The potential for inhibitors of nuclear factor-kB (NF-kB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-kB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-kB inhibitor SN50 (18 mM) attenuated the expression of 20S proteasome a-subunits, two subunits of the 19S regulator MSS1 and p42, and the ubiquitinconjugating enzyme, E2 14k , as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-kB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 mM) and resveratrol (30 mM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin -proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k . However, curcumin (150 and 300 mg kg À1 ) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg À1 ) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-kB DNAbinding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-kB may prove useful for the treatment of muscle wasting in cancer cachexia.

The leucine metabolite ␤-hydroxy-␤-methylbutyrate (HMB) prevents muscle protein degradation in cancer-induced weight loss through attenuation of the ubiquitin-proteasome proteolytic pathway. To investigate the mechanism of this effect, the action of HMB on protein breakdown and intracellular signaling leading to increased proteasome expression by the tumor factor proteolysis-inducing factor (PIF) has been studied in vitro using murine myotubes as a surrogate model of skeletal muscle. A comparison has been made of the effects of HMB and those of eicosapentaenoic acid (EPA), a known inhibitor of PIF signaling. At a concentration of 50 mol/L, EPA and HMB completely attenuated PIF-induced protein degradation and induction of the ubiquitin-proteasome proteolytic pathway, as determined by the "chymotrypsin-like" enzyme activity, as well as protein expression of 20S proteasome ␣-and ␤-subunits and subunit p42 of the 19S regulator. The primary event in PIF-induced protein degradation is thought to be release of arachidonic acid from membrane phospholipids, and this process was attenuated by EPA, but not HMB, suggesting that HMB might act at another step in the PIF signaling pathway. EPA and HMB at a concentration of 50 mol/L attenuated PIF-induced activation of protein kinase C and the subsequent degradation of inhibitor B␣ and nuclear accumulation of nuclear factor B. EPA and HMB also attenuated phosphorylation of p42/44 mitogen-activated protein kinase by PIF, thought to be important in PIF-induced proteasome expression. These results suggest that HMB attenuates PIF-induced activation and increased gene expression of the ubiquitin-proteasome proteolytic pathway, reducing protein degradation.

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-kB (NFkB) in regulating muscle protein degradation and expression of the ubiquitin -proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C 2 C 12 myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-kBa, an NF-kB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-kBa, nuclear accumulation of NF-kB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-kBa proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-kB. Proteolysis-inducing factor also induced increased expression of the ubiquitinproteasome pathway, as determined by 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the b-subunits of the proteasome, protein expression of 20S a-subunits and the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme, E2 14k , in cells containing wild-type, but not mutant, I-kBa. The ability of mutant I-kBa to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin -proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-kB.

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2 14k , has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2 14k were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2 14k mRNA and protein levels in gastrocnemius muscle with increases of 6 -8-fold for C2 and two-fold for E2 14k between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25 -27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18 -20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2 14k mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss.

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