The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-alpha (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14 kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19 S regulatory complex of the 26 S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub-proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.

-The ubiquitin-proteasome proteolytic pathway has recently been reported to be of major importance in the breakdown of skeletal muscle proteins. The

The influence of cancer cachexia and chemotherapy and subsequent recovery of skeletal muscle protein mass and turnover was investigated in mice. Cancer cachexia was induced using colon 26 adenocarcinoma, which is characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C(6)H(12)CIN(3)O(4)S). Reduced food intake was not a factor in these studies. Three days after cachexia began, healthy and tumor-bearing mice were given a single intraperitoneal injection of cystemustine (20 mg/kg). Skeletal muscle mass in tumor-bearing mice was 41% lower (P < 0.05) than in healthy mice 2 wk after cachexia began. Skeletal muscle wasting was mediated initially by decreased protein synthesis (-38%; P < 0.05) and increased degradation (+131%; P < 0.05); later wasting resulted solely from decreased synthesis (~-54 to -69%; P < 0.05). Acute cytotoxicity of chemotherapy did not appear to have an important effect on skeletal muscle protein metabolism in either healthy or tumor-bearing mice. Recovery began 2 days after treatment; skeletal muscle mass was only 11% lower than in healthy mice 11 days after chemotherapy. Recovery of skeletal muscle mass was affected initially by decreased protein degradation (-80%; P < 0.05) and later by increased protein synthesis (+46 to +73%; P < 0.05) in cured compared with healthy mice. This study showed that skeletal muscle wasted from cancer cachexia and after chemotherapeutic treatment is able to generate a strong anabolic response by making powerful changes to protein synthesis and degradation.

The central role of the ubiquitin‐proteasome system in the loss of skeletal muscle protein in many wasting conditions has been well established. However, it is unclear what factors are responsible for the suppression of this system during periods of protein gain. Thus, the aim of these studies was to examine the short‐term effects of insulin release and nutrients on skeletal muscle protein turnover in young rats starved for 48 h, and then infused intravenously with amino acids (AA), or fed an oral diet. Forty‐eight hours of starvation (i.e. prolonged starvation in young rats) decreased muscle protein synthesis and increased proteasome‐dependent proteolysis. Four‐hour AA infusion and 4 h of refeeding increased plasma insulin release and AA concentrations, and stimulated muscle protein synthesis, but had no effect on either total or proteasome‐dependent proteolysis, despite decreased plasma corticosterone concentrations. Both muscle proteasome‐dependent proteolysis and the rate of ubiquitination of muscle proteins were not suppressed until 10 h of refeeding. The temporal response of these two measurements correlated with the normalised expression of the 14‐kDa E2 (a critical enzyme in substrate ubiquitination in muscle) and the expression of the MSS1 subunit of the 19S regulatory complex of the 26S proteasome. In contrast, the starvation‐induced increase in mRNA levels for 20S proteasome subunits was normalised by refeeding within 24 h in muscle, and 6 h in jejunum, respectively. In conclusion, unlike protein synthesis, skeletal muscle proteasome‐dependent proteolysis is not acutely responsive in vivo to insulin, AA, and/or nutrient intake in refed starved rats. This suggests that distinct and perhaps independent mechanisms are responsible for the nutrient‐dependent regulation of protein synthesis and ubiquitin‐proteasome‐dependent proteolysis following a prolonged period of catabolism. Furthermore, factors other than the expression of ubiquitin‐proteasome pathway components appear to be responsible for the suppression of skeletal muscle proteasome‐dependent proteolysis by nutrition.

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-α (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19S regulatory complex of the 26S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub—proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.

The ubiquitin-proteasome proteolytic pathway is of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitinylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyze recent findings in the regulation of ubiquitinylation of protein substrates and of their subsequent proteasome-dependent degradation in animal models of cancer cachexia. In particular, we discuss the influence of various mediators (anorexia, hormones, prostaglandins, cytokines, and proteolysis-inducing factor) in signaling the activation of ubiquitin-proteasome proteolysis in skeletal muscle. These findings have lead to new concepts that are starting to be used for preventing cachexia in cancer and other wasting diseases.

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