Mechanisms of tumour-induced cachexia

The effect of cancer cachexia on host metabolism has been studied in mice transplanted with either the MAC16 adenocarcinoma which induces profound loss of host body weight and depletion of lipid stores or, the MAC13 adenocarcinoma which is of the same histological type, but which grows without an effect on host body weight. Oxidation of D-[U-14C]glucose was elevated in both tumour-bearing states irrespective of cachexia, when compared with non tumour-bearing controls. Both the MAC16 and MAC13 tumours in vivo utilised glucose at the expense of the brain, where its use was partially replaced by 3-hydroxybutyrate, a ketone body. Oxidation of both [U-14C]palmitic acid and [1-14C]triolein was significantly increased in MAC16 tumour-bearing animals and decreased in MAC13 tumour-bearing animals when compared with non tumour-bearing controls, suggesting that in cachectic tumour-bearing animals, mobilisation of body lipids is accompanied by an increased utilisation by the host. Weight loss in MAC16 tumour-bearing animals is associated with the production of a lipolytic factor. Injection of this partially purified lipolytic factor induced weight loss in recipient animals which could be maintained over time in tumour-bearing animals. This suggests that the tumour acts as a sink for the free fatty acids liberated as a result of the mobilisatation of adipose stores. Lipids are important as an energy source in cachectic animals because of their high calorific value and because glucose is being diverted away from host tissues to support tumour growth. Their importance is further demonstrated by the evidence of a MAC16 tumour-associated lipolytic factor. This lipolytic factor is the key to understanding the alterations in host metabolism that occur in tumour-induced cachexia, and may provide future alternatives for the reversal of cachexia and the treatment of cancer itself.

The Role of protein kinase C modulation in the antiproliferative effects of bistratene A, bryostatin 1 and phorbol esters

PKC-mediated signalling pathways are important in cell growth and differentiation, and aberrations in these pathways are implicated in tumourigenesis. The objective of this project was to clarify the link between cell growth inhibition and PKC modulation.The PKC activators bryostatin 1 and 12-0-tetradecanoylphorbol-13-acetate (TPA) inhibited growth in A549 and MCF-7 adenocarcinoma cells with great potency, and induced HL-60 leukaemia cell differentiation. Bistratene A affected these cells similarly. Experiments were conducted to test the hypotheses that bistratene A exerts its effects via PKC modulation and that characteristics of cytostasis induced by bryostatin 1 and TPA depend upon PKC isozyme-specific events. After incubation of A549 cells with TPA or bistratene A, 2D phosphoprotein electrophoretograrns revealed three proteins phosphorylated by both agents. However, bistratene A was unable to induce the formation of cellular networks on the basement membrane substitute Matrigel, and staurosporine was unable to reverse bistratene A-induced [3H]thymidine uptake inhibition, unlike TPA. Bistratene A did not induce PKC translocation or downregulation, activate or inhibit A549 and MCF-7 cell cytosolic PKC or compete for phorbol ester receptors. Western blot analysis and hydroxylapatite chromatography identified PKC α, ε and ζ in these cells. Bistratene A was unable to activate any of these isoforms. Therefore the agent does not exert its antiproliferative effects by modulation of PKC activity. The abilities of bryostatin 1 and TPA (10nM-1μM) to induce PKC isoform translocation and downregulation were compared with antiproliferative effects. Both agents induced dose-dependent downregulation and translocation of PKC α and ε to particulate and nuclear cell fractions. PKC ζ was translocated to the particulate fraction by both agents in MCF-7 cells. The similarity of PKC isoform redistribution by these agents did not explain their divergent effects on cell growth, and the role of nuclear translocation of PKC in cytostasis was not confirmed by these studies. Alternative factors governing the characteristics of growth inhibition induced by these agents are discussed.

Polyunsaturated fatty acids in tumour-induced cachexia

A transplantable murine colon adenocarcinoma (MAC16) was utilised as a model of human cancer cachexia. This tumour has been found to produce extensive weight loss, characterised by depletion of host body protein and lipid stores at a small tumour burden. This weight loss has been found to be associated with production by the tumour of a lipolytic factor, activity of which was inhibited in vitro by the polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA). EPA has also been shown to possess anti-tumour and anti-cachectic activity in vivo, leading to the hypothesis that fatty acids mobilised by the lipolytic factor supply a growth requirement of the MAC16 tumour. In this study mobilisation and sequestration of fatty acids by the tumour was found to be non-specific, although a relationship between weight loss and arachidonic acid (AA) concentration was found in both tumour-bearing mice, and human cancer patients. The anti-tumour effect of EPA, which was found to be associated with an increase in cell loss, but not its anti-cachectic activity, was reversed by the administration of the PUFAs oleic acid (OA) and linoleic acid (LA). LA was also found to be capable of stimulating tumour growth. Inhibition of either the cyclooxygenase or lipoxygenase pathways was found to result in reduction of tumour growth, leading to the implication of one of the metabolites of LA or AA in tumour growth and cachexia. The ethyl ester of EPA was found to be inactive against the growth and cachexia of the MAC16 tumour, due to its retarded uptake compared with the free acid. The anti-proliferative agent 5-fluorouracil was found to cause tumour growth inhibition, and when given in combination with EPA, reduced the phase of tumour regrowth observed after 4 to 5 days of treatment with EPA.

Tumour associated proteolysis and protein metabolism

The effect of cancer cachexia on protein metabolism has been studied in mice transplanted with the MAC16 adenocarcinoma. The progressive cachexia induced by the MAC16 tumour was characterised by a reduction in carcass nitrogen between 16-30% weight loss and a reciprocal increase in tumour nitrogen content. Carcass nitrogen loss was accompanied by a concomitant decrease in gastrocnemius muscle weight and nitrogen content and also by a decrease in liver nitrogen content. The loss of gastrocnemius muscle throughout the progression of cachexia was attributable to a 60% decrease in the rate of protein synthesis and a 240% increase in the rate of protein degradation. The loss of skeletal muscle protein that may be partially mediated by an increased rate of protein degradation has been correlated with a circulatory catabolic factor present only in cachectic tumour-bearing animals, that degrades host muscle in vitro. The proteolysis-inducing factor was found to be heat stable, not a serine protease and was inhibited by indomethacin and eicosapentaenoic acid (EPA) in a dose-related manner. The proteolytic factor induced prostaglandin E2 formation in the gastrocnemius muscle of non tumour-bearing animals and this effect was inhibited by indomethacin and EPA. In vivo studies show EPA (2.0g/kg-1 by gavage) to effectively reverse the decrease in body weight in animals bearing the MAC16 tumour with a concomitant reduction in tumour growth. Muscle from animals treated with EPA showed a decrease (60%) in protein degradation without an effect on protein synthesis. In vivo studies show branched chain amino acid treatment to be ineffective in moderating the cachectic effect of the MAC16 tumour. The action of the factor was largely mimicked by triarachidonin and trilinoleia. The increased serum levels of arachidonic acid in cachectic tumour-bearing animals may thus be responsible for increased protein degradation through prostanoid metabolism. The understanding of protein metabolism and catabolic factors in the cachectic animal may provide future avenues for the reversal of cachexia and the treatment of cancer.metabolism and catabolicmetabolism and cat

Inhibitors of lipolysis in tumor-induced cachexia

The MAC16 tumour produces a factor which exhibits lipid-mobilizing activity in vitro in addition to causing extensive depletion of host lipid stores. The mechanism of the anti-lipolytic effect of two anti-cachectic agents, eicosapentaenoic acid, an ω-3 polyunsaturated fatty acid (PUFA), and N-(3-phenoxycinnamyl)acetohydroxamic acid (BW A4C), a 5-lipoxygenase inhibitor, has been investigated. These two agents reduce tumour growth and reverse the weight loss which accompanies transplantation of the MAC16 murine colon adenocarcinoma into NMRI mice. Mice transplanted with the MAC16 tumour exhibited weight loss which was directly proportional to the serum lipolytic activity measured in vitro up to a weight loss corresponding to 16% of the original body weight. After this time, an inverse relationship between weight loss and lipolytic activity was observed. Body composition analysis revealed a large decrease in body fat relative to other body compartments. The anti-tumour/anti-cachectic effect of EPA did not appear to be due to its ability to inhibit the production of prostaglandin E2. The MAC16 lipolytic factor increased adenylate cyclase activity in adipocyte plasma membranes in a concentration-dependent manner. EPA inhibited the production of cAMP attributed to this lipid-mobilizing factor. EPA produced alterations in Gi , the guanine nucleotide binding protein which mediates hormonal inhibition of adenylate cyclase, in addition to altering cAMP production in adipocyte plasma membranes in response to hormonal stimulation. The alterations in adenylate cyclase activity were complex and not specific to EPA. EPA stimulated adenylate cyclase activity when in a relatively high fatty acid : membrane ratio and inhibited activity when this ratio was lowered. The inhibitory effect of EPA on adenylate cyclase activity may be the underlying mechanism which explains its anti-lipolytic and anti-cachectic effect. The inability of the related ω-3 PUFA, docosahexaenoic acid (DHA), to inhibit cachexia may be due to a difference in the metabolic fates of these two fatty acids. BW A4C inhibited lipolysis in isolated adipocytes which suggests that this compound may possess the potential for an anti-cachectic effect which is independent of its inhibitory effect on tumour growth.

Mechanism of protein catobolism in skeletal muscle during cancer cachexia

Cancer cachexia is characterised by selective depletion of skeletal muscle protein reserves. The ubiquitin-proteasome proteolytic pathway has been shown to be responsible for muscle wasting in a range of cachectic conditions including cancer cachexia. To establish the importance of this pathway in muscle wasting during cancer (and sepsis), a quantitative competitive RT-PCR (QcRT-PCR) method was developed to measure the mRNA levels of the proteasome sub units C2a and C5ß and the ubiquitin-conjugating enzyme E214k. Western blotting was also used to measure the 20S proteasome and E214k protein expression. In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma (MAC16) demonstrated the effect of progressive weight loss on the mRNA and protein expression for 20S proteasome subunits, as well as the ubiquitin-conjugating enzyme, E214k, in gastrocnemius and pectoral muscles. QcRT-PCR measurements showed a good correlation between expression of the proteasome subunits (C2 and CS) and the E214k enzyme mRNA and weight loss in gastrocnemius muscle, where expression increased with increasing weight loss followed by a decrease in expression at higher weight losses (25-27%). Similar results were obtained in pectoral muscles, but with the expression being several fold lower in comparison to that in gastrocnemius muscle, reflecting the different degrees of protein degradation in the two muscles during the process of cancer cachexia. Western blot analysis of 20S and E214k protein expression followed a similar pattern with respect to weight loss as that found with mRNA. In addition, mRNA and protein expression of the 20S proteasome subunits and E214k enzyme was measured in biopsies from cachectic cancer patients, which also showed a good correlation between weight loss and proteasome expression, demonstrating a progressive increase in expression of the proteasome subunits and E214k mRNA and protein in cachectic patients with progressively increasing weight loss.The effect of the cachexia-inducing tumour product PIF (proteolysis inducing factor) and 15-hydroxyeicosatetraenoic acid (15-HETE), the arachidoinic acid metabolite (thought to be the intracellular transducer of PIF action) has also been determined. Using a surrogate model system for skeletal muscle, C2C12 myotubes in vitro, it was shown that both PIF and 15-HETE increased proteasome subunit expression (C2a and C5ß) as well as the E214k enzyme. This increase gene expression was attenuated by preincubation with EPA or the 15-lipoxygenase inhibitor CV-6504; immunoblotting also confirmed these findings. Similarly, in sepsis-induced cachexia in NMRI mice there was increased mRNA and protein expression of the 20S proteasome subunits and the E214k enzyme, which was inhibited by EPA treatment. These results suggest that 15-HETE is the intracellular mediator for PIF induced protein degradation in skeletal muscle, and that elevated muscle catabolism is accomplished through upregulation of the ubiquitin-proteasome-proteolytic pathway. Furthermore, both EPA and CV -6504 have shown anti-cachectic properties, which could be used in the future for the treatment of cancer cachexia and other similar catabolic conditions.

Decreased NADPH oxidase expression and antioxidant activity in cachectic skeletal muscle

Background - Cancer cachexia is the progressive loss of skeletal muscle protein that contributes significantly to cancer morbidity and mortality. Evidence of antioxidant attenuation and the presence of oxidised proteins in patients with cancer cachexia indicate a role for oxidative stress. The level of oxidative stress in tissues is determined by an imbalance between reactive oxygen species production and antioxidant activity. This study aimed to investigate the superoxide generating NADPH oxidase (NOX) enzyme and antioxidant enzyme systems in murine adenocarcinoma tumour-bearing cachectic mice. Methods - Superoxide levels, mRNA levels of NOX enzyme subunits and the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidise (GPx) and catalase was measured in the skeletal muscle of mice with cancer and cancer cachexia. Protein expression levels of NOX enzyme subunits and antioxidant enzyme activity was also measured in the same muscle samples. Results - Superoxide levels increased 1.4-fold in the muscle of mice with cancer cachexia, and this was associated with a decrease in mRNA of NOX enzyme subunits, NOX2, p40phox and p67phox along with the antioxidant enzymes SOD1, SOD2 and GPx. Cancer cachexia was also associated with a 1.3-fold decrease in SOD1 and 2.0-fold decrease in GPx enzyme activity. Conclusion - Despite increased superoxide levels in cachectic skeletal muscle, NOX enzyme subunits, NOX2, p40phox and p67phox, were downregulated along with the expression and activity of the antioxidant enzymes. Therefore, the increased superoxide levels in cachectic skeletal muscle may be attributed to the reduction in the activity of endogenous antioxidant enzymes.

Comparison of the anticatabolic effects of leucine and Ca-β-hydroxy-β-methylbutyrate in experimental models of cancer cachexia

Objective: Loss of skeletal muscle is the most debilitating feature of cancer cachexia, and there are few treatments available. The aim of this study was to compare the anticatabolic efficacy of L-leucine and the leucine metabolite β-hydroxy-β-methylbutyrate (Ca-HMB) on muscle protein metabolism, both invitro and invivo. Methods: Studies were conducted in mice bearing the cachexia-inducing murine adenocarcinoma 16 tumor, and in murine C2 C12 myotubes exposed to proteolysis-inducing factor, lipopolysaccharide, and angiotensin II. Results: Both leucine and HMB were found to attenuate the increase in protein degradation and the decrease in protein synthesis in murine myotubes induced by proteolysis-inducing factor, lipopolysaccharide, and angiotensin II. However, HMB was more potent than leucine, because HMB at 50 μM produced essentially the same effect as leucine at 1 mM. Both leucine and HMB reduced the activity of the ubiquitin-proteasome pathway as measured by the functional (chymotrypsin-like) enzyme activity of the proteasome in muscle lysates, as well as Western blot quantitation of protein levels of the structural/enzymatic proteasome subunits (20 S and 19 S) and the ubiquitin ligases (MuRF1 and MAFbx). Invivo studies in mice bearing the murine adenocarcinoma 16 tumor showed a low dose of Ca-HMB (0.25 g/kg) tobe 60% more effective than leucine (1 g/kg) in attenuating loss of body weight over a 4-d period. Conclusion: These results favor the clinical feasibility of using Ca-HMB over high doses of leucine for the treatment of cancer cachexia. © 2014 Elsevier Inc.

Aspirin and aspirin analogs on esophageal cancer

Background: Esophageal cancer is the eighth most common cancer seen worldwide and is the sixth most common cause of death from cancer. The UK alone has over 8,000 new cases of esophageal cancer every year. Epidemiological studies have shown that low-dose daily intake of aspirin can decrease the incidence of esophageal cancer. However, its use as an anti-cancer drug has been restrained because of its side effects exerted through inhibition of cyclooxygenase (COX) enzymes. In our study, we have investigated the effects of a number of novel aspirin analogs on esophageal cancer cell lines. Methods: The effects of aspirin and its analogs on the viability of esophageal cancer cell lines were tested using the MTT assay. ApoSense and flow cytometric analysis were performed to examine whether aspirin analog-mediated tumor cell death is due to apoptosis or necrosis. Colorimetric assays measuring peroxidase component of cyclooxygenases were employed to screen aspirin analogs for COX inhibition. Results: Our data suggests that the anti-proliferative property of certain aspirin analogs is greater than that of aspirin itself. Benzoylsalicylates and fumaroyl diaspirin were more effective than aspirin against the oe21 squamous cell carcinoma cells and oe33 esophageal adenocarcinoma cells. Flo-1 esophageal adenocarcinoma cells showed resistance to aspirin and most of the aspirin analogs other than the benzoylsalicylates. Both diaspirin and benzoylsalicylates inhibited metabolic activity in all these esophageal cells. However, apoptosis was induced in only a small proportion. We have also shown that these aspirin analogs do not appear to inhibit COX enzymes. Conclusion: We have synthesized and characterized a number of novel aspirin analogs that are more effective against esophageal cancer cell lines than aspirin. These compounds do not exert their anti-proliferative effect through induction of apoptosis. Moreover, these analogs inability to inhibit COX enzymes suggests that they may cause fewer or no side effects compared to aspirin.