Association of Selenoprotein and Selenium Pathway Genotypes with Risk of Colorectal Cancer and Interaction with Selenium Status.

Selenoprotein genetic variations and suboptimal selenium (Se) levels may contribute to the risk of colorectal cancer (CRC) development. We examined the association between CRC risk and genotype for single nucleotide polymorphisms (SNPs) in selenoprotein and Se metabolic pathway genes. Illumina Goldengate assays were designed and resulted in the genotyping of 1040 variants in 154 genes from 1420 cases and 1421 controls within the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Multivariable logistic regression revealed an association of 144 individual SNPs from 63 Se pathway genes with CRC risk. However, regarding the selenoprotein genes, only TXNRD1 rs11111979 retained borderline statistical significance after adjustment for correlated tests (PACT = 0.10; PACT significance threshold was P < 0.1). SNPs in Wingless/Integrated (Wnt) and Transforming growth factor (TGF) beta-signaling genes (FRZB, SMAD3, SMAD7) from pathways affected by Se intake were also associated with CRC risk after multiple testing adjustments. Interactions with Se status (using existing serum Se and Selenoprotein P data) were tested at the SNP, gene, and pathway levels. Pathway analyses using the modified Adaptive Rank Truncated Product method suggested that genes and gene x Se status interactions in antioxidant, apoptosis, and TGF-beta signaling pathways may be associated with CRC risk. This study suggests that SNPs in the Se pathway alone or in combination with suboptimal Se status may contribute to CRC development.

Expression profiling indicating low selenium-sensitive microRNA levels linked to cell cycle and cell stress response pathways in the CaCo-2 cell line.

Se is an essential micronutrient for human health, and fluctuations in Se levels and the potential cellular dysfunction associated with it may increase the risk for disease. Although Se has been shown to influence several biological pathways important in health, little is known about the effect of Se on the expression of microRNA (miRNA) molecules regulating these pathways. To explore the potential role of Se-sensitive miRNA in regulating pathways linked with colon cancer, we profiled the expression of 800 miRNA in the CaCo-2 human adenocarcinoma cell line in response to a low-Se (72 h at <40 nm) environment using nCounter direct quantification. These data were then examined using a range of in silico databases to identify experimentally validated miRNA-mRNA interactions and the biological pathways involved. We identified ten Se-sensitive miRNA (hsa-miR-93-5p, hsa-miR-106a-5p, hsa-miR-205-5p, hsa-miR-200c-3p, hsa-miR-99b-5p, hsa-miR-302d-3p, hsa-miR-373-3p, hsa-miR-483-3p, hsa-miR-512-5p and hsa-miR-4454), which regulate 3588 mRNA in key pathways such as the cell cycle, the cellular response to stress, and the canonical Wnt/ß-catenin, p53 and ERK/MAPK signalling pathways. Our data show that the effects of low Se on biological pathways may, in part, be due to these ten Se-sensitive miRNA. Dysregulation of the cell cycle and of the stress response pathways due to low Se may influence key genes involved in carcinogenesis.

Metabolic programming of a beige adipocyte phenotype by genistein.

SCOPE: Promoting the development of brown or beige adipose tissue may protect against obesity and related metabolic features, and potentially underlies protective effects of genistein in mice. METHODS AND RESULTS: We observed that application of genistein to 3T3-L1 adipocytes changed the lipid distribution from large droplets to a multilocular distribution, reduced mRNAs indicative of white adipocytes (ACC, Fasn, Fabp4, HSL, chemerin, and resistin) and increased mRNAs that are a characteristic feature of brown/beige adipocytes (CD-137 and UCP1). Transcripts with a role in adipocyte differentiation (Cebpß, Pgc1α, Sirt1) peaked at different times after application of genistein. These responses were not affected by the estrogen receptor (ER) antagonist fulvestrant, revealing that this action of genistein is not through the classical ER pathway. The Sirt1 inhibitor Ex-527 curtailed the genistein-mediated increase in UCP1 and Cebpß mRNA, revealing a role for Sirt1 in mediating the effect. Baseline oxygen consumption and the proportional contribution of proton leak to maximal respiratory capacity was greater for cells exposed to genistein, demonstrating greater mitochondrial uncoupling. CONCLUSIONS: We conclude that genistein acts directly on adipocytes or on adipocyte progenitor cells to programme the cells metabolically to adopt features of beige adipocytes. Thus, this natural dietary agent may protect against obesity and related metabolic disease.

Selenium status is associated with colorectal cancer risk in the European prospective investigation of cancer and nutrition cohort.

Suboptimal intakes of the micronutrient selenium (Se) are found in many parts of Europe. Low Se status may contribute to colorectal cancer (CRC) development. We assessed Se status by measuring serum levels of Se and Selenoprotein P (SePP) and examined the association with CRC risk in a nested case-control design (966 CRC cases; 966 matched controls) within the European Prospective Investigation into Cancer and Nutrition. Se was measured by total reflection X-ray fluorescence and SePP by immunoluminometric sandwich assay. Multivariable incidence rate ratios (IRRs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression. Respective mean Se and SePP levels were 84.0 µg/L and 4.3 mg/L in cases and 85.6 µg/L and 4.4 mg/L in controls. Higher Se concentrations were associated with a non-significant lower CRC risk (IRR = 0.92, 95% CI: 0.82-1.03 per 25 µg/L increase). However, sub-group analyses by sex showed a statistically significant association for women (p(trend) = 0.032; per 25 µg/L Se increase, IRR = 0.83, 95% CI: 0.70-0.97) but not for men. Higher SePP concentrations were inversely associated with CRC risk (p(trend) = 0.009; per 0.806 mg/L increase, IRR = 0.89, 95% CI: 0.82-0.98) with the association more apparent in women (p(trend) = 0.004; IRR = 0.82, 95% CI: 0.72-0.94 per 0.806 mg/L increase) than men (p(trend) = 0.485; IRR = 0.98, 95% CI: 0.86-1.12 per 0.806 mg/L increase). The findings indicate that Se status is suboptimal in many Europeans and suggest an inverse association between CRC risk and higher serum Se status, which is more evident in women.

The single-nucleotide polymorphism (GPX4c718t) in the glutathione peroxidase 4 gene influences endothelial cell function: interaction with selenium and fatty acids.

SCOPE: Selenium (Se) is incorporated into selenoproteins as selenocysteine, which requires structures in the 3'-untranslated region (3'-UTR) of selenoprotein mRNAs. The functional consequences of a single nucleotide polymorphism (SNP) within the 3'-UTR of the selenoprotein GPX4 gene (GPX4c718t) was assessed in human umbilical vein endothelial cells (HUVECs) and monocytes from human volunteers. METHODS AND RESULTS: HUVEC and monocytes homozygous for the T- or C-variant of the GPX4c718t SNP were assessed for monocyte-endothelial cell adhesion, expression of VCAM-1 and sensitivity to oxidative challenge. Interaction of the SNP with Se and different PUFA and effects on selenoprotein expression were also investigated. HUVEC and monocytes homozygous for the T-variant showed elevated adhesion levels compared to cells of the C-variant. This effect was modified by Se and PUFA. HUVEC homozygous for the T-variant showed elevated levels of VCAM-1 protein in the presence of arachidonic acid, were more sensitive to oxidative challenge and showed Se-dependant changes in lipid peroxide levels and expression of additional selenoproteins. CONCLUSION: These findings demonstrate functional effects of the GPX4c718t SNP in endothelial cells and may suggest that individuals with the TT genotype have impaired endothelial function and are at greater risk of vascular disease compared to individuals with the CC genotype.

Selenium in human health and disease.

This review covers current knowledge of selenium in the environment, dietary intakes, metabolism and status, functions in the body, thyroid hormone metabolism, antioxidant defense systems and oxidative metabolism, and the immune system. Selenium toxicity and links between deficiency and Keshan disease and Kashin-Beck disease are described. The relationships between selenium intake/status and various health outcomes, in particular gastrointestinal and prostate cancer, cardiovascular disease, diabetes, and male fertility, are reviewed, and recent developments in genetics of selenoproteins are outlined. The rationale behind current dietary reference intakes of selenium is explained, and examples of differences between countries and/or expert bodies are given. Throughout the review, gaps in knowledge and research requirements are identified. More research is needed to improve our understanding of selenium metabolism and requirements for optimal health. Functions of the majority of the selenoproteins await characterization, the mechanism of absorption has yet to be identified, measures of status need to be developed, and effects of genotype on metabolism require further investigation. The relationships between selenium intake/status and health, or risk of disease, are complex but require elucidation to inform clinical practice, to refine dietary recommendations, and to develop effective public health policies.

Genetic variants in selenoprotein genes increase risk of colorectal cancer.

Low selenium (Se) status correlates with increased risk of colorectal cancer (CRC). Since Se exerts its biological roles through the selenoproteins, genetic variations in selenoprotein genes may influence susceptibility to CRC. This study analysed 12 single-nucleotide polymorphisms (SNPs) in selenoprotein genes [glutathione peroxidase 1 (GPX1), GPX4, 15 kDa selenoprotein (SEP15), selenoprotein S (SELS), selenoprotein P (SEPP1) and thioredoxin reductase 2 (TXNRD2)] and in genes that code for a key protein in Se incorporation [SECIS-binding protein 2 (SBP2)] and in antioxidant defence [superoxide dismutase 2 (SOD2)] in relation to sporadic CRC incidence. CRC patients (832) and controls (705) from the Czech Republic were genotyped using allele specific PCR. Logistic regression analysis showed that three SNPs were significantly associated with an altered risk of CRC: rs7579 (SEPP1), rs713041 (GPX4) and rs34713741 (SELS). The association of these SNPs with disease risk remained after data stratification for diagnosis and adjustments for lifestyle factors and sex. Significant two-loci interactions were observed between rs4880 (SOD2), rs713041 (GPX4) and rs960531 (TXNRD2) and between SEPP1 and either SEP15 or GPX4. The results indicate that SNPs in SEPP1, GPX4 and SELS influence risk of CRC. We hypothesize that the two-loci interactions reflect functional interactions between the gene products. We propose that these variants play a role in cancer development and represent potential biomarkers of CRC risk.

Inter-individual variation in DNA damage and base excision repair in young, healthy non-smokers: effects of dietary supplementation and genotype.

Diets rich in fruits and vegetables are associated with lower risk of cancer which may be conferred in part by the antioxidant properties of these foods. However, antioxidant supplementation or increased consumption of antioxidant-rich foods has been reported to have inconsistent effects on DNA damage. The present work (the DART study) investigated the extent of inter-individual variation in DNA damage, the capacity for base excision repair (BER) and the responses of both variables to supplementation with an antioxidant supplement for 6 weeks. There was a wide inter-individual variation in endogenous lymphocyte DNA strand breaks (8-fold variation), in damage after a challenge with H2O2 (16-fold variation) and in DNA repair (41-fold variation) measured using the comet assay. When stratified into tertiles according to the pre-supplementation level of endogenous DNA damage, there was a statistically significant decrease in DNA damage after supplementation in the tertile with the highest pre-supplementation level of damage. There was no effect of supplementation on BER. Endogenous DNA damage level before supplementation was significantly different (P = 0.037) between the three genotypes for the Val16Ala single nucleotide polymorphism in manganese superoxide dismutase (rs4880) with individuals homozygous/wild type showing less damage than those carrying the alanine variant.

Relative abundance of selenoprotein P isoforms in human plasma depends on genotype, se intake, and cancer status.

Selenium (Se), a dietary trace metal essential for human health, is incorporated into selenoproteins as selenocysteine. Selenoprotein P (SePP), the major plasma selenoprotein, has both transport and antioxidant functions. In humans, it exists in plasma as two isoforms of approximately 50 and 60 kDa. This study investigated the effect of polymorphisms in the SEPP-1 gene, Se supplementation, and disease status on the proportions of SePP plasma isoforms. SePP was isolated from plasma from healthy volunteers, before and after a 6-week supplementation with 100 microg sodium selenite, and from colon cancer patients and controls. SePP isoform distribution was analysed by Western blot. In healthy volunteers, the relative abundance of each isoform depended on two SEPP-1 polymorphisms: rs3877899, predicted to cause an Ala-to-Thr amino acid change at position 234, and rs7579, located in the 3'-untranslated region of SEPP-1 mRNA. The difference between genotypes disappeared after Se supplementation. A genotype-dependent reduction was seen in the proportion of the 60-kDa isoform in patients with colorectal cancer compared with controls. We conclude that functional polymorphisms in the SEPP-1 gene influence the proportion of SePP isoforms in plasma. An elevated proportion of the 60-kDa isoform of SePP may increase selenoprotein synthesis and reduce colorectal cancer risk.

Inter-individual variation in nucleotide excision repair in young adults: effects of age, adiposity, micronutrient supplementation and genotype.

Nucleotide excision repair (NER) is responsible for repairing bulky helix-distorting DNA lesions and is essential for the maintenance of genomic integrity. Severe hereditary impairment of NER leads to cancers such as those in xeroderma pigmentosum, and more moderate reductions in NER capacity have been associated with an increased cancer risk. Diet is a proven modifier of cancer risk but few studies have investigated the potential relationships between diet and NER. In the present study, the plasmid-based host cell reactivation assay was used to measure the NER capacity in peripheral blood mononuclear cells from fifty-seven volunteers aged 18-30 years before and after 6 weeks of supplementation with micronutrients (selenium and vitamins A, C and E). As a control, nine individuals remained unsupplemented over the same period. Volunteers were genotyped for the following polymorphisms in NER genes: ERCC5 Asp1104His (rs17655); XPC Lys939Gln (rs2228001); ERCC2 Lys751Gnl (rs13181); XPC PAT (an 83 bp poly A/T insertion-deletion polymorphism in the XPC gene). NER capacity varied 11-fold between individuals and was inversely associated with age and endogenous DNA strand breaks. For the first time, we observed an inverse association between adiposity and NER. No single polymorphism was associated with the NER capacity, although significant gene-gene interactions were observed between XPC Lys939Gln and ERCC5 Asp1104His and XPC Lys939Gln and ERCC2 Lys751Gnl. While there was no detectable effect of micronutrient supplementation on NER capacity, there was evidence that the effect of fruit intake on the NER capacity may be modulated by the ERCC2 Lys751Gnl single nucleotide polymorphism.

Targeting of transcripts encoding membrane proteins in polarized epithelia: RNA-protein binding studies of the SGLT1 3'-UTR.

mRNA stability, mRNA translation and spatial localization of mRNA species within a cell can be governed by signals in the 3'-UTR (3'-untranslated region). Local translation of proteins is essential for the development of many eukaryotic cell types, such as the Drosophila embryo, where the spatial and temporal localization of bicoid and gurken mRNAs, among others, is required to establish morphogen gradients. More recent studies have suggested that mRNA localization also occurs with transcripts coding for membrane-based or secreted proteins, and that localization at organelles such as the endoplasmic reticulum directs translation more efficiently to specific subdomains, so as to aid correct protein localization. In human epithelial cells, the mRNA coding for SGLT1 (sodium-glucose co-transporter 1), an apical membrane protein, has been shown to be localized apically in polarized cells. However, the nature of the signals and RNA-binding proteins involved are unknown. Ongoing work is aimed at identifying the localization signals in the SGLT1 3'-UTR and the corresponding binding proteins. Using a protein extract from polarized Caco-2 cells, both EMSAs (electrophoretic mobility-shift assays) and UV cross-linking assays have shown that a specific protein complex is formed with the first 300 bases of the 3'-UTR sequence. MFold predictions suggest that this region folds into a complex structure and ongoing studies using a series of strategic deletions are being carried out to identify the precise nature of the motif involved, particularly the role of the sequence or RNA secondary structure, as well as to identify the main proteins present within the complex. Such information will provide details of the post-transcriptional events that lead to apical localization of the SGLT1 transcript and may reveal mechanisms of more fundamental importance in the apical localization of proteins in polarized epithelia.

Supplementation of healthy volunteers with nutritionally relevant amounts of selenium increases the expression of lymphocyte protein biosynthesis genes.

BACKGROUND: Selenium is incorporated into 25 selenoproteins in humans. Low dietary selenium has deleterious effects on health and may result in cancer, cardiovascular disease, and immune dysfunction. The underlying mechanisms are not fully understood. Lymphocytes are a target tissue; they can be assessed in healthy persons, and their response has not been explored by using global gene expression profiling techniques. OBJECTIVES: The objectives of the study were to assess the overall effect of selenium supplementation within a normal physiological range on the pattern of lymphocyte gene expression and to identify downstream processes affected by selenium intake. DESIGN: Gene expression was assessed in lymphocytes isolated from 39 healthy persons before and after a 6-wk supplementation with 100 microg Se/d as sodium selenite. Presupplementation and postsupplementation RNA samples from 16 subjects were chosen at random for microarray analysis. Differential gene expression was analyzed by using individual labeling and hybridization with human whole-genome microarrays. Array data were validated by quantitative real-time reverse transcriptase-polymerase chain reaction. RESULTS: The study subjects had an average 19% increase in plasma selenium concentration, which was within a normal range. Fold changes in gene expression were small, but data analysis using biological process identification showed that selenium predominantly affected the genes that encode proteins functioning in protein biosynthesis. Gene expression changes were confirmed by quantitative polymerase chain reaction for 3 representative target genes (RPL37A, RPL30, and EEF1E1). CONCLUSIONS: Ribosomal protein and translation factor genes were up-regulated in response to increased selenium intake. We hypothesize that this up-regulation is linked to increased selenoprotein production and enhanced lymphocyte function.

Genetic polymorphisms in the human selenoprotein P gene determine the response of selenoprotein markers to selenium supplementation in a gender-specific manner (the SELGEN study).

Selenium (Se), a micronutrient essential for human health, is incorporated into at least 25 selenoproteins including selenoprotein P (SePP), which transports Se within the body. This research identified two single nucleotide polymorphisms (SNPs) in the SePP gene, one in the coding region (position 24731, causing an Ala to Thr change) and one in the 3'untranslated region (position 25191). Their frequency was similar in Caucasian, Chinese, and South Asian populations. Prospectively genotyped volunteers were supplemented for 6 wk with 100 microg sodium selenite/day. Blood samples were analyzed for plasma Se and selenoprotein biomarkers at baseline, after supplementation, and during a washout period. Plasma Se, SePP, and glutathione peroxidase 3 (GPx3) levels increased with supplementation. Baseline plasma Se content depended on both SePP genotypes and body mass index. Presupplementation SePP concentration was associated with gender and genotype at SNP 24731 and postsupplementation concentration with SNP 25191. Both SNPs and gender were associated with differences in GPx3 activity, plasma, and erythrocyte thioredoxin reductase 1 concentrations and lymphocyte glutathione peroxidase 1 and 4 activities and concentrations. In conclusion, the data reveal two common functional SNPs within the human SePP gene that may predict behavior of biomarkers of Se status and response to supplementation and thus susceptibility to disease.

The biological revolution: understanding the impact of SNPs on diet-cancer interrelationships.

Evidence is accumulating that individual risk of neoplasia depends on complex interactions among genetic inheritance, a range of exposures both in utero and in postnatal life, and the play of chance. Knowledge of the portfolio of genetic variants that confer susceptibility or resistance to cancer is limited, and there is potential for genome-wide scans and hypothesis-driven studies to reveal novel polymorphisms and haplotypes that modify risk. There is only fragmentary evidence of the scale and nature of diet-gene interactions that modulate risk of neoplasia, but it seems probable that such interactions will play a significant role as they do in other complex diseases including cardiovascular disease and type 2 diabetes. All existing evidence about diet-gene interactions and cancer risk comes from observational studies, and it will be necessary to undertake intervention studies to test the hypotheses generated by epidemiologic investigations. Because it is very unlikely that primary cancer will be an endpoint in dietary intervention studies in the foreseeable future, development of robust surrogate endpoints is a high priority. Emerging biological science using epigenomics, proteomics, and other molecular technologies appears to offer novel approaches to the discovery and validation of surrogate endpoints.