Andrew J. Farquharson

Conjugated linoleic acids (CLAs) regulate the expression of key apoptotic genes in human breast cancer cells

Conjugated linoleic acid (CLA) reduces mammary tumorigenesis in rodent models, induces apoptosis in rodent mammary tumor cell lines, and decreases expression of antiapoptotic bcl‐2 in rat mammary tissue. This investigation focused on the cell mechanisms underlying the antitumor effects of CLA. Changes (mRNA, protein) in expression of major proapoptotic p53, p21WAF1/CIP1, bax, bcl‐Xs genes, and the antiapoptotic bcl‐2 gene were observed in malignant MCF‐7 and MDA‐MB‐231 cells and in benign MCF‐10a human mammary tumor cells in culture. CLA, but not linoleic acid (LA), inhibited proliferation in all cells; CLA mix was most effective. CLA increased DNA damage (apoptosis). CLA increased mRNA expression of p53 and p21WAF1/CIP1 (three‐ to fivefold and twofold, respectively) but either decreased bcl‐2 by 20–30% or had no effect in MCF‐7 and MCF‐10a cells, respectively; protein expression reflected mRNA values. In MDA‐MBA‐231 (mutant p53) cells, mRNA for p53 was not changed, but p21WAF1/CIP1 and bcl‐2 mRNA was increased. Protein expression largely reflected mRNA changes but, surprisingly, CLA completely suppressed mutant p53 protein in MDA‐MB‐231 cells. Apparent antiapoptotic effects of increased bcl‐2 expression in MDAMBA‐231 cells were countered by increased proapoptotic p21WAF1/CIP1, Bax, and Bcl‐Xs proteins. Findings indicate that CLA elicits mainly proapoptotic effects in human breast tumor cells through both p53‐dependent and p53‐independent pathways, according to cell type.

Olive oil phenolics: effects on DNA oxidation and redox enzyme mRNA in prostate cells

Hydroxytyrosol, tyrosol and caffeic acid effects on hydrogen peroxide-induced DNA damage, hydroperoxide generation and redox enzyme gene expression were studied in oxidative-stress-sensitive human prostate cells (PC3). Hydroxytyrosol led to lower levels of hydroperoxides, DNA damage, and mRNA levels of classic glutathione peroxidase (GPx) for all the studied concentrations. Only hydroxytyrosol was effective at low concentrations (10 microm). Tyrosol reduced DNA oxidation only at high (>50 microm) concentrations and increased hydroperoxides, GPx and phospholipid hydroperoxide GPx mRNA levels. Caffeic acid elicited effects between those of the other two phenolics. Results indicate that hydroxytyrosol is the only significant antioxidant phenolic in olive oil and may be the major component accounting for its beneficial properties. Tyrosol appeared to exhibit pro-oxidant effects (only at high concentrations) and caffeic acid was neutral. Both number and position of hydroxyl groups appear to play a role in the cellular effects of hydroxytyrosol.

High-Fat Diet Alters Gene Expression in the Liver and Colon: Links to Increased Development of Aberrant Crypt Foci

BackgroundObesity is associated with an increased risk of colon cancer. High-fat diets that lead to obesity may be a contributing factor, but the mechanisms are unknown.AimsThis study examines susceptibility to azoxymethane (AOM)-induced precancerous lesions in mice in response to consumption of either a low or a high-fat diet and associated molecular changes in the liver and colon.MethodsGene markers of xenobiotic metabolism, leptin-regulated inflammatory cytokines and proliferation were assessed in liver and colon in response to high-fat feeding to determine links with increased sensitivity to AOM.ResultsHigh-fat feeding increased development of AOM-induced precancerous lesions and was associated with increased CYP2E1 gene expression in the liver, but not the colon. Leptin receptors and the colon stem cell marker (Lgr5) were down-regulated in the proximal colon, with a corresponding up-regulation of the inflammatory cytokine (IL6) in response to high-fat feeding. Notably in the distal colon, where aberrant crypt foci develop in response to AOM, the proliferative stem cell marker, Lgr5, was significantly up-regulated with high-fat feeding.ConclusionsThe current study provides evidence that high-fat diets can alter regulation of molecular markers of xenobiotic metabolism that may expose the colon to carcinogens, in parallel with activation of β-catenin-regulated targets regulating colon epithelial cells. High-fat diets associated with obesity may alter multiple molecular factors that act synergistically to increase the risk of colon cancer associated with obesity.

Leptin up‐regulates pro‐inflammatory cytokines in discrete cells within mouse colon

Dysregulation of leptin associated with obesity is implicated in obesity‐related colon cancer, but mechanisms are elusive. Increased adiposity and elevated plasma leptin are associated with perturbed metabolism in colon and leptin receptors are expressed on colon epithelium. We hypothesise that obesity increases the sensitivity of the colon to cancer by disrupting leptin‐regulated gene targets within colon tissues. PCR arrays were used to firstly identify leptin responsive genes and secondly to identify responses to leptin challenge in wild‐type mice, or those lacking leptin (ob/ob). Leptin‐regulated genes were localised in the colon using in situ hybridisation. IL6, IL1β and CXCL1 were up‐regulated by leptin and localised to discrete cells in gut epithelium, lamina propria, muscularis and at the peritoneal serosal surface. Leptin regulates pro‐inflammatory genes such as IL6, IL1β and CXCL1, and might increase the risk of colon cancer among obese individuals. J. Cell. Physiol. 226: 2123–2130, 2011.

Custom Design of a GeXP Multiplexed Assay Used to Assess Expression Profiles of Inflammatory Gene Targets in Normal Colon, Polyp, and Tumor Tissue

Colon cancers are characterized by aberrant gene expression signatures associated with disease initiation and progression. Identification of aberrant gene expression associated with colon carcinogenesis has increased significantly with application of gene array technologies. Downstream processing of these data has been hindered by the lack of robust multiplexed gene quantitative technologies facilitating study of the identified multiple gene targets. The GenomeLab Genetic Analysis System presents a novel technology platform for quantitative multiplexed gene expression analysis. This report describes the custom design of a GeXP multiplexed assay used to assess expression profiles of 14 inflammatory gene targets in normal, polyp, and tumor tissue. Characteristic normal, polyp, and tumor tissue gene expression profiles were obtained. Statistical analysis confirmed comparable relative quantitation of gene expression using the GeXP, macroarray, and single-plex real-time polymerase chain reaction assays. GeXP assays may be usefully applied in clinical and regulatory studies of multiple gene targets. This system permits custom-design options for relative quantification of multiple gene target expression, simultaneously in a single reaction, using nanogram quantities of total RNA template. The system provides an approach to advance the study of multiple targets identified from gene array analysis with potential for characterizing gene expression signatures in clinical diagnostics.

Possible mechanisms for the putative antiatherogenic and antitumorigenic effects of conjugated polyenoic fatty acids

Conjugated polyenoic fatty acids (CPFA) are derived from the parent polyenoic acid by oxidation or hydrogenation reactions in vivo or in vitro. They occur naturally in ruminant-derived foods, due to bacterial hydrogenation in the tureen, and in chemically hydrogenated vegetable fats. The most common CPFA are those derived from linoleic acid (CLA) which occur as a mixture of cis, trans and trans, cis 9,11 and 10,12 isomers; derivatives of other PFA also occur. Originally regarded as detrimental lipid oxidation products, CLA were recently attributed with antiatherogenic and antitumorigenic properties (1). The cell mechanisms underlying these effects are not known. Our finding that CLA induced glutathione peroxidase (GPx) activity in human umbilical vein endothelial cells (HUVEC) could be important (2). This study highlights other possible beneficial mechanisms elicited by CLA in HUVEC and cancer cells. Methods and results. HUVEC and human prostate cancer cells (PC3) were treated with 0-150 mM CLA (9, 11 and 10, 12 mix) for 24 h. mRNA expression of GPx, phospholipid GPx (PHGPx) and the adhesion molecules ICAM-1 and ESelectin was assessed by Northern analysis; PHGPx activity by spectral analysis using an NADPH-coupled assay, and diacylglycerol (DAG) by thin-layer chromatography after incubation with 14C arachidonic acid. In HUVEC, GPx and PHGPx mRNA were increased circa twofold with increasing CLA concentrations although enzyme activity was unchanged. CLA (10 mM) inhibited cytokine-induced adhesion molecules (ICAM-1 and E-Selectin) by 50%. In PC3 cells, CLA increased GPx mRNA by about 50% and reduced DAG to circa 50% of control at 100 mM. Conclusions. Induction of intrinsic antioxidant enzyme mRNA and suppression of adhesion molecule mRNA by CLA in vascular and/or cancer cells suggests these fatty acids are potent modulators of gene expression. These findings also emphasize the possibility that such gene modulations are important mechanisms whereby CLA elicit their reported antiatherogenic and antitumorigenic effects. Increased redox enzyme activity protects against free radical-induced cell damage and is implicated in attenuation of stress protein expression like adhesion molecules, upregulation of which are implicated in increased vascular disease. Enhanced DAG production by malignant prostate tissue was regarded as a tumor-promoting factor through protein kinase C activation similar to phorbol ester action. Inhibition of DAG in PC3 cells by CLA could explain, at least in part, their anticancer effects.

Novel sites of cytosolic glutathione peroxidase expression in colon

Glutathione peroxidases (Gpx) are important moderators of oxidative stress that is implicated in the pathogenesis of numerous diseases including colon cancer. Previous studies report limited examinations of cytosolic glutathione peroxidase location of expression in colon tissue. This study reports evidence of both common sites of Gpx1 and Gpx2 expression in rat colon and sites that are exclusive to each isoform. Semi‐quantitative PCR performed previously demonstrated RNA expression of Gpx1 and Gpx2 in proximal, transverse and distal colon. Mapping the distribution throughout the entire colon has revealed specific, novel sites of glutathione peroxidase expression in colon lymphatic tissue. In situ hybridisation and immunohistochemistry confirmed micro‐anatomical location of Gpx1 within lymphatic tissue and the lamina propria, sub‐mucosa, muscularis and serosa, but not the lumenal epithelium. In situ hybridisation and immunohistochemistry were consistent with reports of microanatomical location of Gpx2 in the lumenal epithelium. Novel sites of Gpx2 expression were also observed in lymphatic tissue. Immunolocalisation in the vicinity of aberrant crypt foci was also examined to further investigate the link between glutathione peroxidases and colon cancer. This did not reveal significant abnormalities, nor did measurement of cytosolic glutathione peroxidase activity or gene expression in colon tissue from rats treated with the colontropic chemical, 1,2‐dimethylhydrazine. These results support the potential for Gpx1 and Gpx2 redundancy in lymphatic tissue, but not in epithelial cells of the colon crypt or in the lamina propria, sub‐mucosa, muscularis or serosa.

Novel multiplex method to assess insulin, leptin and adiponectin regulation of inflammatory cytokines associated with colon cancer

The role of altered levels of insulin, leptin and adiponectin in contributing to the observed increased risk of colon cancer associated with obesity remains to be determined. Elevated insulin and leptin associated with obesity are linked to inflammatory responses. Conversely, adiponectin levels are reduced in obese individuals and this hormone is generally associated with anti-inflammatory responses. Inflammatory cytokines are key components of processes linked with carcinogenesis. Insulin, leptin and adiponectin receptor expression profiles were assessed in human normal, adenomatous polyp and tumour tissue. Insulin, leptin and adiponectin regulation of inflammatory cytokines previously identified as being associated with early events in colon carcinogenesis were further investigated here using a surrogate colon epithelial cell line and a custom designed GeXP assay of the inflammatory cytokines (CCL20, CXCL1, CXCL2, CXCL3, CXCL11, IL1RN, CXCL4, IL8, CCL19, CCL21, CCL23, CCL5, IL10RB and TNFRSF1A). Mean insulin, leptin and adiponectin receptor expression levels were lower in adenomatous polyp samples in comparison with normal and tumour tissue. In contrast to leptin, insulin significantly reduced CCL20 and CXCL11 and increased CXCL3 expression. Full length adiponectin, but not globular adiponectin, induced CCL5, CXCL1, CXCL3 and CCL20 gene expression. GeXP assay permitted measurement of changes in gene expression of cytokines in response to insulin and adiponectin, indicating the potential for insulin and adiponectin regulation of mediators of inflammation associated with early events in colon carcinogenesis.

Impact of obesity and leptin on protein expression profiles in mouse colon.

BackgroundElevated leptin levels in obesity are associated with increased risk of colon pathology, implicating leptin signaling in colon disease. However, leptin-regulated processes in the colon are currently uncharacterized. Previously, we demonstrated that leptin receptors are expressed on colon epithelium and that increased adiposity and elevated plasma leptin in rats are associated with perturbed metabolism in colon tissue. Thus, we hypothesize that obesity disrupts expression of proteins regulated by leptin in the colon.MethodsA proteomic analysis was conducted to investigate firstly, differences in the colon of mice lacking leptin and leptin signaling (ob/ob and db/db, respectively) by comparing protein expression profiles with wild-type mice. Secondly, responses to leptin challenge in wild-type mice and ob/ob mice were compared to identify leptin-regulated proteins and associated cellular processes.ResultsForty proteins were identified with significantly altered expression patterns associated with differences in leptin status in comparisons between all groups of mice. These proteins are associated with calcium binding, cell cycle, cell proliferation, electron transport chain, energy metabolism, protein folding and transport, redox regulation, structural proteins, and proteins involved in transport and regulation of mucus production.ConclusionsThis study provides evidence that obesity and leptin significantly alter protein profiles of a number of proteins linked to cellular processes in colon tissues that may be linked to the increased risk of colon pathology associated with obesity.

Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding

The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D.