alpha-chymotrypsin and Cachexia

Cancer-cachexia causes severe weight loss, particularly from the wasting of skeletal muscle, which occurs due to increased protein catabolism and/or decreased protein synthesis. The muscle protein degradation observed in cancer patients is mediated by a specific cytokine, proteolysis-inducing factor (PIF), which is produced by the tumour. This protein increases the ubiquitin-proteasome pathway activity, and the synthesis of muscle protein in these patients can be affected by several factors, including nutrient-related signalling. Some nutrients, such as leucine, can decrease the ubiquitin-proteasome pathway activity and increase the skeletal muscle protein content in cachectic animals. In this study, we investigated the effects of leucine on cell viability, morphology, functional proteasome activity, enzymatic activity, and protein synthesis and degradation in C2C12 myotubes exposed to the proteolysis-inducing factor (PIF)-like protein purified from Walker tumour-bearing rats. Walker factor (WF) had no cytotoxic effects on myotube cells and morphological characteristics were not altered in the presence of WF and/or leucine. However, increased alkaline phosphatase activity was observed. At higher WF concentrations, chymotrypsin-like activity, cathepsin B activity and 20S proteasome gene expression increased. Treating myotubes with leucine before exposure to WF causes leads to a decrease in proteasome activity as well as the activity of chymotrypsin and cathepsin enzymes. Total protein synthesis decreased in WF-treated cells concomitantly as protein degradation increased. After leucine exposure, the observed effects of WF were minimal or even reverted in some cases. Taken together, these results suggest an important modulatory effect for leucine on the effects of WF in C2C12 myotube cells.

Topics: Alkaline Phosphatase; Animals; Cachexia; Carcinoma 256, Walker; Cathepsin B; Cell Survival; Chymotrypsin; Gene Expression; Leucine; Male; Muscle Fibers, Skeletal; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteoglycans; Proteolysis; Rats; Rats, Wistar

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

Topics: Animals; Blood Proteins; Cachexia; Cells, Cultured; Chymotrypsin; Curcumin; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme Inhibitors; I-kappa B Proteins; Male; Mice; Mice, Inbred Strains; Muscle Fibers, Skeletal; Muscle, Skeletal; Neoplasms, Experimental; NF-kappa B; NF-KappaB Inhibitor alpha; Proteasome Endopeptidase Complex; Proteoglycans; Resveratrol; Stilbenes; Ubiquitins; Weight Loss

Treatment of C(2)C(12) myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2, -3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 microM eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA. Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the anti-apoptotic protein bcl-2, which were both attenuated by 50 microM EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C(2)C(12) myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blood Proteins; Blotting, Western; Cachexia; Cell Line; Cells, Cultured; Chymotrypsin; Cysteine Endopeptidases; Cytochromes c; Cytosol; DNA Fragmentation; Dose-Response Relationship, Drug; Eicosapentaenoic Acid; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Mice; Multienzyme Complexes; Muscle, Skeletal; Nucleosomes; Proteasome Endopeptidase Complex; Proteoglycans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C(2)C(12) myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 20S proteasome alpha-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 microM). At a concentration of 4 nM, PIF induced a transient decrease in IkappaBalpha levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IkappaBalpha, an NF-kappaB inhibitory protein, returned to normal after 60 min. Depletion of IkappaBalpha from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-kappaB/IkappaB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-kappaB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-kappaB inhibitor peptide SN50, suggesting that NF-kappaB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-kappaB accumulation in the nucleus.

Topics: Animals; Blood Proteins; Blotting, Western; Cachexia; Cells, Cultured; Chymotrypsin; Colonic Neoplasms; Cysteine Endopeptidases; DNA Primers; Eicosapentaenoic Acid; Electrophoretic Mobility Shift Assay; Female; Fungal Proteins; Gene Expression Regulation; I-kappa B Proteins; Mice; Mice, Knockout; Multienzyme Complexes; Muscle Fibers, Skeletal; NF-kappa B; NF-KappaB Inhibitor alpha; Proteasome Endopeptidase Complex; Proteins; Proteoglycans; Transcription Factors; Ubiquitins

An improved technique of two dimensional immunoelectrophoresis of serum proteins on gelatinized cellulose acetate for routine evaluation of protein nutritional state is described. The method, which allows the estimation of more than 30 protein fractions, proves extremely useful in monitoring the effectiveness of total parenteral nutrition and protein metabolic conditions. Sera samples from healthy subjects and cachectical neoplastic patients have been examined and preliminary results are reported.

Topics: alpha-Macroglobulins; Beta-Globulins; Cachexia; Chymotrypsin; Gastrointestinal Neoplasms; Humans; Immunoelectrophoresis; Lipoproteins, HDL; Lipoproteins, LDL; Orosomucoid; Prealbumin; Protein-Energy Malnutrition; Serum Albumin; Transferrin

Topics: Alcoholism; Blood Protein Disorders; Cachexia; Carbohydrate Metabolism; Chymotrypsin; Deficiency Diseases; Dietary Proteins; Gastrectomy; Humans; Jejunum; Lipase; Male; Middle Aged; Nutrition Disorders; Pancreas; Pancreatin; Trypsin