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Papers by Steven Russell
Cancers, 2021
Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open acce... more Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department Structural and Functional Biology, Institute of Biology, UNICAMP, Campinas 13083-970, SP, Brazil; bread.cruz@gmail.com (B.C.); ago_oliveira@yahoo.com (A.O.); lala.viana311088@gmail.com (L.R.V.); leisaaguiar@yahoo.com.br (L.L.-A.); colomberamaiara@gmail.com (M.C.C.); rafaelrossiphd@gmail.com (R.R.V.) 2 Department Cancer Physiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA; rafaelcanevarolo@gmail.com 3 Nano-Cell Interactions Lab., Department Biochemistry and Tissue Biology, Institute of Biology, UNICAMP, Campinas 13083-970, SP, Brazil; fefossa@gmail.com (F.G.-F.); dejesus@unicamp.br (M.B.d.J.) 4 Biochemistry and Tissue Biology, Institute of Biology, UNICAMP, Campinas 13083-970, SP, Brazil; l...
Oncology Reports, 2014
Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NS... more Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.
Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. ... more Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2 14k , has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2 14k were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2 14k mRNA and protein levels in gastrocnemius muscle with increases of 6-8-fold for C2 and twofold for E2 14k between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25-27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased twofold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18-20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2 14k mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss.
British Journal of Cancer, 2004
The potential for inhibitors of nuclear factor-kB (NF-kB) activation to act as inhibitors of musc... more The potential for inhibitors of nuclear factor-kB (NF-kB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-kB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-kB inhibitor SN50 (18 mM) attenuated the expression of 20S proteasome a-subunits, two subunits of the 19S regulator MSS1 and p42, and the ubiquitinconjugating enzyme, E2 14k , as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-kB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 mM) and resveratrol (30 mM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k. However, curcumin (150 and 300 mg kg À1) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg À1) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-kB DNAbinding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-kB may prove useful for the treatment of muscle wasting in cancer cachexia.
In vivo (Athens, Greece)
n-3 fatty acids are increasingly being administered to cancer patients for the treatment of cache... more n-3 fatty acids are increasingly being administered to cancer patients for the treatment of cachexia, and it is thus important to know of any potential interactions with ongoing cytotoxic drug therapy. For this reason eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were administered to mice bearing the cachexia-inducing MAC16 colon adenocarcinoma, and the effect of epothilone, gemcitabine, 5-fluorouracil and cyclophosphamide on tumour growth and body weight determined. Epothilone alone had a minimal effect on tumour growth rate, but this was potentiated by DH4, while for 5-fluorouracil and cyclophosphamide tumour growth inhibition was enhanced by EPA. The antitumour effect of gemcitabine was not altered by either fatty acid. EPA arrested the development of cachexia, while DHA had no effect and the same was true for their effect on tumour growth rate. The anticachectic effect of EPA was only seen in combination with 5-fluorouracil. These results suggest that n-3 fatty acid...
Endocrinology, 2010
Zinc-α2-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpo... more Zinc-α2-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpose of this study was to evaluate the ability of recombinant human ZAG to attenuate type 2 diabetes in the ob/ob mouse model. ZAG (50 μg daily, iv) induced a progressive loss of body weight (3.5g in 5 d), without an effect on food or water intake but with a 0.4 C rise in body temperature, suggesting an increased energy expenditure. Despite an increased plasma glycerol, indicative of increased lipolysis, levels of glucose, triglycerides, and nonesterified fatty acids were decreased by 17, 25, and 62%, respectively, due to an increased use of both glucose and lipids by muscle and brown adipose tissue. The weight of the latter increased 2-fold, and there was increased expression of uncoupling proteins-1 and -3. Plasma insulin levels were reduced by 36%, whereas pancreatic insulin was increased 4-fold, and there was a 53% decrease in the total area under the glucose curve in the glucose toler...
Cancer Chemotherapy and Pharmacology, 2008
D-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrop... more D-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrophy in a murine cachexia model through an increase in protein synthesis and a decrease in degradation. The mechanism of this effect has been investigated in murine myotubes using a range of catabolic stimuli, including proteolysis-inducing factor (PIF), angiotensin II (Ang II), lipopolysaccharide, and tumour necrosis factor- / interferon-. At a concentration of 100M AT was found to attenuate both the induction of protein degradation and depression of protein synthesis in response to all stimuli. The effect on protein degradation was accompanied by attenuation of the increased expression and activity of the ubiquitinproteasome pathway. This suggests that AT inhibits a signalling step common to all four agents. This target has been shown to be activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) and the subsequent phosphorylation of eukaryotic initiation factor 2 on the -subunit, together with downstream signalling pathways leading to protein degradation. AT also inhibited activation of caspase-3/-8, which is thought to lead to activation of PKR. The mechanism of this effect may be related to the ability of AT to chelate divalent metal ions, since the attenuation of the increased activity of the ubiquitin-proteasome pathway by PIF and Ang II, as well as the depression of protein synthesis by PIF, were reversed by increasing concentrations of Zn 2+. The ability of AT to attenuate muscle atrophy by a range of stimuli suggests that it may be effective in several conditions.
Biochemical Journal, 2007
In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a signific... more 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 eIF2α (eukaryotic initiation factor 2α) 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 phospho...
Biochemical Journal, 2008
In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor)... more In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser473 within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin–proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin–proteasome pathway through activation of NF-κB (nuclear factor κB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or u...
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2012
Short page-heading: Zinc-2-glycotprotein binds activates 3and 2-adrenergic receptors, but not ... more Short page-heading: Zinc-2-glycotprotein binds activates 3and 2-adrenergic receptors, but not b1, and its effect on body weight and insulin sensitivity arise through this interaction.
American Journal of Physiology-Endocrinology and Metabolism, 2008
Both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) and angiotensin II (ANG II) induced an ... more Both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) and angiotensin II (ANG II) induced an increase in total protein degradation in murine myotubes, which was completely attenuated by treatment with β-hydroxy-β-methylbutyrate (HMB; 50 μM). There was an increase in formation of reactive oxygen species (ROS) within 30 min, as well as an increase in the activity of both caspase-3 and -8, and both effects were attenuated by HMB. Moreover, inhibitors of caspase-3 and -8 completely attenuated both ROS formation and total protein degradation induced by TNF-α/IFN-γ and ANG II. There was an increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR), which was attenuated by the specific caspase-3 and -8 inhibitors. Neither ROS formation or protein degradation occurred in myotubes expressing a catalytically inactive PKR variant, PKRΔ6, in response to TNF-α/IFN-γ, compared with myotubes expressing wild-type PKR, although there was still activation of caspase-3 and...
American Journal of Physiology-Endocrinology and Metabolism, 2008
β-Hydroxy-β-methylbutyrate (HMB; 50 μM) has been shown to attenuate the depression in protein syn... more β-Hydroxy-β-methylbutyrate (HMB; 50 μM) has been shown to attenuate the depression in protein synthesis in murine myotubes in response to lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) with or without interferon-γ (IFN-γ), and angiotensin II (ANG II). The mechanism for the depression of protein synthesis by all three agents was the same and was attributed to activation of double-stranded RNA-dependent protein kinase (PKR) with the subsequent phosphorylation of eukaryotic initiation factor 2 (eIF2) on the α-subunit as well as increased phosphorylation of the elongation factor (eEF2). Myotubes expressing a catalytically inactive PKR variant, PKRΔ6, showed no depression of protein synthesis in response to either LPS or TNF-α, confirming the importance of PKR in this process. There was no effect of any of the agents on phosphorylation of mammalian target of rapamycin (mTOR) or initiation factor 4E-binding protein (4E-BP1), and thus no change in the amount of eIF4E bound to 4E...
American Journal of Physiology-Endocrinology and Metabolism, 2007
To investigate the mechanism by which β-hydroxy-β-methylbutyrate (HMB) attenuates the depression ... more To investigate the mechanism by which β-hydroxy-β-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25–50 μM). HMB (50 μM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70S6k), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1–2 h) stimulation of phosphorylation of mTOR and p70S6k. However, in the presence of HMB, phosphorylation of mTOR, p70S6k, and 4E-BP1 was increased, and inactive 4E-BP1-eIF...
Endocrinology, 2010
The mechanism by which the adipokine zinc-α2-glycoprotein (ZAG) increases the mass of gastrocnemi... more The mechanism by which the adipokine zinc-α2-glycoprotein (ZAG) increases the mass of gastrocnemius, but not soleus muscle of diabetic mice, has been evaluated both in vivo and in vitro. There was an increased phosphorylation of both double-stranded RNA-dependent protein kinase and its substrate, eukaryotic initiation factor-2α, which was attenuated by about two-thirds in gastrocnemius but not soleus muscle of ob/ob mice treated with ZAG (50 μg, iv daily) for 5 d. ZAG also reduced the expression of the phospho forms of p38MAPK and phospholipase A2, as well as expression of the ubiquitin ligases (E3) muscle atrophy F-box/atrogin-1 and muscle RING finger protein, and the increased activity of both caspase-3 and casapse-8 to values found in nonobese controls. ZAG also increased the levels of phospho serine-threonine kinase and mammalian target of rapamycin in gastrocnemius muscle and reduced the phosphorylation of insulin receptor substrate-1 (Ser307) associated with insulin resistance...
Endocrinology, 2012
Zinc-2-glycoprotein (ZAG) is an adipokine with the potential as a therapeutic agent in the treatm... more Zinc-2-glycoprotein (ZAG) is an adipokine with the potential as a therapeutic agent in the treatment of obesity and type 2diabetes.. In this study we show that human ZAG which is a 41kDa protein, when administered to ob/ob mice at 50 g day-1 po in the drinking water produced a progressive loss of body weight (5g after 8 days treatment), together with a 0.5 o C increase in rectal temperature, and a 40% reduction in urinary excretion of glucose. There was also a 33% reduction in the area under the curve during an oral glucose tolerance test and an increased sensitivity to insulin. These results were similar to those after iv administration of ZAG. However, tryptic digestion was shown to inactivate ZAG. There was no evidence of human ZAG in the serum, but a 2-fold elevation of murine ZAG, which was also observed in target tissues such as white adipose tissue. To determine whether the effect was due to interaction of the human ZAG with the-adrenoreceptor (-AR) in the gastrointestinal tract before digestion, ZAG was co-administered to ob/ob mice together with propanolol (40mgkg-1), a non-specific-AR antagonist. The effect of ZAG on body weight, rectal temperature, urinary glucose excretion, improvement in glucose disposal and increased insulin sensitivity were attenuated by propanolol, as was the increase in murine ZAG in the serum. These results suggest that oral administration of ZAG increases serum levels through interaction with a-AR in the upper gastrointestinal tract, and gene expression studies showed this to be in the oesophagus.
Cancers, 2020
Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle i... more Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted...
Future medicinal chemistry
The abnormalities of lipid metabolism observed in cancer cachexia may be induced by a lipid-mobil... more The abnormalities of lipid metabolism observed in cancer cachexia may be induced by a lipid-mobilizing factor produced by adenocarcinomas. The specific molecules and metabolic pathways that mediate the actions of lipid-mobilizing factor are not known. The mitochondrial uncoupling proteins-1,-2 and-3 are suggested to play essential roles in energy dissipation and disposal of excess lipid. Here, we studied the effects of lipid-mobilizing factor on the expression of uncoupling proteins-1,-2 and-3 in normal mice. Lipid-mobilizing factor isolated from the urine of cancer patients was injected intravenously into mice over a 52-h period, while vehicle was similarly given to controls. Lipid-mobilizing factor caused significant reductions in body weight (710%, P=0.03) and fat mass (720%, P50.01) accompanied by a marked decrease in plasma leptin (759%, P50.01) and heavy lipid deposition in the liver. In brown adipose tissue, uncoupling protein-1 mRNA levels were elevated in lipid-mobilizing factor-treated mice (+96%, P50.01), as were uncoupling proteins-2 and-3 (+57% and +37%, both P50.05). Lipid-mobilizing factor increased uncoupling protein-2 mRNA in both skeletal muscle (+146%, P50.05) and liver (+142%, P=0.03). The protein levels of uncoupling protein-1 in brown adipose tissue and uncoupling protein-2 in liver were also increased with lipid-mobilizing factor administration (+49% and +67%, both P=0.02). Upregulation by lipid-mobilizing factor of uncoupling proteins-1,-2 and-3 in brown adipose tissue, and of uncoupling protein-2 in skeletal muscle and liver, suggests that these uncoupling proteins may serve to utilize excess lipid mobilized during fat catabolism in cancer cachexia.
Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipoc... more Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10 77-10 75 M) of the specific b3adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOK1 cells transfected with the human b3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a b3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the b3-adrenoceptor showed a high affinity binding site with a Kd value 78+45 nM and a B max value (282+1 fmole mg protein 71) comparable with that of other b3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a b3-adrenoceptor.
Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of ca... more Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of cachectic cancer patients, caused a significant increase in glucose oxidation to CO 2, compared with control mice receiving phosphate buffered saline. Glucose utilisation by various tissues was determined by the 2-deoxyglucose tracer technique and shown to be elevated in brain, heart, brown adipose tissue and gastrocnemius muscle. The tissue glucose metabolic rate was increased almost threefold in brain, accounting for the ability of lipid mobilising factor to decrease blood glucose levels. Lipid mobilising factor also increased overall lipid oxidation, as determined by the production of 14 CO 2 from [ 14 C carboxy] triolein, being 67% greater than phosphate buffered saline controls over a 24 h period. There was a significant increase in [ 14 C] lipid accumulation in plasma, liver and white and brown adipose tissue after administration of lipid mobilising factor. These results suggest that changes in carbohydrate metabolism and loss of adipose tissue, together with an increased whole body fatty acid oxidation in cachectic cancer patients, may arise from tumour production of lipid mobilising factor.
Cancers, 2021
Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open acce... more Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department Structural and Functional Biology, Institute of Biology, UNICAMP, Campinas 13083-970, SP, Brazil; bread.cruz@gmail.com (B.C.); ago_oliveira@yahoo.com (A.O.); lala.viana311088@gmail.com (L.R.V.); leisaaguiar@yahoo.com.br (L.L.-A.); colomberamaiara@gmail.com (M.C.C.); rafaelrossiphd@gmail.com (R.R.V.) 2 Department Cancer Physiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA; rafaelcanevarolo@gmail.com 3 Nano-Cell Interactions Lab., Department Biochemistry and Tissue Biology, Institute of Biology, UNICAMP, Campinas 13083-970, SP, Brazil; fefossa@gmail.com (F.G.-F.); dejesus@unicamp.br (M.B.d.J.) 4 Biochemistry and Tissue Biology, Institute of Biology, UNICAMP, Campinas 13083-970, SP, Brazil; l...
Oncology Reports, 2014
Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NS... more Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.
Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. ... more Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2 14k , has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2 14k were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2 14k mRNA and protein levels in gastrocnemius muscle with increases of 6-8-fold for C2 and twofold for E2 14k between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25-27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased twofold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18-20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2 14k mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss.
British Journal of Cancer, 2004
The potential for inhibitors of nuclear factor-kB (NF-kB) activation to act as inhibitors of musc... more The potential for inhibitors of nuclear factor-kB (NF-kB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-kB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-kB inhibitor SN50 (18 mM) attenuated the expression of 20S proteasome a-subunits, two subunits of the 19S regulator MSS1 and p42, and the ubiquitinconjugating enzyme, E2 14k , as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-kB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 mM) and resveratrol (30 mM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k. However, curcumin (150 and 300 mg kg À1) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg À1) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-kB DNAbinding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-kB may prove useful for the treatment of muscle wasting in cancer cachexia.
In vivo (Athens, Greece)
n-3 fatty acids are increasingly being administered to cancer patients for the treatment of cache... more n-3 fatty acids are increasingly being administered to cancer patients for the treatment of cachexia, and it is thus important to know of any potential interactions with ongoing cytotoxic drug therapy. For this reason eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were administered to mice bearing the cachexia-inducing MAC16 colon adenocarcinoma, and the effect of epothilone, gemcitabine, 5-fluorouracil and cyclophosphamide on tumour growth and body weight determined. Epothilone alone had a minimal effect on tumour growth rate, but this was potentiated by DH4, while for 5-fluorouracil and cyclophosphamide tumour growth inhibition was enhanced by EPA. The antitumour effect of gemcitabine was not altered by either fatty acid. EPA arrested the development of cachexia, while DHA had no effect and the same was true for their effect on tumour growth rate. The anticachectic effect of EPA was only seen in combination with 5-fluorouracil. These results suggest that n-3 fatty acid...
Endocrinology, 2010
Zinc-α2-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpo... more Zinc-α2-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpose of this study was to evaluate the ability of recombinant human ZAG to attenuate type 2 diabetes in the ob/ob mouse model. ZAG (50 μg daily, iv) induced a progressive loss of body weight (3.5g in 5 d), without an effect on food or water intake but with a 0.4 C rise in body temperature, suggesting an increased energy expenditure. Despite an increased plasma glycerol, indicative of increased lipolysis, levels of glucose, triglycerides, and nonesterified fatty acids were decreased by 17, 25, and 62%, respectively, due to an increased use of both glucose and lipids by muscle and brown adipose tissue. The weight of the latter increased 2-fold, and there was increased expression of uncoupling proteins-1 and -3. Plasma insulin levels were reduced by 36%, whereas pancreatic insulin was increased 4-fold, and there was a 53% decrease in the total area under the glucose curve in the glucose toler...
Cancer Chemotherapy and Pharmacology, 2008
D-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrop... more D-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrophy in a murine cachexia model through an increase in protein synthesis and a decrease in degradation. The mechanism of this effect has been investigated in murine myotubes using a range of catabolic stimuli, including proteolysis-inducing factor (PIF), angiotensin II (Ang II), lipopolysaccharide, and tumour necrosis factor- / interferon-. At a concentration of 100M AT was found to attenuate both the induction of protein degradation and depression of protein synthesis in response to all stimuli. The effect on protein degradation was accompanied by attenuation of the increased expression and activity of the ubiquitinproteasome pathway. This suggests that AT inhibits a signalling step common to all four agents. This target has been shown to be activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) and the subsequent phosphorylation of eukaryotic initiation factor 2 on the -subunit, together with downstream signalling pathways leading to protein degradation. AT also inhibited activation of caspase-3/-8, which is thought to lead to activation of PKR. The mechanism of this effect may be related to the ability of AT to chelate divalent metal ions, since the attenuation of the increased activity of the ubiquitin-proteasome pathway by PIF and Ang II, as well as the depression of protein synthesis by PIF, were reversed by increasing concentrations of Zn 2+. The ability of AT to attenuate muscle atrophy by a range of stimuli suggests that it may be effective in several conditions.
Biochemical Journal, 2007
In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a signific... more 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 eIF2α (eukaryotic initiation factor 2α) 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 phospho...
Biochemical Journal, 2008
In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor)... more In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser473 within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin–proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin–proteasome pathway through activation of NF-κB (nuclear factor κB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or u...
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2012
Short page-heading: Zinc-2-glycotprotein binds activates 3and 2-adrenergic receptors, but not ... more Short page-heading: Zinc-2-glycotprotein binds activates 3and 2-adrenergic receptors, but not b1, and its effect on body weight and insulin sensitivity arise through this interaction.
American Journal of Physiology-Endocrinology and Metabolism, 2008
Both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) and angiotensin II (ANG II) induced an ... more Both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) and angiotensin II (ANG II) induced an increase in total protein degradation in murine myotubes, which was completely attenuated by treatment with β-hydroxy-β-methylbutyrate (HMB; 50 μM). There was an increase in formation of reactive oxygen species (ROS) within 30 min, as well as an increase in the activity of both caspase-3 and -8, and both effects were attenuated by HMB. Moreover, inhibitors of caspase-3 and -8 completely attenuated both ROS formation and total protein degradation induced by TNF-α/IFN-γ and ANG II. There was an increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR), which was attenuated by the specific caspase-3 and -8 inhibitors. Neither ROS formation or protein degradation occurred in myotubes expressing a catalytically inactive PKR variant, PKRΔ6, in response to TNF-α/IFN-γ, compared with myotubes expressing wild-type PKR, although there was still activation of caspase-3 and...
American Journal of Physiology-Endocrinology and Metabolism, 2008
β-Hydroxy-β-methylbutyrate (HMB; 50 μM) has been shown to attenuate the depression in protein syn... more β-Hydroxy-β-methylbutyrate (HMB; 50 μM) has been shown to attenuate the depression in protein synthesis in murine myotubes in response to lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) with or without interferon-γ (IFN-γ), and angiotensin II (ANG II). The mechanism for the depression of protein synthesis by all three agents was the same and was attributed to activation of double-stranded RNA-dependent protein kinase (PKR) with the subsequent phosphorylation of eukaryotic initiation factor 2 (eIF2) on the α-subunit as well as increased phosphorylation of the elongation factor (eEF2). Myotubes expressing a catalytically inactive PKR variant, PKRΔ6, showed no depression of protein synthesis in response to either LPS or TNF-α, confirming the importance of PKR in this process. There was no effect of any of the agents on phosphorylation of mammalian target of rapamycin (mTOR) or initiation factor 4E-binding protein (4E-BP1), and thus no change in the amount of eIF4E bound to 4E...
American Journal of Physiology-Endocrinology and Metabolism, 2007
To investigate the mechanism by which β-hydroxy-β-methylbutyrate (HMB) attenuates the depression ... more To investigate the mechanism by which β-hydroxy-β-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25–50 μM). HMB (50 μM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70S6k), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1–2 h) stimulation of phosphorylation of mTOR and p70S6k. However, in the presence of HMB, phosphorylation of mTOR, p70S6k, and 4E-BP1 was increased, and inactive 4E-BP1-eIF...
Endocrinology, 2010
The mechanism by which the adipokine zinc-α2-glycoprotein (ZAG) increases the mass of gastrocnemi... more The mechanism by which the adipokine zinc-α2-glycoprotein (ZAG) increases the mass of gastrocnemius, but not soleus muscle of diabetic mice, has been evaluated both in vivo and in vitro. There was an increased phosphorylation of both double-stranded RNA-dependent protein kinase and its substrate, eukaryotic initiation factor-2α, which was attenuated by about two-thirds in gastrocnemius but not soleus muscle of ob/ob mice treated with ZAG (50 μg, iv daily) for 5 d. ZAG also reduced the expression of the phospho forms of p38MAPK and phospholipase A2, as well as expression of the ubiquitin ligases (E3) muscle atrophy F-box/atrogin-1 and muscle RING finger protein, and the increased activity of both caspase-3 and casapse-8 to values found in nonobese controls. ZAG also increased the levels of phospho serine-threonine kinase and mammalian target of rapamycin in gastrocnemius muscle and reduced the phosphorylation of insulin receptor substrate-1 (Ser307) associated with insulin resistance...
Endocrinology, 2012
Zinc-2-glycoprotein (ZAG) is an adipokine with the potential as a therapeutic agent in the treatm... more Zinc-2-glycoprotein (ZAG) is an adipokine with the potential as a therapeutic agent in the treatment of obesity and type 2diabetes.. In this study we show that human ZAG which is a 41kDa protein, when administered to ob/ob mice at 50 g day-1 po in the drinking water produced a progressive loss of body weight (5g after 8 days treatment), together with a 0.5 o C increase in rectal temperature, and a 40% reduction in urinary excretion of glucose. There was also a 33% reduction in the area under the curve during an oral glucose tolerance test and an increased sensitivity to insulin. These results were similar to those after iv administration of ZAG. However, tryptic digestion was shown to inactivate ZAG. There was no evidence of human ZAG in the serum, but a 2-fold elevation of murine ZAG, which was also observed in target tissues such as white adipose tissue. To determine whether the effect was due to interaction of the human ZAG with the-adrenoreceptor (-AR) in the gastrointestinal tract before digestion, ZAG was co-administered to ob/ob mice together with propanolol (40mgkg-1), a non-specific-AR antagonist. The effect of ZAG on body weight, rectal temperature, urinary glucose excretion, improvement in glucose disposal and increased insulin sensitivity were attenuated by propanolol, as was the increase in murine ZAG in the serum. These results suggest that oral administration of ZAG increases serum levels through interaction with a-AR in the upper gastrointestinal tract, and gene expression studies showed this to be in the oesophagus.
Cancers, 2020
Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle i... more Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted...
Future medicinal chemistry
The abnormalities of lipid metabolism observed in cancer cachexia may be induced by a lipid-mobil... more The abnormalities of lipid metabolism observed in cancer cachexia may be induced by a lipid-mobilizing factor produced by adenocarcinomas. The specific molecules and metabolic pathways that mediate the actions of lipid-mobilizing factor are not known. The mitochondrial uncoupling proteins-1,-2 and-3 are suggested to play essential roles in energy dissipation and disposal of excess lipid. Here, we studied the effects of lipid-mobilizing factor on the expression of uncoupling proteins-1,-2 and-3 in normal mice. Lipid-mobilizing factor isolated from the urine of cancer patients was injected intravenously into mice over a 52-h period, while vehicle was similarly given to controls. Lipid-mobilizing factor caused significant reductions in body weight (710%, P=0.03) and fat mass (720%, P50.01) accompanied by a marked decrease in plasma leptin (759%, P50.01) and heavy lipid deposition in the liver. In brown adipose tissue, uncoupling protein-1 mRNA levels were elevated in lipid-mobilizing factor-treated mice (+96%, P50.01), as were uncoupling proteins-2 and-3 (+57% and +37%, both P50.05). Lipid-mobilizing factor increased uncoupling protein-2 mRNA in both skeletal muscle (+146%, P50.05) and liver (+142%, P=0.03). The protein levels of uncoupling protein-1 in brown adipose tissue and uncoupling protein-2 in liver were also increased with lipid-mobilizing factor administration (+49% and +67%, both P=0.02). Upregulation by lipid-mobilizing factor of uncoupling proteins-1,-2 and-3 in brown adipose tissue, and of uncoupling protein-2 in skeletal muscle and liver, suggests that these uncoupling proteins may serve to utilize excess lipid mobilized during fat catabolism in cancer cachexia.
Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipoc... more Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10 77-10 75 M) of the specific b3adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOK1 cells transfected with the human b3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a b3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the b3-adrenoceptor showed a high affinity binding site with a Kd value 78+45 nM and a B max value (282+1 fmole mg protein 71) comparable with that of other b3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a b3-adrenoceptor.
Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of ca... more Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of cachectic cancer patients, caused a significant increase in glucose oxidation to CO 2, compared with control mice receiving phosphate buffered saline. Glucose utilisation by various tissues was determined by the 2-deoxyglucose tracer technique and shown to be elevated in brain, heart, brown adipose tissue and gastrocnemius muscle. The tissue glucose metabolic rate was increased almost threefold in brain, accounting for the ability of lipid mobilising factor to decrease blood glucose levels. Lipid mobilising factor also increased overall lipid oxidation, as determined by the production of 14 CO 2 from [ 14 C carboxy] triolein, being 67% greater than phosphate buffered saline controls over a 24 h period. There was a significant increase in [ 14 C] lipid accumulation in plasma, liver and white and brown adipose tissue after administration of lipid mobilising factor. These results suggest that changes in carbohydrate metabolism and loss of adipose tissue, together with an increased whole body fatty acid oxidation in cachectic cancer patients, may arise from tumour production of lipid mobilising factor.