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

Pooling and suppression in human spatial vision

A distinct feature of several recent models of contrast masking is that detecting mechanisms are divisively inhibited by a broadly tuned ‘gain pool’ of narrow-band spatial pattern mechanisms. The contrast gain control provided by this ‘cross-channel’ architecture achieves contrast normalisation of early pattern mechanisms, which is important for keeping them within the non-saturating part of their biological operating characteristic. These models superseded earlier ‘within-channel’ models, which had supposed that masking arose from direct stimulation of the detecting mechanism by the mask. To reveal the extent of masking, I measured the levels produced with large ranges of pattern spatial relationships that have not been explored before. Substantial interactions between channels tuned to different orientations and spatial frequencies were found. Differences in the masking levels produced with single and multiple component mask patterns provided insights into the summation rules within the gain pool. A widely used cross-channel masking model was tested on these data and was found to perform poorly. The model was developed and a version in which linear summation was allowed between all components within the gain pool but with the exception of the self-suppressing route typically provided the best account of the data. Subsequently, an adaptation paradigm was used to probe the processes underlying pooled responses in masking. This delivered less insight into the pooling than the other studies and areas were identified that require investigation for a new unifying model of masking and adaptation. In further experiments, levels of cross-channel masking were found to be greatly influenced by the spatio-temporal tuning of the channels involved. Old masking experiments and ideas relying on within-channel models were re-elevated in terms of contemporary cross-channel models (e.g. estimations of channel bandwidths from orientation masking functions) and this led to different conclusions than those originally arrived at. The investigation of effects with spatio-temporally superimposed patterns is focussed upon throughout this work, though it is shown how these enquiries might be extended to investigate effects across spatial and temporal position.

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.

Subspace modelling for structured noise suppression

The problem of structured noise suppression is addressed by i)modelling the subspaces hosting the components of the signal conveying the information and ii)applying a nonlin- ear non-extensive technique for effecting the right separation. Although the approach is applicable to all situations satisfying the hypothesis of the proposed framework, this work is motivated by a particular scenario, namely, the cancellation of low frequency noise in broadband seismic signals.

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.

Orientation masking and cross-orientation suppression (XOS):implications for estimates of channel bandwidth

Most contemporary models of spatial vision include a cross-oriented route to suppression (masking from a broadly tuned inhibitory pool), which is most potent at low spatial and high temporal frequencies (T. S. Meese & D. J. Holmes, 2007). The influence of this pathway can elevate orientation-masking functions without exciting the target mechanism, and because early psychophysical estimates of filter bandwidth did not accommodate this, it is likely that they have been overestimated for this corner of stimulus space. Here we show that a transient 40% contrast mask causes substantial binocular threshold elevation for a transient vertical target, and this declines from a mask orientation of 0° to about 40° (indicating tuning), and then more gently to 90°, where it remains at a factor of ∼4. We also confirm that cross-orientation masking is diminished or abolished at high spatial frequencies and for sustained temporal modulation. We fitted a simple model of pedestal masking and cross-orientation suppression (XOS) to our data and those of G. C. Phillips and H. R. Wilson (1984) and found the dependency of orientation bandwidth on spatial frequency to be much less than previously supposed. An extension of our linear spatial pooling model of contrast gain control and dilution masking (T. S. Meese & R. J. Summers, 2007) is also shown to be consistent with our results using filter bandwidths of ±20°. Both models include tightly and broadly tuned components of divisive suppression. More generally, because XOS and/or dilution masking can affect the shape of orientation-masking curves, we caution that variations in bandwidth estimates might reflect variations in processes that have nothing to do with filter bandwidth.

Interocular suppression in normal and amblyopic vision:spatio-temporal properties

We measured the properties of interocular suppression in strabismic amblyopes and compared these to dichoptic masking in binocularly normal observers. We used a dichoptic version of the well-established probed-sinewave paradigm that measured sensitivity to a brief target stimulus (one of four letters to be discriminated) in the amblyopic eye at different times relative to a suppression-inducing mask in the fixing eye. This was done using both sinusoidal steady state and transient approaches. The suppression-inducing masks were either modulations of luminance or contrast (full field, just overlaying the target, or just surrounding the target). Our results were interpreted using a descriptive model that included contrast gain control and spatio-temporal filtering prior to excitatory binocular combination. The suppression we measured, other than in magnitude, was not fundamentally different from normal dichoptic masking: lowpass spatio-temporal properties with similar contributions from both surround and overlay suppression.

A common rule for integration and suppression of luminance contrast across eyes, space, time, and pattern

Visual perception begins by dissecting the retinal image into millions of small patches for local analyses by local receptive fields. However, image structures extend well beyond these receptive fields and so further processes must be involved in sewing the image fragments back together to derive representations of higher order (more global) structures. To investigate the integration process, we also need to understand the opposite process of suppression. To investigate both processes together, we measured triplets of dipper functions for targets and pedestals involving interdigitated stimulus pairs (A, B). Previous work has shown that summation and suppression operate over the full contrast range for the domains of ocularity and space. Here, we extend that work to include orientation and time domains. Temporal stimuli were 15-Hz counter-phase sine-wave gratings, where A and B were the positive and negative phases of the oscillation, respectively. For orientation, we used orthogonally oriented contrast patches (A, B) whose sum was an isotropic difference of Gaussians. Results from all four domains could be understood within a common framework in which summation operates separately within the numerator and denominator of a contrast gain control equation. This simple arrangement of summation and counter-suppression achieves integration of various stimulus attributes without distorting the underlying contrast code.

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.

Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers

Aim - The aim of the study was to determine the potential for KV1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia. Methods and results - Blood vessels were obtained from patients or mice and studied in culture. Reverse transcriptasepolymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function.  KV1.3 was unique among the  KV1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of  KV1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia. Conclusion - KV1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events. © 2010 The Author.

Suppression of spatiotemporal chaos in the oscillatory CO oxidation on Pt(110) by focused laser light

Chemical turbulence in the oscillatory catalytic CO oxidation on Pt(110) is suppressed by means of focused laser light. The laser locally heats the platinum surface which leads to a local increase of the oscillation frequency, and to the formation of a pacemaker which emits target waves. These waves slowly entrain the medium and suppress the spatiotemporal chaos present in the absence of laser light. Our experimental results are confirmed by a detailed numerical analysis of one- and two-dimensional media using the Krischer-Eiswirth-Ertl model for CO oxidation on Pt110. Different control regimes are identified and the dispersion relation of the system is determined using the pacemaker as an externally tunable wave source.

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.