186 Table 3 Enzyme activity and H2O2 levels in skeletal muscle of mice with cancer and cancer cachexia SOD1 (U/mg protein) SOD2 (U/mg protein) Catalase (mmol/min/mg protein) GPx (mmol/min/mg protein) H2O2 (μM) The values represent the mean±SEM *P<0.003, statistically significant differences 4 Discussion Cancer 23.3±0.8 11.2±0.8 4.1±0.4 1.48±0.08 1.11±0.07 Cachexia 17.8±0.9* 9.9±0.8 3.5±0.2 0.74±0.22* 1.15±0.04 Levels of O2− were increased in cancer-induced skeletal muscle cachexia. Evidence of this was found with increased levels of O2− detected by DHE fluorescence in the skeletal muscle of cachectic mice, when compared to non-cachectic mice. However, the NOX enzyme systems do not appear to be the source of increased O2− in cancer-induced skeletal muscle cachexia. In fact, we observed a decrease in the expression of the NOX2 enzyme subunits, NOX2, p40phox and p67phox in cachectic skeletal muscle. Although this study would imply an additional source of O2− production in cancer-induced skeletal muscle cachexia, what may be of greater importance is the decrease in antioxidant enzyme function, primarily responsible for O2− dismutation. While this study may be indicative of a lack of compensation by SOD1 and GPx, it may give evidence for their depression, in response to cancer induction and lack of dismutation of even normal cellular O2− production. The decrease in antioxidant gene expression and activity that function as part of the cellular defence system, for the elimination of O2− and H2O2, may indicate antioxidant dysfunction in cachectic skeletal muscle, rather than an increase in ROS production. Cancer induces changes in skeletal muscle that lead to an imbalance in protein synthesis and degradation, resulting in muscle protein loss and function [1]. Interestingly, not all cancer patients develop this secondary condition, and it is this phenomena that has made the development of cancer cachexia relatively undefined [1, 2]. In order to investigate this complex condition, we investigated a model that utilises two similar MAC models, only one of which induces the secondary muscle wasting condition of cancer cachexia. The significant decline in body weight (15–25%) and skeletal muscle mass that was observed in mice bearing the MAC16 tumour, demonstrated the development of cancer-induced skeletal muscle cachexia. While the MAC16 cancer cachectic model is well-established in NMRI mice [4], this study mimics the cachectic condition in MAC16 tumour-bearing nude mice. This model of cancer and cancer cachexia establishes a direct comparison between J Cachexia Sarcopenia Muscle (2011) 2:181–188 a cancer control and cancer cachectic mouse model. Contrasting studies have implicated the importance of calorie restriction in the development of cancer cachexia; however, our study, like others [18], did not observe any change in food intake from cachectic mice. This study therefore suggests a more complex catabolic-mediated response to protein degeneration, in the development of cancer cachexia. The physical changes in skeletal muscle were mirrored by significant cellular oxidative changes in response to MAC16 induction and cachectic development. In particular, the significant increase in O2− levels observed in cachectic skeletal muscle indicates that O2− is implicated in the cancer cachectic condition. Previous studies have proposed a central mediating role for ROS in the development of cancer cachexia [5, 18]; however, the source of ROS in this process remains to be elucidated. The knowledge that the NOX enzyme system functions primarily to produce O2− [19], recognises NOX-generated O2− as more than a by- product of cellular metabolism, but rather a product of a regulated response to stimuli for a physiological purpose. This function of NOX makes this enzyme system the potential source of O2−, vulnerable to changes associated with the cancer condition and development of cachexia. To our knowledge, this study is the first to investigate the NOX enzymes in cancer cachectic skeletal muscle. Inter- estingly, the NOX2 enzyme has consistently been shown to increase in degenerative conditions [19]; however, this study does not support an increase in the NOX2 enzyme in cancer cachexia. It is evident, however, from this study and others, that the absence of a functioning antioxidant enzyme system to dismutate O2− is present in cancer cachectic skeletal muscle [20]. Cellular oxidative stress has been implicated in skeletal muscle wasting conditions with marked attenuation following antioxidant induction [21]. Despite the increase in O2- in cachectic skeletal muscle that is indicative of a need for SOD activity, we observed a depression in SOD1 activity levels. Furthermore, our SOD gene expression data would appear to suggest that the inability for SOD to compensate for the increase in O2− is regulated at the gene level. Alterations in this important protective system can lead to oxidative imbalance, and induce critical changes to cellular structure and function [20]. Antioxidant enzymes are valuable indicators of ROS production as well as changes in cellular redox signalling and evidence of cell functionality [13]. The consequences of SOD antioxidant enzyme modifications have been demonstrated in studies investigating SOD knockout models. Muller et al. [22] demonstrated an age-dependent loss of muscle mass in mice lacking SOD1, as well as a significant decrease in their average life span. Furthermore, Sun et al. [23] investigated the effects of SOD1 and SOD2 overexpression in Drosophila that demonstrated a decrease in cumulative