Catabolic factors in tumour-induced cachexia

A transplantable colon adenocarcinoma of the mouse (MAC16) was utilized as a model of human cancer cachexia. The MAC16 tumour produced extensive weight loss in the host at small tumour burdens and without a reduction in either food or fluid intake. The weight loss was characterised by a decrease in both carcass fat and muscle mass which were directly proportional to the weight of the tumour. The weight loss has been correlated with the production of circulatory catabolic factors by the tumour, which degrade host muscle and adipose tissue in vitro. These factors were further characterised and have been shown to be distinct and separable by gel exclusion chromatography. The proteolytic factors (molecular weight > 150k daltons) were distinguishable from the lipolytic factors which appeared related with molecular weights of approximately 3.0, 1.5 and 0.7k daltons. Lipolytic factors of the same molecular weights were identified in other tumour models and in the body fluids of tumour-bearing animals and cancer patients. These factors were not present in healthy individuals or in patients with other weight-losing conditions. Various temperatures studied reversed the weight loss seen in the cachexia induced by the MAC16 adenocarcinoma in vivo. The effects of these treatments could be linked in vitro to the inhibition of the catabolic factors produced by the tumour. These results suggest that these factors may be responsible for the cachexia the tumour confers on its host. These factors may be useful in the understanding and therapy of cancer cachexia.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Investigation of the antiproliferative properties of tumour promoting phorbol esters and related compounds

Tumour promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) exert a multitude of biological effects on many cellular systems, many of which are believed to be mediated via the activation of the enzyme protein kinase C (PKC). TPA and other biologically active phorbol esters inhibited the proliferation of the A549 human lung carcinoma cell line. However, after 5-6 days culture in the continued presence of the phorbol ester cells began to proliferate at a rate similar to that of untreated cells. Resistance to TPA was lost following subculturing, although subculture in the presence of 10 nM TPA for more than 9 weeks resulted in a more resistant phenotype. The selection of a TPA-resistant subpopulation was not responsible for the observed resistance. The antiproliferative properties of other PKC activators were investigated. Mezerein induced the same antiproliferative effects as TPA but synthetic diacylglycerols (DAGs), the presumed physiological ligands of PKC, exerted only a non-specific cytotoxic influence on growth. Bryostatins 1 and 2 were able to induce transient growth arrest of A549 cells in a manner similar to phorbol esters at nanomolar concentrations, but at higher concentrations blocked both their own antiproliferative action and also that of phorbol esters and mezerein. Fourteen compounds synthesized to mimic features of the phorbol ester pharmacophore and/or DAGs did not mimic the antiproliferative properties of TPA in A549 cells and exerted only a DAG-like non-specific cytotoxicity at high concentrations. The subcellular distribution and activity of PKC was determined following partial purification by non-denaturing polyacrylamide gel electrophoresis. Treatment with TPA, mezerein or bryostatins resulted in a concentration-dependent shift of PKC activity from the cytosol to cellular membranes within 30 min. Significant translocation was not observed on treatment with DAGs. Chronic exposure of cells to TPA caused a time- and concentration dependent down-regulation of functional PKC activity. A complete loss of PKC activity was also observed on treatment with growth-inhibitory concentrations of bryostatins. No PKC activity was detected in cells resistant to the growth-inhibitory influence of TPA. Measurement of intracellular Ca2+ concentrations using A549 cells cultured on Cytodex 1 microcarrier beads revealed that TPA, mezerein and the bryostatins induced a similar rapid rise in intracellular Ca2+ levels.

Studies of the stability and metabolism of tumour inhibitory tetrazinones

Temozolomide is an imidazotetrazinone with antineoplastic properties. It is structurally related to dacarbazine. Temozolomide was not metabolized in vitro by liver fractions. Chemical decomposition appears to play an important r^ole in its in vitro and in vivo disposition. In contrast, 3-methylbenzotriazinone, a structural analogue, was metabolized by hepatic microsomes to afford benzotriazinone and a hydrophilic metabolite. The cytotoxicity of temozolomide, dacarbazine, 5-[3-(hydroxy-methyl-3-methyl-triazen-1-yl]imidazole-5-carboxamide (HMMTIC) and 3-monomethyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) were investigated in TLX5 murine lymphoma cells. Unlike dacarbazine, which was not toxic, MTIC, HMMTIC and temozolomide were cytotoxic in the absence of microsomes. Decarbazine was only cytotoxic in the presence of microsomes. The formation of MTIC from dacarbazine, HMMTIC and temozolomide was determined by reversed phase high performance liquid chromatography in mixtures incubated under conditions identical to those described before. MTIC was generated chemically from temozolomide and HMMTIC metabolically from dacarbazine. Using [14C]temozolomide, it was found that, in mice, the major route of excretion of the drug is via the kidneys. An acidic metabolite (metabolite I) was found in the urine of mice which had received temozolomide but its identity has not been established. 1H NMR, UV and chemical analyses revealed that Metabolite I possesses an intact NNN linkage and the site of metabolism is at the N3 methyl group. A further acidic metabolite (metabolite II) was found in the urine of patients. Metabolite II was unambiguously identified as the 8-carboxylic acid derivative of temozolomide. In vitro cytotoxicity assay showed that ony metabolite II is cytotoxic but not metabolite I. Pharmacokinetic studies of temozolomide and MTIC in vivo were performed on mice bearing TLX5 tumour. Temozolomide was eliminated from the plasma monophasically with a t1/2 of 0.7hr. MTIC was identified as a product of decomposition. MTIC was eliminated rapidly with a t1/2 of 2min. Though temozolomide shares many biochemical and biological similarities with clinically used dacarbazine, the results obtained in this study show that it differs markedly in its pharmacokinetic properties from dacarbazine, as temozolomide produced relatively sustained plasma levels which were reflected by drug concentrations in the tumour.

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.

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

Discriminating antigen and non-antigen using proteome dissimilarity III:tumour and parasite antigens

Computational genome analysis enables systematic identification of potential immunogenic proteins within a pathogen. Immunogenicity is a system property that arises through the interaction of host and pathogen as mediated through the medium of a immunogenic protein. The overt dissimilarity of pathogenic proteins when compared to the host proteome is conjectured by some to be the determining principal of immunogenicity. Previously, we explored this idea in the context of Bacterial, Viral, and Fungal antigen. In this paper, we broaden and extend our analysis to include complex antigens of eukaryotic origin, arising from tumours and from parasite pathogens. For both types of antigen, known antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. In contrast to our previous results, both visual inspection and statistical evaluation indicate a much wider range of homologues and a significant level of discrimination; but, as before, we could not determine a viable threshold capable of properly separating non-antigen from antigen. In concert with our previous work, we conclude that global proteome dissimilarity is not a useful metric for immunogenicity for presently available antigens arising from Bacteria, viruses, fungi, parasites, and tumours. While we see some signal for certain antigen types, using dissimilarity is not a useful approach to identifying antigenic molecules within pathogen genomes.

The role of zinc in the anti-tumour and anti-cachectic activity of D-myo-inositol 1,2,6-triphosphate

Background: D-myo-inositol-1,2,6-triphosphate (a-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 ZnSO4. Results: At a dose of 40mgkg-1, AT effectively attenuated both weight loss and growth of the MAC16 tumour, and both effects were attenuated by co-administration of Zn2+. 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 ZnSO4. Conclusion: These results suggest that zinc is important in both tumour growth and cachexia in this animal model.

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

BACKGROUND: Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. METHODS: In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. RESULTS: WF decreased the viability of C2C12 myotubes, especially at concentrations of 20-25 mug.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. CONCLUSION: These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model. © 2008 Yano et al; licensee BioMed Central Ltd.

The role of TG2 in regulating S100A4-mediated mammary tumour cell migration

The importance of S100A4, a Ca2+-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2) a Ca2+-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector) and highly metastatic KP1 cells (R37 cells transfected with S100A4), we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression). Inhibition was paralleled by a decrease in S100A4 polymer formation. co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and a5ß1 integrin co-signalling pathways linked by activation of PKCa in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models

Solid tumours display a complex drug resistance phenotype that involves inherent and acquired mechanisms. Multicellular resistance is an inherent feature of solid tumours and is known to present significant barriers to drug permeation in tumours. Given this barrier, do acquired resistance mechanisms such as P-glycoprotein (P-gp) contribute significantly to resistance? To address this question, the multicellular tumour spheroid (MCTS) model was used to examine the influence of P-gp on drug distribution in solid tissue. Tumour spheroids (TS) were generated from either drug-sensitive MCF7WT cells or a drug-resistant, P-gp-expressing derivative MCF7Adr. Confocal microscopy was used to measure time courses and distribution patterns of three fluorescent compounds; calcein-AM, rhodamine123 and BODIPY-taxol. These compounds were chosen because they are all substrates for P-gp-mediated transport, exhibit high fluorescence and are chemically dissimilar. For example, BODIPY-taxol and rhodamine 123 showed high accumulation and distributed extensively throughout the TSWT, whereas calcein-AM accumulation was restricted to the outermost layers. The presence of P-gp in TSAdr resulted in negligible accumulation, regardless of the compound. Moreover, the inhibition of P-gp by nicardipine restored intracellular accumulation and distribution patterns to levels observed in TSWT. The results demonstrate the effectiveness of P-gp in modulating drug distribution in solid tumour models. However, the penetration of agents throughout the tissue is strongly determined by the physico-chemical properties of the individual compounds.

Inhibition of activation of dsRNA-dependent protein kinase and tumour growth inhibition

Inhibition of dsRNA-activated protein kinase (PKR), not only attenuates muscle atrophy in a murine model of cancer cachexia (MAC16), but it also inhibits tumour growth. In vitro the PKR inhibitor maximally inhibited growth of MAC16 tumour cells at a concentration of 200 nM, which was also maximally effective in attenuating phosphorylation of PKR and of eukaryotic initiation factor (eIF)2 on the a-subunit. There was no effect on the growth of the MAC13 tumour, which does not induce cachexia, even at concentrations up to 1,000 nM. There was constitutive phosphorylation of PKR and eIF2a in the MAC16, but not in the MAC13 tumour, while levels of total PKR and eIF2a were similar. There was constitutive upregulation of nuclear factor-?B (NF-?B) in the MAC16 tumour only, and this was attenuated by the PKR inhibitor, suggesting that it arose from activation of PKR. In MAC16 alone the PKR inhibitor also attenuated expression of the 20S proteasome. The PKR inhibitor potentiated the cytotoxicity of both 5-fluorouracil and gemcitabine to MAC16 cells in vitro. These results suggest that inhibitors of PKR may be useful therapeutic agents against tumours showing increased expression of PKR and constitutive activation of NF-?B, and may also prove useful in sensitising tumours to standard chemotherapeutic agents.