n = 4) and lost approximately 15–25% of their original body weight were used in this study. Mouse body weight and skeletal muscle weights were recorded at tissue collection and evaluated as a measure of body mass and skeletal muscle cachexia for all mice. Body weight and quadricep weights were significantly less in cachectic (MAC16 tumour-bearing mice) when compared to mice with cancer alone (bearing MAC13 tumours) (p < 0.001; p = 0.003, Table 1). To account for any differences in calorie intake between the groups, food intake was recorded throughout the course of the study and showed no differences between cachectic and cancer mice (Table 1). 3.2 O2− levels in skeletal muscle of cancer and cancer cachectic mice O2− levels were measured by histological DHE fluores- cence in skeletal muscle sections from mice with cancer Table 1 Mean body weight, quadriceps weight and food intake of mice with cancer and cancer cachexia Weight Body weight (g) Quadriceps (mg) Food intake (g) The values represent the mean±SEM *P<0.003, statistically significant differences Cancer 22.3±0.3 117.0±4.2 3.6±0.3 Cachexia 17.1±0.3* 90.1±2.1* 3.6±0.5 J Cachexia Sarcopenia Muscle (2011) 2:181–188 and cachexia. Skeletal muscle O2− levels were significantly higher in cachectic mice (9.36 ± 2.67 AU) when com- pared to mice with cancer (6.47 ± 0.77 AU) alone (p = 0.001, Fig. 1). 3.3 mRNA expression of pro-oxidant and antioxidant enzymes in skeletal muscle of cancer and cancer cachectic mice The mRNA level of the NOX2 and NOX4 enzyme systems previously shown to express in skeletal muscle to generate O2− were measured in mice with cancer and cancer cachexia. The mRNA of the regulatory and catalytic NOX2 enzyme subunits NOX2, p40phox and p67phox were lower in the skeletal muscle of cachectic mice when compared to mice with cancer alone (p = 0.031; p < 0.001; p = 0.002, Table 2). However, the mRNA expression of the additional NOX enzyme subunits, NOX4, p22phox and p47phox and Rac1 were similar in skeletal muscle from mice bearing with cancer and cancer cachexia (Table 2). The mRNA level of SOD antioxidant enzymes responsible for the dismutation of O2− were measured in mice with cancer and cancer cachexia. The mRNA level of the SOD1 and SOD2 were significantly lower in the skeletal muscle from cachectic mice when compared to mice with cancer alone (p = 0.007; p < 0.001, Table 2). Similarly, the mRNA level of the H2O2 scavenging antioxidant enzyme GPx was lower in skeletal muscle from cachectic mice compared to mice with (A) (B) 15 f o s e tin cn10 u e c y s tirrab lrfeou 5 r 0 A * Cancer Cachexia Cancer * Cachexia Fig. 1 O2− levels in the skeletal muscle of mice with cancer and cancer cachexia. The values represent the mean±SEM. Statistically significant difference *P=0.001. a O2− levels detected by dihydroe- thidium (DHE) fluorescence expressed as arbitrary units of fluores- cence . The values represent the mean±SEM. Statistically significant difference *P=0.001. b Histological DHE fluorescence in the skeletal muscle of mice with cancer and cancer cachexia