The role of glucocorticoids in the induction of zinc-α2-glycoprotein expression in adipose tissue in cancer cachexia

Loss of adipose tissue in cancer cachexia in mice bearing the MAC16 tumour arises from an increased lipid mobilisation through increased expression of zinc-α2-glycoprotein (ZAG) in white (WAT) and brown (BAT) adipose tissue. Glucocorticoids have been suggested to increase ZAG expression, and this study examines their role in cachexia and the mechanisms involved. In mice bearing the MAC16 tumour, serum cortisol concentrations increased in parallel with weight loss, and the glucocorticoid receptor antagonist RU38486 (25 mg kg-1) attenuated both the loss of body weight and ZAG expression in WAT. Dexamethasone (66 μg kg-1) administration to normal mice produced a six-fold increase in ZAG expression in both WAT and BAT, which was also attenuated by RU38486. In vitro studies using 3T3-L1 adipocytes showed dexamethasone (1.68 μM) to stimulate lipolysis and increase ZAG expression, and both were attenuated by RU38486 (10 μM), anti-ZAG antibody (1 μ gml-1), and the β3-adrenoreceptor (β3-AR) antagonist SR59230A (10 μM). Zinc-α2-glycoprotein also increased its own expression and this was attenuated by SR59230A, suggesting that it was mediated through the β3-AR. This suggests that glucocorticoids stimulate lipolysis through an increase in ZAG expression, and that they are responsible for the increase in ZAG expression seen in adipose tissue of cachectic mice. © 2005 Cancer Research UK.

Mechanisms of Cancer Cachexia

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the a-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NF?B. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-a, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment. Copyright © 2009 the American Physiological Society

Changes in nucleic acid and protein levels in atrophying skeletal muscle in cancer cachexia

Background: To investigate factors responsible for muscle loss in cachexia changes in nucleic acid and protein levels have been determined and compared with those induced by a tumour-produced cachectic factor, proteolysis-inducing factor (PIF). Materials and Methods: Mice were transplanted with the MAC16 tumour, while non-tumour bearing mice received PIF (1.5 mg/kg; i.v.) over a 24 h period. Results: There was an exponential decrease in RNA and protein in gastrocnemius muscle with weight loss without an effect on the DNA content. Levels of myosin followed the decrease in total protein, while actin levels remained constant. There was also a significant loss of protein from soleus muscle and spleen, but not from heart, liver and kidney. PIF also produced a significant loss of RNA and protein in spleen and reduced the protein content of soleus muscle. Conclusion: This suggests that PIF may be responsible for changes in protein and RNA content of tissues with the development of cachexia.

Double-blind, placebo-controlled, randomized study of eicosapentaenoic acid diester in patients with cancer cachexia

Purpose: Eicosapentaenoic acid (EPA) has been proposed to have specific anticachectic effects. This trial compared EPA diethyl ester with placebo in cachectic cancer patients for effects on weight and lean body mass. Patients and Methods: Five hundred eighteen weight-losing patients with advanced gastrointestinal or lung cancer were studied in a multicenter, double-blind, placebo controlled trial. Patients were randomly assigned to receive a novel preparation of pure EPA at a dose of 2 g or 4 g daily or placebo (2g EPA, n = 175; 4 g EPA, n = 172; placebo, n = 171). Patients were assessed at 4 weeks and 8 weeks. Results: The groups were well balanced at baseline. Mean weight loss at baseline was 18% (n = 518). Over the 8-week treatment period, both intention-to-treat analysis and per protocol analysis revealed no statistically significant improvements in survival, weight, or other nutritional variables. There was, however, a trend in favor of EPA with analysis of the primary end point, weight, at 8 weeks showing a borderline, nonsignificant treatment effect (P = .066). Relative to placebo, mean weight increased by 1.2 kg with 2 g EPA (95% CI, 0 kg to 2.3 kg) and by 0.3 kg with 4g EPA (-0.9 kg to 1.5 kg). Conclusion: The results indicate no statistically significant benefit from single agent EPA in the treatment of cancer cachexia. Future studies should concentrate on other agents or combination regimens. © 2006 by American Society of Clinical Oncology.

Molecular pathways leading to cancer cachexia

Loss of body weight in cancer patients strongly influences morbidity and mortality. Recent studies have suggested that both tumor and host factors play a major role in tissue catabolism in cachexia, leading to upregulation of degradative pathways in both skeletal muscle and adipose tissue. ©2005 Int. Union Physiol. Sci./Am. Physiol. Soc.

Effect of a protein and energy dense n-3 fatty acid enriched oral supplement on loss of weight and lean tissue in cancer cachexia:A randomised double blind trial

Aim: N-3 fatty acids, especially eicosapentaenoic acid (EPA), may possess anticachectic properties. This trial compared a protein and energy dense supplement enriched with n-3 fatty acids and antioxidants (experimental: E) with an isocaloric isonitrogenous control supplement (C) for their effects on weight, lean body mass (LBM), dietary intake, and quality of life in cachectic patients with advanced pancreatic cancer. Methods: A total of 200 patients (95 E; 105 C) were randomised to consume two cans/day of the E or C supplement (480 ml, 620 kcal, 32 g protein ± 2.2 g EPA) for eight weeks in a multicentre, randomised, double blind trial. Results: At enrolment, patients' mean rate of weight loss was 3.3 kg/month. Intake of the supplements (E or C) was below the recommended dose (2 cans/day) and averaged 1.4 cans/day. Over eight weeks, patients in both groups stopped losing weight (Δweight E: -0.25 kg/month versus C: -0.37 kg/month; p=0.74) and LBM (ΔLBM E: +0.27 kg/month versus C: +0.12 kg/month; p=0.88) to an equal degree (change from baseline E and C, p<0.001). In view of evident non-compliance in both E and C groups, correlation analyses were undertaken to examine for potential dose-response relationships. E patients demonstrated significant correlations between their supplement intake and weight gain (r=0.50, p<0.001) and increase in LBM (r=0.33, p=0.036). Such correlations were not statistically significant in C patients. The relationship of supplement intake with change in LBM was significantly different between E and C patients (p=0.043). Increased plasma EPA levels in the E group were associated with weight and LBM gain (r=0.50, p<0.001; r=0.51, p=0.001). Weight gain was associated with improved quality of life (p<0.01) only in the E group. Conclusion: Intention to treat group comparisons indicated that at the mean dose taken, enrichment with n-3 fatty acids did not provide a therapeutic advantage and that both supplements were equally effective in arresting weight loss. Post hoc dose-response analysis suggests that if taken in sufficient quantity, only the n-3 fatty acid enriched energy and protein dense supplement results in net gain of weight, lean tissue, and improved quality of life. Further trials are required to examine the potential role of n-3 enriched supplements in the treatment of cancer cachexia.

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia. © 2003 Wiley-Liss, Inc.

Cachexia in cancer patients

Cachexia — the massive (up to 80%) loss of both adipose tissue and skeletal muscle mass — is a significant factor in the poor performance status and high mortality rate of cancer patients. Although this metabolic defect has been known since cancer was first studied, it is only recently that major advances have been made in the identification of catabolic factors that act to destroy host tissues during the cachectic process. Although anorexia is frequently present, depression of food intake alone seems not to be responsible for the wasting of body tissues, as nutritional supplementation or pharmacological manipulation of appetite is unable to reverse the catabolic process — particularly with respect to skeletal muscle. However, recent clinical studies in cancer patients have shown that nutritional supplementation can be effective when combined with agents that attenuate the action of tumour factors and modifiers of the central effects on appetite might also show promise.

Effect of a fish oil-enriched nutritional supplement on metabolic mediators in patients with pancreatic cancer cachexia

Weight loss in advanced cancer patients is refractory to conventional nutritional support. This may be due to metabolic changes mediated by proinflammatory cytokines, hormones, and tumor-derived products. We previously showed that a nutritional supplement enriched with fish oil will reverse weight loss in patients with pancreatic cancer cachexia. The present study examines the effect of this supplement on a number of mediators thought to play a role in cancer cachexia. Twenty weight-losing patients with pancreatic cancer were asked to consume a nutritional supplement providing 600 kcal and 2 g of eicosapentaenoic acid per day. At baseline and after 3 wk, patients were weighed and samples were collected to measure serum concentrations of interleukin (IL)-6 and its soluble receptor tumor necrosis factor receptors I and II, cortisol, insulin, and leptin, peripheral blood mononuclear cell production of IL-1 beta, IL-6, and tumor necrosis factor, and urinary excretion of proteolysis inducing factor. After 3 wk of consumption of the fish oil-enriched nutritional supplement, there was a significant fall in production of IL-6 (from median 16.5 to 13.7 ng/ml, P = 0.015), a rise in serum insulin concentration (from 3.3 to 5.0 mU/l, P = 0.0064), a fall in the cortisol-to-insulin ratio (P = 0.0084), and a fall in the proportion of patients excreting proteolysis inducing factor (from 88% to 40%, P = 0.008). These changes occurred in association with weight gain (median 1 kg, P = 0.024). Various mediators of catabolism in cachexia are modulated by administration of a fish oil-enriched nutritional supplement in pancreatic cancer patients. This may account for the reversal of weight loss in patients consuming this supplement.

Cachexia in MAC16 adenocarcinoma:Suppression of hunger despite normal regulation of leptin, insulin and hypothalamic neuropeptide Y

Weight loss normally stimulates hunger, through mechanisms that include falls in circulating leptin and insulin, leading to stimulation of hypothalamic neuropeptide Y (NPY). Here, we investigated the leptin, insulin and NPY to clarify why hunger is suppressed in mice with severe cachexia due to the MAC16 adenocarcinoma. MAC16-bearing mice progressively lost weight (19% below controls) and fat (-61%) over 16 days after tumour transplantation, while total food intake fell by 10%. Pair-fed mice showed less wasting, with final weight being 9% and fat mass 25% below controls. Plasma leptin fell by 85% in MAC16 and 51% in pair-fed mice, in proportion to loss of fat. Plasma insulin was also reduced by 49% in MAC16 and 53% in pair-fed groups. Hypothalamic leptin receptor (OB-Rb) mRNA was significantly increased in both MAC16 (+223%) and pair-fed (+192%) mice. Hypothalamic NPY mRNA was also significantly raised in MAC16 (+152%) and pair-fed (+99%) groups, showing negative correlations with plasma leptin and insulin, and a positive association with OB-Rb mRNA. In MAC16-induced cachexia, leptin production and hypothalamic OB-Rb and NPY expression are regulated appropriately in response to fat depletion. Therefore, suppression of hunger is probably due to tumour products that inhibit NPY transport or release, or that interfere with neuronal targets downstream of NPY.

Modulation of adipocyte G-protein expression in cancer cachexia by a lipid-mobilizing factor (LMF)

Adipocytes isolated from cachectic mice bearing the MAC 16 tumour showed over a 3-fold increase in lipolytic response to both low concentrations of isoprenaline and a tumour-derived lipid mobilizing factor (LMF). This was reflected by an enhanced stimulation of adenylate cyclase in plasma membrane fractions of adipocytes in the presence of both factors. There was no up-regulation of adenylate cyclase in response to forskolin, suggesting that the effect arose from a change in receptor number or G-protein expression. Immunoblotting of adipocyte membranes from mice bearing the MAC16 tumour showed an increased expression of Gαs up to 10% weight loss and a reciprocal decrease in Gα. There was also an increased expression of Gαs and a decrease in Gα in adipose tissue from a patient with cancer-associated weight loss compared with a non-cachectic cancer patient. The changes in G-protein expression were also seen in adipose tissue of normal mice administered pure LMF as well as in 3T3L1 adipocytes in vitro. The changes in G-protein expression induced by LMF were attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). This suggests that this tumour-derived lipolytic factor acts to sensitize adipose tissue to lipolytic stimuli, and that this effect is attenuated by EPA, which is known to preserve adipose tissue in cancer cachexia. © 2001 Cancer Research Campaign.

Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia

A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with a related tumour (MAC13), which does not induce cachexia. Using specific peptide substrates, there was an increase of 54% in the proteolytic activity of caspase-1, 84% of caspase-8, 98% of caspase-3 151% to caspase-6 and 177% of caspase-9, in the gastrocnemius muscle of animals bearing the MAC16 tumour (up to 25% weight loss), in relation to muscle from animals bearing the MAC13 tumour (1-5% weight loss). The dual pattern of 89 kDa and 25 kDa fragmentation of poly (ADP-ribose) polymerase (PARP) occurred in the muscle samples from animals bearing the MAC16 tumour and with a high amount of caspase-like activity. Cytochrome c was present in the cytosolic fractions of gastrocnemius muscles from both groups of animals, suggesting that cytochrome c release from mitochondria may be involved in caspase activation. There was no evidence for DNA fragmentation into a nucleosomal ladder typical of apoptosis in the muscles of either group of mice. This data supports a role for caspases in the catabolic events in muscle involved in the cancer cachexia syndrome. © 2001 Cancer Research Campaign.

Mechanism of attenuation of skeletal muscle protein catabolism in cancer cachexia by eicosapentaenoic acid

Cancer cachexia is characterized by selective depletion of skeletal muscle protein reserves. Soleus muscles from mice bearing a cachexia-inducing tumor (MAC16) showed an increased protein degradation in vitro, as measured by tyrosine release, when compared with muscles from nontumor-bearing animals. After incubation under conditions that modify different proteolytic systems, lysosomal, calcium-dependent, and ATP-dependent proteolysis were found to contribute to the elevated protein catabolism. Treatment of mice bearing the MAC16 tumor with the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), attenuated loss of body weight and significantly suppressed protein catabolism in soleus muscles through an inhibition of an ATP-dependent proteolytic pathway. The ATP-ubiquitin-dependent proteolytic pathway is considered to play a major role in muscle catabolism in cachexia, and functional proteasome activity, as determined by “chymotrypsin-like” enzyme activity, was significantly elevated in gastrocnemius muscle of mice bearing the MAC16 tumor as weight loss progressed. When animals bearing the MAC16 tumor were treated with EPA, functional proteasome activity was completely suppressed, together with attenuation of the expression of 20S proteasome a-subunits and the p42 regulator, whereas there was no effect on the expression of the ubiquitin-conjugating enzyme (E214k). These results suggest that EPA induces an attenuation of the up-regulation of proteasome expression in cachectic mice, and this was correlated with an increase in myosin expression, confirming retention of contractile proteins. EPA also inhibited growth of the MAC16 tumor in a dose-dependent manner, and this correlated with suppression of the expression of the 20S proteasome a-subunits in tumor cells, suggesting that this may be the mechanism of tumor growth inhibition. Thus EPA antagonizes loss of skeletal muscle proteins in cancer cachexia by down-regulation of proteasome expression, and this may also be the mechanism for inhibition of tumor growth.

Attenuation of skeletal muscle atrophy in cancer cachexia by d-myo-inositol 1,2,6-triphosphate

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.