Cachexia-inducing Tumour Research Articles

Effect of branched-chain amino acids on muscle atrophy in cancer cachexia.

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.

Adipose atrophy in cancer cachexia: morphologic and molecular analysis of adipose tissue in tumour-bearing mice

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/EBPα), 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/EBPα 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.

Dermcidin expression in hepatic cells improves survival without N-glycosylation, but requires asparagine residues

Proteolysis-inducing factor, a cachexia-inducing tumour product, is an N-glycosylated peptide with homology to the unglycosylated neuronal survival peptide Y-P30 and a predicted product of the dermcidin gene, a pro-survival oncogene in breast cancer. We aimed to investigate whether dermcidin is pro-survival in liver cells, in which proteolysis-inducing factor induces catabolism, and to determine the role of potentially glycosylated asparagine residues in this function. Reverse cloning of proteolysis-inducing factor demonstrated ∼100% homology with the dermcidin cDNA. This cDNA was cloned into pcDNA3.1+ and both asparagine residues removed using site-directed mutagenesis. In vitro translation demonstrated signal peptide production, but no difference in molecular weight between the products of native and mutant vectors. Immunocytochemistry of HuH7 cells transiently transfected with V5-His-tagged dermcidin confirmed targeting to the secretory pathway. Stable transfection conferred protection against oxidative stress. This was abrogated by mutation of both asparagines in combination, but not by mutation of either asparagine alone. These findings suggest that dermcidin may function as an oncogene in hepatic as well as breast cells. Glycosylation does not appear to be required, but the importance of asparagine residues suggests a role for the proteolysis-inducing factor core peptide domain.

Tumor–host interactions

A number of malignant tumors interact with the host to cause a syndrome of cachexia, characterized by extensive loss of adipose tissue and skeletal muscle mass, but with preservation of proteins in visceral tissues. Although anorexia is frequently present, the body composition changes in cancer cachexia cannot be explained by nutritional deprivation alone. Loss of skeletal muscle mass is a result of depression in protein synthesis and an increase in protein degradation. The main degradative pathway that has been found to have increased expression and activity in the skeletal muscle of cachectic patients is the ubiquitin-proteasome proteolytic pathway. Cachexia-inducing tumors produce catabolic factors such as proteolysis-inducing factor (PIF), a 24 kDa sulfated glycoprotein, which inhibit protein synthesis and stimulate degradation of intracellular proteins in skeletal muscle by inducing an increased expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. While the oligosaccharide chains in PIF are required to initiate protein degradation the central polypeptide core may act as a growth and survival factor. Only cachexia-inducing tumors are capable of elaborating fully glycosylated PIF, and the selectivity of production possibly rests with the acquisition of the necessary glycosylating enzymes, rather than expressing the gene for the polypeptide core. Loss of adipose tissue is probably the result of an increase in catabolism rather than a defect in anabolism. A lipid mobilizing factor (LMF), identical with the plasma protein Zn-alpha2-glycoprotein (ZAG) is found in the urine of cachectic cancer patients and is produced by tumors causing a decrease in carcass lipid. LMF causes triglyceride hydrolysis in adipose tissue through a cyclic AMP-mediated process by interaction with a beta3-adrenoreceptor. Thus, by producing circulating factors certain malignant tumors are able to interfere with host metabolism even without metastasis to that particular site.

Effect of cancer cachexia on the activity of tripeptidyl-peptidase II in skeletal muscle

The ubiquitin–proteasome proteolytic pathway plays a major role in degradation of myofibrillar proteins in skeletal muscle during cancer cachexia. The end-product of this pathway is oligopeptides and these are degraded by the extralysomal peptidase tripeptidyl-peptidase II (TPPII) together with various aminopeptidases to form tripeptides and amino acids. To investigate if a relationship exists between the activity of the proteasome and TPPII, functional activities have been measured in gastrocnemius muscle of mice bearing the MAC16 tumour, and with varying extents of weight loss. TPPII activity was quantitated using the specific substrate Ala-Ala-Phe-7-amido-4-methylcoumarin, while proteasome activity was determined as the ‘chymotrypsin-like’ enzyme activity. Both proteasome proteolytic activity and TPPII activity increased in parallel with increasing weight loss, reaching a maximum at 16% weight loss, after which there was a progressive decrease in activity for both proteases with increasing weight loss. In murine myotubes, proteolysis-inducing factor, which is a sulphated glycoprotein produced by cachexia-inducing tumours, induced an increase in activity of both proteasome and TPPII, with an identical dose–response curve, and both activities were inhibited by eicosapentaenoic acid. These results suggest that the activities of both the proteasome and TPPII are regulated in a parallel manner in cancer cachexia, and that both are induced by the same factor and probably have the same intracellular signalling pathways and transcription factors.

Role of protein kinase C and NF-kappaB in proteolysis-inducing factor-induced proteasome expression in C(2)C(12) myotubes.

Proteolysis-inducing factor (PIF) is a sulphated glycoprotein produced by cachexia-inducing tumours, which initiates muscle protein degradation through an increased expression of the ubiquitin–proteasome proteolytic pathway. The role of kinase C (PKC) in PIF-induced proteasome expression has been studied in murine myotubes as a surrogate model of skeletal muscle. Proteasome expression induced by PIF was attenuated by 4α-phorbol 12-myristate 13-acetate (100 nM) and by the PKC inhibitors Ro31-8220 (10 μM), staurosporine (300 nM), calphostin C (300 nM) and Gö 6976 (200 μM). Proteolysis-inducing factor-induced activation of PKCα, with translocation from the cytosol to the membrane at the same concentration as that inducing proteasome expression, and this effect was attenuated by calphostin C. Myotubes transfected with a constitutively active PKCα (pCO2) showed increased expression of proteasome activity, and a longer time course, compared with their wild-type counterparts. In contrast, myotubes transfected with a dominant-negative PKCα (pKS1), which showed no activation of PKCα in response to PIF, exhibited no increase in proteasome activity at any time point. Proteolysis-inducing factor-induced proteasome expression has been suggested to involve the transcription factor nuclear factor-κB (NF-κB), which may be activated through PKC. Proteolysis-inducing factor induced a decrease in cytosolic I-κBα and an increase in nuclear binding of NF-κB in pCO2, but not in pKS1, and the effect in wild-type cells was attenuated by calphostin C, confirming that it was mediated through PKC. This suggests that PKC may be involved in the phosphorylation and degradation of I-κBα, induced by PIF, necessary for the release of NF-κB from its inactive cytosolic complex.

Role of lipid-mobilising factor (LMF) in protecting tumour cells from oxidative damage.

Lipid-mobilising factor (LMF) is produced by cachexia-inducing tumours and is involved in the degradation of adipose tissue, with increased oxidation of the released fatty acids through an induction of uncoupling protein (UCP) expression. Since UCP-2 is thought to be involved in the detoxification of free radicals if LMF induced UCP-2 expression in tumour cells, it might attenuate free radical toxicity. As a model system we have used MAC13 tumour cells, which do not produce LMF. Addition of LMF caused a concentration-dependent increase in UCP-2 expression, as determined by immunoblotting. This effect was attenuated by the β3 antagonist SR59230A, suggesting that it was mediated through a β3 adrenoreceptor. Co-incubation of LMF with MAC13 cells reduced the growth-inhibitory effects of bleomycin, paraquat and hydrogen peroxide, known to be free radical generators, but not chlorambucil, an alkylating agent. There was no effect of LMF alone on cellular proliferation. These results indicate that LMF antagonises the antiproliferative effect of agents working through a free radical mechanism, and may partly explain the unresponsiveness to the chemotherapy of cachexia-inducing tumours.

Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes.

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin–proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression.

TNF-alpha is involved in activating DNA fragmentation in skeletal muscle.

Intraperitoneal administration of 100 μg kg−1 (body weight) of tumour necrosis factor-α to rats for 8 consecutive days resulted in a significant decrease in protein content, which was concomitant with a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumour necrosis factor-α-treated animals as compared with the non-treated controls. Analysis of muscle DNA fragmentation clearly showed enhanced laddering in the skeletal muscle of tumour necrosis factor-α-treated animals, suggesting an apoptotic phenomenon. In a different set of experiments, mice bearing a cachexia-inducing tumour (the Lewis lung carcinoma) showed an increase in muscle DNA fragmentation (9.8-fold) as compared with their non-tumour-bearing control counterparts as previously described. When gene-deficient mice for tumour necrosis factor-α receptor protein I were inoculated with Lewis lung carcinoma, they were also affected by DNA fragmentation; however the increase was only 2.1-fold. These results suggest that tumour necrosis factor-α partly mediates DNA fragmentation during experimental cancer-associated cachexia.British Journal of Cancer (2002) 86, 1012–1016. DOI: 10.1038/sj/bjc/6600167 www.bjcancer.com© 2002 Cancer Research UK

Facteurs lipolytiques et protéolytiques de la cachexie cancéreuse

Patients with cancer cachexia experience a profound loss of both adipose tissue and skeletal muscle mass. Depletion of adipose tissue arises primarily from an increased hydrolysis of triglycerides mediated by a tumour factor, lipid mobilizing factor (LMF). LMF induces stimulation of adipocyte adenylate cyclase in a GTP-dependent process through binding to a β-adrenergic receptor, as for lipolytic hormones, resulting in specific depletion of carcass fat. Loss of muscle mass results from a decrease in protein synthesis and an increase in protein degradation, the latter being mediated through an increased expression of the components of the ubiquitin-proteasome pathway. Both murine and human cachexia-inducing tumours have been shown to secrete a proteolysis-inducing factor (PIF), a 24 kDa sulphated glycoprotein capable of inducing catabolism of skeletal muscle proteins both in vitro and in vivo. PIF initiates muscle protein catabolism by upregulation of the expression of the ubiquitin-proteasome pathway, possibly through the mediation of 15-hydroxyeicosatetraenoic acid (15-HETE). This process is attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which has also been shown to completely stabilize weight loss in patients with unresectable pancreatic carcinoma. This suggests that knowledge of the catabolic mediators involved in cancer cachexia well lead to effective therapeutic intervention.

Cancer anorexia and cachexia

Patients with cancer cachexia experience a profound wasting of adipose tissue and lean body mass. Anorexia, although often present, is insufficient to account for tissue wasting because 1) cachexia involves massive depletion of skeletal muscle that does not occur during anorexia, 2) nutritional supplementation cannot replenish the loss of lean body mass, 3) cachexia can occur without anorexia, and 4) food intake might be normal for the lower weight of the cancer patient. Anorexia can arise from 1) decreased taste and smell of food, 2) early satiety, 3) dysfunctional hypothalamic membrane adenylate cyclase, 4) increased brain tryptophan, and 5) cytokine production. Appetite stimulants such as cyproheptadine, medroxyprogesterone acetate, and megestrol acetate do not significantly improve lean body mass. Tumor products might be more important in the development of cachexia. Cachectic patients excrete in their urine a lipid-mobilizing factor that directly stimulates lipolysis in a cyclic AMP–dependent manner and increases energy expenditure. Loss of skeletal muscle in cachexia is caused by upregulation of the ubiquitin–proteasome catabolic pathway. Cachexia-inducing tumors elaborate a sulfated glycoprotein, which directly initiates protein catabolism in skeletal muscle. The action of this proteolysis-inducing factor is attenuated by the polyunsaturated fatty acid eicosapentaenoic acid, which is also effective in preventing loss of skeletal muscle in cancer patients. Antagonists of tumor catabolic factors will provide important new agents in the treatment of cancer cachexia.

Effect of a cachectic factor on carbohydrate metabolism and attenuation by eicosapentaenoic acid.

The effect of a proteolysis-inducing factor (PIF), produced by cachexia-inducing tumours on glucose utilization by different tissues and the effect of pretreatment with the polyunsaturated fatty acid eicosapentaenoic acid (EPA), has been determined using the 2-deoxyglucose tracer technique. Mice receiving PIF showed a profound depression of body weight (2.3 g) over a 24-h period, which was completely abolished by pretreatment with a monoclonal antibody to PIF or by 3 days pretreatment with EPA at 500 mg kg−1. Animals receiving PIF exhibited a marked hypoglycaemia, which was effectively reversed by both antibody and EPA pretreatment. There was an increase in glucose utilization by brain, heart and brown fat, but a decrease by kidney, white fat, diaphragm and gastrocnemius muscle after administration of PIF. Changes in organ glucose consumption were attenuated by either monoclonal antibody, EPA, or both. There was a decrease in 2-deoxyglucose uptake by C2 C12 myoblasts in vitro, which was attenuated by EPA. This suggests a direct effect of PIF on glucose uptake by skeletal muscle. These results suggest that in addition to a direct catabolic effect on skeletal muscle PIF has a profound effect on glucose utilization during cachexia. © 1999 Cancer Research Campaign

Induction of muscle protein degradation by a tumour factor.

An antigen of apparent molecular weight of 24,000, reactive with a murine monoclonal antibody, has been isolated from a cachexia-inducing tumour (MAC 16) and has been shown to initiate muscle protein degradation in vitro using isolated soleus muscle. Administration of this material to female NMRI mice (20 g) produced a pronounced depression in body weight (2.72 +/- 0.14 g; P<0.005 from control) over a 24 h period. This weight loss was attenuated in mice pretreated with the monoclonal antibody (0.06 +/- 0.26 g over 24 h) and occurred without a reduction in food and water intake. There was no change in body water composition, and the major contribution to the decrease in body weight was a decrease in the non-fat carcass dry weight (mainly lean body mass). The plasma levels of glucose and most amino acids were also significantly depressed. The decrease in lean body mass was accounted for by an increase (by 50%) in protein degradation and a decrease (by 50%) in protein synthesis in gastrocnemius muscle. Protein degradation was significantly decreased and protein synthesis increased to control values in mice pretreated with the monoclonal antibody. Protein degradation initiated in vitro with the proteolysis-inducing factor was abolished in mice pretreated with eicosapentaenoic acid (EPA), which had been shown to prevent muscle wastage in mice bearing the MAC16 tumour. Protein degradation was associated with a significant elevation of prostaglandin E2 production by isolated soleus muscle, which was inhibited by both the monoclonal antibody and EPA. These results suggest that this material may be the humoral factor mediating changes in skeletal muscle protein homeostasis during the process of cancer cachexia in animals bearing the MAC16 tumour, and could potentially be involved in other cases of cachexia.

5385740 Biologically active material characterized by catabolic activity generally associated with cachexia-inducing tumors, preparations, production a : Tisdale Michael J; Beck Susan A Claverdon, United Kingdom Assigned to Cancer Research Campaign Technology Limited

5385740 Biologically active material characterized by catabolic activity generally associated with cachexia-inducing tumors, preparations, production a : Tisdale Michael J; Beck Susan A Claverdon, United Kingdom Assigned to Cancer Research Campaign Technology Limited

Comparison of the effectiveness of eicosapentaenoic acid administered as either the free acid or ethyl ester as an anticachectic and antitumour agent

A comparison has been made of the effectiveness of eicosapentaenoic (EPA) acid administered as either the free acid or the ethyl ester as an anticachectic and antitumour agent in mice bearing an experimental cachexia-inducing tumour (MAC16 colon adenocarcinoma). While the free acid of EPA was effective in reversing host body weight loss and inhibiting tumour growth the ethyl ester was ineffective in either respect at the same dose level, even when administered with a high fat diet. The lack of effectiveness of the ethyl ester correlated with the inability to reach effective plasma and tumour concentrations of EPA over the initial time period. Whereas effective plasma concentrations of EPA were achieved within 24 h after administration of the free acid, a time lapse of 96 h was required with the ethyl ester, even when combined with a high fat diet. Due to the acuteness of the MAC16 model this time is too long for a therapeutic benefit to be realized.

Tumour-associated hypoglycaemia in a murine cachexia model.

Animals bearing a cachexia-inducing tumour, the MAC16 adenocarcinoma, showed a progressive decrease in blood glucose levels with increasing weight loss, while animals bearing a histologically similar tumour, the MAC13 adenocarcinoma, showed no change in either body weight or blood glucose levels with growth of the tumour. The effect of the MAC16 tumour on blood glucose levels appeared to be unrelated to food intake, glucose consumption by the tumour, or to the production of increased levels of IGF-I and IGF-II mRNA by the tumour cells. The relationship between the induction of cachexia and alteration in blood glucose levels remains unknown.

Effect of megestrol acetate on weight loss induced by tumour necrosis factor alpha and a cachexia-inducing tumour (MAC16) in NMRI mice

The effect of the synthetic progesterone, megestrol acetate, on weight loss induced by both tumour necrosis factor alpha (TNF) as a model for the cachexia accompanying the acquired immunodeficiency syndrome and by a cachexia-inducing tumour (MAC16) has been studied in NMRI mice. Megestrol acetate was effective in preventing weight loss in both model systems with treated animals having an increase in intake of both food and water. Megestrol acetate was unable to prevent loss of body weight in animals pair-fed with TNF treated animals, suggesting that the increase in food and water intake was responsible for the increase in body weight. Analysis of body composition showed that the major contribution to the increase in body weight in animals treated with megestrol acetate was an increase in water content, although there was also an increase in carcass fat in animals bearing the MAC16 tumour given the high dose of megestrol acetate. Animals bearing the MAC16 tumour had a significant increase in tumour weight after treatment with megestrol acetate, possibly owing to the increased plasma glucose levels. These results suggest that an increase in appetite and weight gain alone are not sufficient to justify the anticachectic effect of a particular agent and that body composition analysis and tumour growth rate are very important parameters.

Comparison of weight loss induced by recombinant tumour necrosis factor with that produced by a cachexia-inducing tumour.

A comparison has been made of the cachectic effects produced by the transplantable murine adenocarcinoma of the mouse colon (MAC16) with tumour necrosis factor-alpha (cachectin). Tumour necrosis factor-alpha (TNF-alpha) produced a dose-related weight reduction that was accompanied by a decrease in both food and water intake. The degree of weight loss was directly proportional to the decreased food and water intake. In contrast weight loss produced by the MAC16 tumour occurred without a reduction in fluid or nutrient intake. Both the MAC16 tumour and TNF-alpha produced hypoglycaemia and a reduction in the circulatory level of free fatty acids (FFA), but had opposite effects on the level of plasma triglycerides with the MAC16 tumour-induced cachexia causing a decrease and TNF-alpha producing an increase. The MAC16 tumour elaborated a lipolytic factor which caused an immediate release of FFA from adipose tissue. In contrast TNF-alpha had no effect on mobilization of adipose triglycerides over a short time period. Both TNF-alpha and extracts from the MAC16 tumour caused an enhanced release of amino acids from mouse diaphragm, which was suppressible with indomethacin and heat labile. No TNF was detected in the MAC16 tumour or in the serum of tumour-bearing animals. Both tumour and non-tumour-bearing animals responded with a similar elevation of their serum TNF levels 90 min after a single injection of endotoxin. It is concluded that weight loss produced by TNF-alpha arises from an anorexic effect and that this differs from the complex metabolic changes associated with cancer cachexia.