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1.
J Frailty Aging ; 4(4): 209-15, 2015.
Article in English | MEDLINE | ID: mdl-27031020

ABSTRACT

OBJECTIVE: Sarcopenia-the loss of muscle mass and functionality occurring with age-is a pervasive problem with few effective treatments beyond exercise. We examined the ability of the green tea catechin, epigallocatechin-3-gallate (EGCg), to impact muscle mass and the molecular pathway involved in muscle atrophy in a rat model of sarcopenia. METHODS: 20-month-old Sprague-Dawley rats were treated for 8 weeks with control diet or control plus 200mg/kg body weight of EGCg diet. RESULTS: EGCg-supplemented animals had significantly greater gastrocnemius muscle mass than the aged controls, and showed a trend for increased muscle fiber cross-sectional areas (CSA) (p=0.06). These changes were associated with significantly lower protein expressions of the intramuscular 19S and 20S proteasome subunits and the MuRF1 and MAFbx ubiquitin ligases in the EGCg-treated animals. Proteasome activity as determined by 'Chymotrypsin-like' enzyme activity was also significantly reduced by EGCg. Muscle mRNA expression of IL-15 and IGF-1 were significantly increased in the EGCg group vs. the aged controls. In comparison to younger adult animals (6 month), the protein expression of 19S, 20S, MuRF1, MAFbx, and myostatin were increased between approximately 4- and 12-fold in the aged controls, but only up to ~2-fold in the aged EGCg animals. CONCLUSIONS: EGCg supplementation was able to preserve muscle in sarcopenic rats, partly through attenuating protein degradation via the ubiquitin-proteasome pathway, together with increased expression of anabolic factors.

2.
Br J Cancer ; 111(5): 903-8, 2014 Aug 26.
Article in English | MEDLINE | ID: mdl-25101564

ABSTRACT

BACKGROUND: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine. METHODS: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes. RESULTS: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody. CONCLUSIONS: These results suggest that the murine and human PIF receptors are identical.


Subject(s)
Muscle Fibers, Skeletal/metabolism , Proteoglycans/metabolism , Receptors, Cell Surface/metabolism , Adenocarcinoma/metabolism , Animals , Cachexia/metabolism , Cell Line , Cell Line, Tumor , Colonic Neoplasms/metabolism , Humans , Melanoma/metabolism , Mice , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitin/metabolism
3.
Cell Signal ; 24(11): 2118-22, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22820507

ABSTRACT

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C(2)C(12) murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca(2+)(i), which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) also induced a rise in Ca(2+)(i), but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca(2+)(i) induced by PIF and AngII was completely attenuated by the Zn(2+) chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn(2+). The Ca(2+)(i) rise induced by PIF was independent of the presence of extracellular Ca(2+), and attenuated by the Ca(2+) pump inhibitor thapsigargin, suggesting that the Ca(2+)(i) rise was due to release from intracellular stores. This rise in Ca(2+)(i) induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca(2+)(i), which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.


Subject(s)
Calcium/metabolism , Proteoglycans/pharmacology , Angiotensin II/pharmacology , Animals , Atrophy/metabolism , Boron Compounds/pharmacology , Cell Line , Chelating Agents/pharmacology , Estrenes/pharmacology , Inositol 1,4,5-Trisphosphate Receptors/antagonists & inhibitors , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Lipopolysaccharides/pharmacology , Mice , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Pyrrolidinones/pharmacology , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism , Zinc/metabolism
4.
Br J Cancer ; 105(1): 83-8, 2011 Jun 28.
Article in English | MEDLINE | ID: mdl-21673682

ABSTRACT

BACKGROUND: Atrophy of skeletal muscle in cancer cachexia has been attributed to a tumour-produced highly glycosylated peptide called proteolysis-inducing factor (PIF). The action of PIF is mediated through a high-affinity membrane receptor in muscle. This study investigates the ability of peptides derived from the 20 N-terminal amino acids of the receptor to neutralise PIF action both in vitro and in vivo. METHODS: Proteolysis-inducing factor was purified from the MAC16 tumour using an initial pronase digestion, followed by binding on DEAE cellulose, and the pronase was inactivated by heating to 80°C, before purification of the PIF using affinity chromatography. In vitro studies were carried out using C(2)C(12) murine myotubes, while in vivo studies employed mice bearing the cachexia-inducing MAC16 tumour. RESULTS: The process resulted in almost a 23,000-fold purification of PIF, but with a recovery of only 0.004%. Both the D- and L-forms of the 20mer peptide attenuated PIF-induced protein degradation in vitro through the ubiquitin-proteosome proteolytic pathway and increased expression of myosin. In vivo studies showed that neither the D- nor the L-peptides significantly attenuated weight loss, although the D-peptide did show a tendency to increase lean body mass. CONCLUSION: These results suggest that the peptides may be too hydrophilic to be used as therapeutic agents, but confirm the importance of the receptor in the action of the PIF on muscle protein degradation.


Subject(s)
Cachexia/etiology , Colonic Neoplasms/pathology , Muscle Proteins/metabolism , Muscular Atrophy/etiology , Peptide Fragments/metabolism , Proteoglycans/metabolism , Animals , Blotting, Western , Cachexia/metabolism , Cachexia/pathology , Cells, Cultured , Colonic Neoplasms/metabolism , Male , Mice , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/metabolism , Proteoglycans/antagonists & inhibitors , Proteoglycans/isolation & purification
5.
Int J Obes (Lond) ; 35(5): 658-65, 2011 May.
Article in English | MEDLINE | ID: mdl-20856251

ABSTRACT

OBJECTIVE: To investigate the anti-obesity effect of the adipokine zinc-α(2)-glycoprotein (ZAG) in rats and the mechanism of this effect. SUBJECTS: Mature male Wistar rats (540 ± 83 g) were administered human recombinant ZAG (50 µg per 100 g body weight given intravenously daily) for 10 days, while control animals received an equal volume of phosphate-buffered saline (PBS). RESULTS: Animals treated with ZAG showed a progressive decrease in body weight, without a decrease in food and water intake, but with a 0.4 °C rise in body temperature. Body composition analysis showed loss of adipose tissue, but an increase in lean body mass. The loss of fat was due to an increase in lipolysis as shown by a 50% elevation of plasma glycerol, accompanied by increased utilization of non-esterified fatty acids, as evidenced by the 55% decrease in plasma levels. Plasma levels of glucose and triglycerides were also reduced by 36-37% and there was increased expression of the glucose transporter 4 in both skeletal muscle and adipose tissue. Expression of the lipolytic enzymes adipose triglyceride lipase and hormone-sensitive lipase in the white adipose tissue (WAT) were increased twofold after ZAG administration. There was almost a twofold increased expression of uncoupling proteins 1 and 3 in brown adipose tissue and WAT, which would contribute to increased substrate utilization. Administration of ZAG increased ZAG expression twofold in the gastrocnemius muscle, BAT and WAT, which was probably necessary for its biological effect. CONCLUSION: These results show that ZAG produces increased lipid mobilization and utilization in the rat.


Subject(s)
Adipose Tissue/drug effects , Anti-Obesity Agents/pharmacology , Body Weight/drug effects , Glucose Transporter Type 4/drug effects , Obesity/drug therapy , Seminal Plasma Proteins/pharmacology , Adipose Tissue/metabolism , Animals , Anti-Obesity Agents/administration & dosage , Body Weight/physiology , Carrier Proteins/metabolism , Glucose Transporter Type 4/metabolism , Lipolysis , Male , Obesity/metabolism , Obesity/physiopathology , Rats , Rats, Wistar , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Seminal Plasma Proteins/administration & dosage , Thinness/etiology , Weight Loss/physiology , Zn-Alpha-2-Glycoprotein
6.
Int J Obes (Lond) ; 35(3): 345-54, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20697416

ABSTRACT

OBJECTIVE: To investigate the mechanism of the lipid depletion by zinc-α(2)-glycoprotein (ZAG). DESIGN: Studies were conducted in the ob/ob mouse, or on isolated adipocytes from these animals or their lean counterparts. RESULTS: Treatment of these animals for 15 days with ZAG (100 µg, intravenously, daily) resulted in a reduction of body weight of 6.55 g compared with phosphate-buffered saline-treated controls, without a change in food or water intake, but with a 0.4 °C rise in rectal temperature. ZAG-treated mice had a 30% reduction in carcass fat mass and a twofold increase in weight of brown adipose tissue. Epididymal adipocytes from ZAG-treated mice showed an increased expression of ZAG and hormone-sensitive lipase (HSL), and this was maintained for a further 3 days in the absence of ZAG. There was an increased lipolytic response to isoproterenol, which was retained for 3 days in vitro in the absence of ZAG. Expression of HSL was also increased in subcutaneous and visceral adipose tissue, as was also adipose triglyceride lipase (ATGL). There was a rapid loss of labelled lipid from epididymal adipose tissue of ZAG-treated mice, but not from the other depots, reflecting the difference in sensitivity to lipolytic stimuli. The increased expression of HSL and ATGL may involve the extracellular signal-regulated kinase (ERK) pathway, as the active (phospho) form was upregulated in all adipose depots after ZAG administration, whereas in vitro studies showed induction of HSL and ATGL by ZAG to be attenuated by PD98059, an inhibitor of the ERK pathway. CONCLUSION: These results suggest that ZAG not only induces direct lipolysis, but also sensitizes adipose tissue to other lipolytic stimuli.


Subject(s)
Adipose Tissue/drug effects , Anti-Obesity Agents/pharmacology , Lipolysis/drug effects , Obesity/drug therapy , Seminal Plasma Proteins/pharmacology , Adipocytes/drug effects , Adipocytes/metabolism , Adipose Tissue/metabolism , Adiposity/drug effects , Animals , Carrier Proteins/metabolism , Lipase/metabolism , Lipolysis/physiology , Male , Mice , Mice, Obese , Obesity/metabolism , Seminal Plasma Proteins/metabolism , Sterol Esterase/metabolism , Zn-Alpha-2-Glycoprotein
7.
Br J Cancer ; 102(5): 833-6, 2010 Mar 02.
Article in English | MEDLINE | ID: mdl-20145616

ABSTRACT

BACKGROUND: D-myo-inositol-1,2,6-triphosphate (alpha-trinositol, AT) is a polyanionic molecule capable of chelating divalent metal ions with anti-tumour and anti-cachectic activity in a murine model. METHODS: To investigate the role of zinc in this process, mice bearing cachexia-inducing MAC16 tumour were treated with AT, with or without concomitant administration of ZnSO(4). RESULTS: At a dose of 40 mg kg(-1), AT effectively attenuated both weight loss and growth of the MAC16 tumour, and both effects were attenuated by co-administration of Zn(2+). The concentration of zinc in gastrocnemius muscle increased with increasing weight loss, whereas administration of AT decreased the levels of zinc in plasma, skeletal muscle and tumour, which were restored back to control values after administration of ZnSO(4). CONCLUSION: These results suggest that zinc is important in both tumour growth and cachexia in this animal model.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cachexia/drug therapy , Inositol Phosphates/pharmacology , Neoplasms, Experimental/drug therapy , Trace Elements/pharmacology , Zinc/pharmacology , Animals , Cachexia/pathology , Disease Models, Animal , Male , Mice , Neoplasms, Experimental/pathology , Weight Loss/drug effects
8.
Cell Signal ; 22(5): 783-90, 2010 May.
Article in English | MEDLINE | ID: mdl-20074639

ABSTRACT

The role of Ca(2+) in the activation of PKR (double-stranded-RNA-dependent protein kinase), which leads to skeletal muscle atrophy, has been investigated in murine myotubes using the cell-permeable Ca(2+) chelator BAPTA/AM (1,2-bis (o-aminphenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester). BAPTA/AM effectively attenuated both the increase in total protein degradation, through the ubiquitin-proteasome pathway, and the depression of protein synthesis, induced by both proteolysis-inducing factor (PIF) and angiotensin II (Ang II). Since both protein synthesis and degradation were attenuated this suggests the involvement of PKR. Indeed BAPTA/AM attenuated both the activation (autophosphorylation) of PKR and the subsequent phosphorylation of eIF2alpha (eukaryotic initiation factor 2alpha) in the presence of PIF, suggesting the involvement of Ca(2+) in this process. PIF also induced an increase in the activity of both caspases-3 and -8, which was attenuated by BAPTA/AM. The increase in caspase-3 and -8 activity was shown to be responsible for the activation of PKR, since the latter was completely attenuated by the specific caspase-3 and -8 inhibitors. These results suggest that Ca(2+) is involved in the increase in protein degradation and decrease in protein synthesis by PIF and Ang II through activation of PKR by caspases-3 and -8.


Subject(s)
Muscular Atrophy/enzymology , Muscular Atrophy/pathology , eIF-2 Kinase/metabolism , Animals , Caspase 3/metabolism , Caspase 8/metabolism , Caspase Inhibitors , Cell Line , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Activation/drug effects , Eukaryotic Initiation Factor-2/metabolism , Mice , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/enzymology , Phosphorylation/drug effects , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis/drug effects , Protein Processing, Post-Translational/drug effects , Protein Subunits/metabolism , Signal Transduction/drug effects
9.
Cancer Chemother Pharmacol ; 64(3): 517-27, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19112551

ABSTRACT

PURPOSE: To determine the effectiveness of the polyanionic, metal binding agent D-myo-inositol-1,2,6-triphosphate (alpha trinositol, AT), and its hexanoyl ester (HAT), in tissue wasting in cancer cachexia. METHODS: The anti-cachexic effect was evaluated in the MAC16 tumour model. RESULTS: Both AT and HAT attenuated the loss of body weight through an increase in the nonfat carcass mass due to an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. The decrease in protein degradation was associated with a decrease in activity of the ubiquitin-proteasome proteolytic pathway and caspase-3 and -8. Protein synthesis was increased due to attenuation of the elevated autophosphorylation of double-stranded RNA-dependent protein kinase, and of eukaryotic initiation factor 2alpha together with hyperphosphorylation of eIF4E-binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2. In vitro, AT completely attenuated the protein degradation in murine myotubes induced by both proteolysis-inducing factor and angiotensin II. CONCLUSION: These results show that AT is a novel therapeutic agent with the potential to alleviate muscle wasting in cancer patients.


Subject(s)
Cachexia/drug therapy , Inositol Phosphates/pharmacology , Muscle Fibers, Skeletal/drug effects , Muscular Atrophy/drug therapy , Neoplasms, Experimental/physiopathology , Adaptor Proteins, Signal Transducing , Animals , Body Weight/drug effects , Cachexia/etiology , Carrier Proteins/drug effects , Carrier Proteins/metabolism , Caspase 3/drug effects , Caspase 3/metabolism , Caspase 8/drug effects , Caspase 8/metabolism , Cell Cycle Proteins , Eukaryotic Initiation Factor-2/drug effects , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factors , Inositol Phosphates/chemistry , Male , Mice , Mice, Inbred Strains , Muscle Fibers, Skeletal/pathology , Muscle Proteins/drug effects , Muscle Proteins/metabolism , Muscular Atrophy/etiology , Phosphoproteins/drug effects , Phosphoproteins/metabolism , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/metabolism , Protein Kinases/drug effects , Protein Kinases/metabolism , RNA, Double-Stranded/metabolism , Ubiquitin/metabolism
10.
Mol Cell Biochem ; 313(1-2): 63-9, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18360789

ABSTRACT

Proteolysis-inducing factor (PIF), a tumour-produced cachectic factor, induced a dose-dependent decrease in protein synthesis in murine myotubes, together with an increase in phosphorylation of eucaryotic initiation factor 2 (eIF2) on the alpha-subunit. Both insulin (1 nM) and insulin-like growth factor I (IGF-I) (13.2 nM) attenuated the depression of protein synthesis by PIF and the increased phosphorylation of eIF2alpha, by inhibiting the activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) by induction of protein phosphatase 1. A low-molecular weight inhibitor of PKR also reversed the depression of protein synthesis by PIF to the same extent, as did insulin and IGF-I. Both insulin and IGF-I-stimulated protein synthesis in the presence of PIF, and this was attenuated by Salubrinal, an inhibitor of phospho eIF2alpha phosphatase, suggesting that at least part of this action was due to their ability to inhibit phosphorylation of eIF2alpha. Both insulin and IGF-I also attenuated the induction of protein degradation in myotubes induced by PIF, this effect was also attenuated by Salubrinal. These results suggest an alternative mechanism involving PKR to explain the effect of insulin and IGF-I on protein synthesis and degradation in skeletal muscle in the presence of catabolic factors.


Subject(s)
Insulin-Like Growth Factor I/pharmacology , Insulin/pharmacology , Muscle, Skeletal/enzymology , Protein Biosynthesis/drug effects , eIF-2 Kinase/metabolism , Animals , Blotting, Western , Cell Line , Mice , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/enzymology , Muscle, Skeletal/drug effects , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Proteoglycans/pharmacology
11.
Br J Cancer ; 98(2): 443-9, 2008 Jan 29.
Article in English | MEDLINE | ID: mdl-18087277

ABSTRACT

Previous studies suggest that the activation (autophosphorylation) of dsRNA-dependent protein kinase (PKR) can stimulate protein degradation, and depress protein synthesis in skeletal muscle through phosphorylation of the translation initiation factor 2 (eIF2) on the alpha-subunit. To understand whether these mediators are important in muscle wasting in cancer patients, levels of the phospho forms of PKR and eIF2alpha have been determined in rectus abdominus muscle of weight losing patients with oesophago-gastric cancer, in comparison with healthy controls. Levels of both phospho PKR and phospho eIF2alpha were significantly enhanced in muscle of cancer patients with weight loss irrespective of the amount and there was a linear relationship between phosphorylation of PKR and phosphorylation of eIF2alpha (correlation coefficient 0.76, P=0.005). This suggests that phosphorylation of PKR led to phosphorylation of eIF2alpha. Myosin levels decreased as the weight loss increased, and there was a linear relationship between myosin expression and the extent of phosphorylation of eIF2alpha (correlation coefficient 0.77, P=0.004). These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer patients.


Subject(s)
Adenocarcinoma/complications , Eukaryotic Initiation Factor-2/metabolism , Gastrointestinal Neoplasms/complications , Muscle, Skeletal/pathology , Muscular Atrophy/etiology , Muscular Atrophy/metabolism , Weight Loss , eIF-2 Kinase/metabolism , Adenocarcinoma/metabolism , Adult , Aged , Aged, 80 and over , Cachexia/etiology , Cachexia/metabolism , Female , Gastrointestinal Neoplasms/metabolism , Humans , Male , Middle Aged , Models, Biological , Phosphorylation , Up-Regulation , Weight Loss/physiology
13.
Cell Signal ; 19(8): 1797-806, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17532611

ABSTRACT

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and D-alpha-tocopherol (10 microM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), through inhibition of phosphorylation of the NF-kappaB inhibitor protein (I-kappaB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 microM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 microM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 microM) and D609 (200 microM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 microM) and NSC23766 (10 microM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 microM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 microM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with D-alpha-tocopherol (1 mg kg(-1)) attenuated protein degradation and increased protein synthesis in skeletal muscle.


Subject(s)
Angiotensin II/pharmacology , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/metabolism , Proteoglycans/pharmacology , Reactive Oxygen Species/metabolism , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Kinetics , Male , Mice , Mice, Inbred Strains , Models, Biological , Neoplasm Transplantation , Proteasome Endopeptidase Complex/analysis , Proteasome Endopeptidase Complex/physiology , Random Allocation , Reactive Oxygen Species/analysis , Time Factors , Transplantation, Homologous
14.
Br J Cancer ; 96(8): 1216-22, 2007 Apr 23.
Article in English | MEDLINE | ID: mdl-17387345

ABSTRACT

Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies in vitro have suggested that activation of the dsRNA-dependent protein kinase (PKR) may be responsible for these changes in protein synthesis and degradation. In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg(-1)) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2alpha phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-kappaB (NF-kappaB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia.


Subject(s)
Cachexia/drug therapy , Muscular Atrophy/drug therapy , Protein Kinase Inhibitors/therapeutic use , eIF-2 Kinase/antagonists & inhibitors , Animals , DNA/metabolism , Male , Mice , NF-kappa B/metabolism , Phosphorylation , Proteins/metabolism , eIF-2 Kinase/metabolism
16.
Br J Cancer ; 95(8): 1028-37, 2006 Oct 23.
Article in English | MEDLINE | ID: mdl-17047651

ABSTRACT

Extensive loss of adipose tissue is a hallmark of cancer cachexia but the cellular and molecular basis remains unclear. This study has examined morphologic and molecular characteristics of white adipose tissue in mice bearing a cachexia-inducing tumour, MAC16. Adipose tissue from tumour-bearing mice contained shrunken adipocytes that were heterogeneous in size. Increased fibrosis was evident by strong collagen-fibril staining in the tissue matrix. Ultrastructure of 'slimmed' adipocytes revealed severe delipidation and modifications in cell membrane conformation. There were major reductions in mRNA levels of adipogenic transcription factors including CCAAT/enhancer binding protein alpha (C/EBPalpha), CCAAT/enhancer binding protein beta, peroxisome proliferator-activated receptor gamma, and sterol regulatory element binding protein-1c (SREBP-1c) in adipose tissue, which was accompanied by reduced protein content of C/EBPalpha and SREBP-1. mRNA levels of SREBP-1c targets, fatty acid synthase, acetyl CoA carboxylase, stearoyl CoA desaturase 1 and glycerol-3-phosphate acyl transferase, also fell as did glucose transporter-4 and leptin. In contrast, mRNA levels of peroxisome proliferators-activated receptor gamma coactivator-1alpha and uncoupling protein-2 were increased in white fat of tumour-bearing mice. These results suggest that the tumour-induced impairment in the formation and lipid storing capacity of adipose tissue occurs in mice with cancer cachexia.


Subject(s)
Adenocarcinoma/complications , Adipose Tissue/pathology , Cachexia/etiology , Adenocarcinoma/physiopathology , Adipose Tissue/metabolism , Adipose Tissue/ultrastructure , Animals , Atrophy , Blotting, Western , Body Weight/physiology , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cachexia/genetics , Cachexia/metabolism , Female , Gene Expression/genetics , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Ion Channels/genetics , Ion Channels/metabolism , Leptin/genetics , Leptin/metabolism , Male , Mice , Mice, Inbred Strains , Microscopy, Electron , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/metabolism , Uncoupling Protein 2
17.
Br J Cancer ; 94(5): 731-6, 2006 Mar 13.
Article in English | MEDLINE | ID: mdl-16495932

ABSTRACT

Gastro-oesophageal cancer is associated with a high incidence of cachexia. Proteolysis-inducing factor (PIF) has been identified as a possible cachectic factor and studies suggest that PIF is produced exclusively by tumour cells. We investigated PIF core peptide (PIF-CP) mRNA expression in tumour and benign tissue from patients with gastro-oesophageal cancer and in gastro-oesophageal biopsies for healthy volunteers. Tumour tissue and adjacent benign tissue were collected from patients with gastric and oesophageal cancer (n=46) and from benign tissue only in healthy controls (n=11). Expression of PIF-CP mRNA was quantified by real-time PCR. Clinical and pathological information along with nutritional status was collected prospectively. In the cancer patients, PIF-CP mRNA was detected in 27 (59%) tumour samples and 31 (67%) adjacent benign tissue samples. Four (36%) gastro-oesophageal biopsies from healthy controls also expressed PIF-CP mRNA. Expression was higher in tumour tissue (P=0.031) and benign tissue (P=0.022) from cancer patients compared with healthy controls. In the cancer patients, tumour and adjacent benign tissue PIF-CP mRNA concentrations were correlated with each other (P<0.0001, r=0.73) but did not correlate with weight loss or prognosis. Although PIF-CP mRNA expression is upregulated in both tumour and adjacent normal tissue in gastro-oesophageal malignancy, expression does not relate to prognosis or cachexia. Post-translational modification of PIF may be a key step in determining the biological role of PIF in the patient with advanced cancer and cachexia.


Subject(s)
Blood Proteins/biosynthesis , Cachexia/physiopathology , Esophageal Neoplasms/complications , Esophageal Neoplasms/genetics , Stomach Neoplasms/complications , Stomach Neoplasms/genetics , Aged , Biopsy , Blood Proteins/physiology , Cachexia/etiology , Cachexia/genetics , Case-Control Studies , Esophageal Neoplasms/pathology , Female , Gene Expression Profiling , Humans , Male , Middle Aged , Polymerase Chain Reaction , Prognosis , Protein Processing, Post-Translational , Proteoglycans , RNA, Messenger/biosynthesis , Stomach Neoplasms/pathology , Up-Regulation
18.
Life Sci ; 78(25): 2898-910, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16343552

ABSTRACT

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 microM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome alpha-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E2(14k), also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-kappaBalpha, and increased nuclear accumulation of nuclear factor-kappaB (NF-kappaB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-kappaB activation by resveratrol (30 microM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-kappaB.


Subject(s)
Muscle, Skeletal/drug effects , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis/drug effects , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/toxicity , Ubiquitin/metabolism , Animals , Cells, Cultured , Mice , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Myoblasts, Skeletal/drug effects , Myoblasts, Skeletal/metabolism , NF-kappa B/metabolism
19.
Br J Cancer ; 93(7): 774-80, 2005 Oct 03.
Article in English | MEDLINE | ID: mdl-16160695

ABSTRACT

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.


Subject(s)
Cachexia/physiopathology , Muscle, Skeletal/physiopathology , Neoplasms, Experimental/physiopathology , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Animals , Base Sequence , Blotting, Western , Cachexia/complications , Cachexia/enzymology , Cachexia/metabolism , DNA Primers , Mice , Muscle, Skeletal/enzymology , Muscle, Skeletal/metabolism , Neoplasms, Experimental/complications , Neoplasms, Experimental/enzymology , Neoplasms, Experimental/metabolism , Reverse Transcriptase Polymerase Chain Reaction
20.
Int J Biochem Cell Biol ; 37(10): 2196-206, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16125116

ABSTRACT

Atrophy of skeletal muscle is common in patients with cancer and results in increased morbidity and mortality. In order to design effective therapy the mechanism by which this occurs needs to be elucidated. Most studies suggest that the ubiquitin-proteasome proteolytic pathway is most important in intracellular proteolysis, although there have been no reports on the activity of this pathway in patients with different extents of weight loss. In this report the expression of the ubiquitin-proteasome pathway in rectus abdominis muscle has been determined in cancer patients with weight loss of 0-34% using a competitive reverse transcriptase polymerase chain reaction to measure expression of mRNA for proteasome subunits C2 and C5, while protein expression has been determined by western blotting. Overall, both C2 and C5 gene expression was increased by about three-fold in skeletal muscle of cachectic cancer patients (average weight loss 14.5+/-2.5%), compared with that in patients without weight loss, with or without cancer. The level of gene expression was dependent on the amount of weight loss, increasing maximally for both proteasome subunits in patients with weight loss of 12-19%. Further increases in weight loss reduced expression of mRNA for both proteasome subunits, although it was still elevated in comparison with patients with no weight loss. There was no evidence for an increase in expression at weight losses less than 10%. There was a good correlation between expression of proteasome 20Salpha subunits, detected by western blotting, and C2 and C5 mRNA, showing that increased gene expression resulted in increased protein synthesis. Expression of the ubiquitin conjugating enzyme, E2(14k), with weight loss followed a similar pattern to that of proteasome subunits. These results suggest variations in the expression of key components of the ubiquitin-proteasome pathway with weight loss of cancer patients, and suggest that another mechanism of protein degradation must be operative for patients with weight loss less than 10%.


Subject(s)
Muscle, Skeletal/metabolism , Proteasome Endopeptidase Complex/metabolism , Weight Loss , Aged , Biopsy , Blotting, Western , Female , Humans , Male , Muscular Atrophy/etiology , Muscular Atrophy/metabolism , Neoplasms/complications , Neoplasms/metabolism , Proteasome Endopeptidase Complex/genetics , Protein Subunits/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods
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