[CANCER RESEARCH 61, 3604 –3609, May 1, 2001] Mechanism of Attenuation of Skeletal Muscle Protein Catabolism in Cancer Cachexia by Eicosapentaenoic Acid 1 Alison S. Whitehouse, Helen J. Smith, Joanne L. Drake, and Michael J. Tisdale 2 Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET, United Kingdom ABSTRACT Cancer cachexia is characterized by selective depletion of skeletal muscle protein reserves. Soleus muscles from mice bearing a cachexia- inducing tumor (MAC16) showed an increased protein degradation in vitro, as measured by tyrosine release, when compared with muscles from nontumor-bearing animals. After incubation under conditions that mod- ify different proteolytic systems, lysosomal, calcium-dependent, and ATP- dependent proteolysis were found to contribute to the elevated protein catabolism. Treatment of mice bearing the MAC16 tumor with the poly- unsaturated fatty acid, eicosapentaenoic acid (EPA), attenuated loss of body weight and significantly suppressed protein catabolism in soleus muscles through an inhibition of an ATP-dependent proteolytic pathway. The ATP-ubiquitin-dependent proteolytic pathway is considered to play a major role in muscle catabolism in cachexia, and functional proteasome activity, as determined by “chymotrypsin-like” enzyme activity, was sig- nificantly elevated in gastrocnemius muscle of mice bearing the MAC16 tumor as weight loss progressed. When animals bearing the MAC16 tumor were treated with EPA, functional proteasome activity was com- pletely suppressed, together with attenuation of the expression of 20S proteasome -subunits and the p42 regulator, whereas there was no effect on the expression of the ubiquitin-conjugating enzyme (E2 14k ). These results suggest that EPA induces an attenuation of the up-regulation of proteasome expression in cachectic mice, and this was correlated with an increase in myosin expression, confirming retention of contractile pro- teins. EPA also inhibited growth of the MAC16 tumor in a dose-dependent manner, and this correlated with suppression of the expression of the 20S proteasome -subunits in tumor cells, suggesting that this may be the mechanism of tumor growth inhibition. Thus EPA antagonizes loss of skeletal muscle proteins in cancer cachexia by down-regulation of protea- some expression, and this may also be the mechanism for inhibition of tumor growth. INTRODUCTION Cancer patients with cachexia show a specific depletion of their skeletal muscle mass, whereas the visceral protein compartment re- mains unchanged (1). This loss of muscle protein forms part of the syndrome of cancer cachexia, which results in the loss of function of the cancer patient and eventually death from hypostatic pneumonia, attributable to the loss of respiratory function. Most treatments that have been investigated to alleviate this termi- nal decline have not succeeded in attenuating the loss of muscle mass. Thus, administration of total parenteral nutrition resulted in a short- term weight gain, suggesting retention of water (2), whereas body composition analysis showed no preservation of lean body mass and a temporary maintenance of fat stores. The appetite stimulant meges- trol acetate has also been shown to induce a weight gain of greater than 5% in 15% of the patients treated (3), although body composition analysis showed no evidence for an increase in lean body mass, and the weight gain appeared to arise from an increase in adipose tissue or an increase in body fluid (4). Another appetite stimulant, the serotonin antagonist cyproheptadine, failed to significantly abate progressive weight loss in cachectic patients (5). Thus, provision of excess calo- ries alone is unable to counteract loss of skeletal muscle in cachexia. These results suggest that metabolic alterations in cachexia are more important than anorexia in mediating loss of skeletal muscle. Whereas various cytokines have been postulated to be responsible for the metabolic changes in cachexia seen in some animal models, there is little evidence that they play a major role in human cancer cachexia (6). We have isolated a PIF 3 produced by cachexia-inducing murine (7) and human tumors (8), which is distinct from the cytokines and is able to induce protein catabolism directly in isolated skeletal muscle (9). PIF was found to be excreted in the urine of patients with carcinomas of the pancreas, liver, rectum, colon, breast, lung, and ovary where the weight loss was greater than or equal to 1 kg/month (10). PIF was detected in the urine of 80% of patients with pancreatic cancer (n = 55; Ref. 11). These patients had a significantly greater total weight loss and rate of weight loss than in patients without PIF in the urine. The activity of PIF is attenuated by the polyunsaturated fatty acid, EPA, in model systems (12), and EPA is able to prevent the weight loss induced by the MAC16 colon adenocarcinoma, with preservation of lean body mass (13). This effect is attributable to inhibition of the increased protein degradation seen in skeletal muscle. EPA alone has been shown to attenuate the development of weight loss in pancreatic cancer patients (14) and, when incorporated into a nutritional supplement, produced significant weight gain (2.5 kg at 7 weeks; Ref. 15). Unlike nutritional supplementation alone, body com- position analysis showed a significant gain in lean body mass with no change in fat mass or percentage body water. This suggests that an EPA-enriched nutritional supplement may be able to reverse cachexia in advanced pancreatic cancer. A recent clinical study (16) in patients with generalized solid tumors also showed EPA to prolong survival. There are three major proteolytic pathways responsible for the catabolism of proteins in skeletal muscle: the lysosomal system, which is predominantly concerned with proteolysis of extracellular proteins and cell surface receptors (17); a cytosolic Ca 2+ -activated system, which may play an important role in tissue injury, necrosis, and autolysis (18) and which is independent of ATP; and an ATP- ubiquitin-dependent proteolytic pathway, which is believed to be responsible for the breakdown of the bulk of the intracellular proteins in skeletal muscle (17). In the present study, the effect of EPA on each of the proteolytic pathways has been examined in skeletal muscle of mice bearing the MAC16 tumor, which produces profound cachexia with small tumor masses (19). Previous studies (20) have shown that the ATP-ubiquitin-dependent proteolytic pathway is responsible for the loss of skeletal muscle in such cachectic mice, and attention has been focused on this catabolic pathway. MATERIALS AND METHODS Animals. Pure strain female NMRI mice were obtained from our own inbred colony and were fed a rat and mouse breeding diet (Special Diet Services, Witham, United Kingdom) and water ad libitum. Animals were Received 11/7/00; accepted 2/27/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Scotia Pharmaceuticals Ltd. (Stirling, United Kingdom) and the World Cancer Research Fund. 2 To whom requests for reprints should be addressed, at Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET, United Kingdom. 3 The abbreviations used are: PIF, proteolysis-inducing factor; EPA, eicosapentaenoic acid. 3604 Research. on December 6, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from