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Supplementary Figure 1 from Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells

Supplementary Figure 2 from Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells

Supplementary Table 1 from Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells

Selenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic 75Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 ± 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G2-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle. Cancer Prev Res; 3(5); 630–9. ©2010 AACR.

Selenium is a fascinating element that has a long history, most of which documents it as a deleterious element to health. In more recent years, selenium has been found to be an essential element in the diet of humans, all other mammals, and many other life forms. It has many health benefits that include, for example, roles in preventing heart disease and certain forms of cancer, slowing AIDS progression in HIV patients, supporting male reproduction, inhibiting viral expression, and boosting the immune system, and it also plays essential roles in mammalian development. Elucidating the molecular biology of selenium over the past 40 years generated an entirely new field of science which encompassed the many novel features of selenium. These features were (1) how this element makes its way into protein as the 21st amino acid in the genetic code, selenocysteine (Sec); (2) the vast amount of machinery dedicated to synthesizing Sec uniquely on its tRNA; (3) the incorporation of Sec into protein; and (4) the roles of the resulting Sec-containing proteins (selenoproteins) in health and development. One of the research areas receiving the most attention regarding selenium in health has been its role in cancer prevention, but further research has also exposed the role of this element as a facilitator of various maladies, including cancer.

Selenophosphate synthetase (SEPHS) was originally discovered in prokaryotes as an enzyme that catalyzes selenophosphate synthesis using inorganic selenium and ATP as substrates. However, in contrast to prokaryotes, two paralogs, SEPHS1 and SEPHS2, occur in many eukaryotes. Prokaryotic SEPHS, also known as SelD, contains either cysteine (Cys) or selenocysteine (Sec) in the catalytic domain. In eukaryotes, only SEPHS2 carries out selenophosphate synthesis and contains Sec at the active site. However, SEPHS1 contains amino acids other than Sec or Cys at the catalytic position. Phylogenetic analysis of SEPHSs reveals that the ancestral SEPHS contains both selenophosphate synthesis and another unknown activity, and that SEPHS1 lost the selenophosphate synthesis activity. The three-dimensional structure of SEPHS1 suggests that its homodimer is unable to form selenophosphate, but retains ATPase activity to produce ADP and inorganic phosphate. The most prominent function of SEPHS1 is that it is implicated in the regulation of cellular redox homeostasis. Deficiency of SEPHS1 leads to the disturbance in the expression of genes involved in redox homeostasis. Different types of reactive oxygen species (ROS) are accumulated in response to SEPHS deficiency depending on cell or tissue types. The accumulation of ROS causes pleiotropic effects such as growth retardation, apoptosis, DNA damage, and embryonic lethality. SEPHS1 deficiency in mouse embryos affects retinoic signaling and other related signaling pathways depending on the embryonal stage until the embryo dies at E11.5. Dysregulated SEPHS1 is associated with the pathogenesis of various diseases including cancer, Crohn's disease, and osteoarthritis.

The selenium field expanded at a rapid rate for about 45 years, from the mid-1970’s until about 2015 (see [...]

Selenoproteins play important roles in many cellular functions and biochemical pathways in mammals. Our previous study showed that the deficiency of the 15 kDa selenoprotein (Selenof) significantly reduced the formation of aberrant crypt foci (ACF) in a mouse model of azoxymethane (AOM)-induced colon carcinogenesis. The objective of this study was to examine the effects of Selenof on inflammatory tumorigenesis, and whether dietary selenium modified these effects. For 20 weeks post-weaning, Selenof-knockout (KO) mice and littermate controls were fed diets that were either deficient, adequate or high in sodium selenite. Colon tumors were induced with AOM and dextran sulfate sodium. Surprisingly, KO mice had drastically fewer ACF but developed a similar number of tumors as their littermate controls. Expression of genes important in inflammatory colorectal cancer and those relevant to epithelial barrier function was assessed, in addition to structural differences via tissue histology. Our findings point to Selenof’s potential role in intestinal barrier integrity and structural changes in glandular and mucin-producing goblet cells in the mucosa and submucosa, which may determine the type of tumor developing.

Summary: Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins. : Yim et al. report that Selenof (15-kDa selenoprotein; Sep15) functions as a gatekeeper of immunoglobulins and, likely, other client proteins en route from the ER to the Golgi apparatus, thereby preventing secretion of dysfunctional proteins and supporting redox quality control. Keywords: Selenof, selenoprotein, oxidoreductase, immunoglobulins, IgM, knockout mouse, endoplasmic reticulum, Sep15, gatekeeper

OBJECTIVES: The goal of this study is to compare the microbiome of domesticated horses with and without probiotic supplementation. METHODS: Our University's Institutional Animal Care and Use Committee has granted an exemption, as the horses were not housed on campus and there was no experimental manipulation to the horses’ feeding implemented. Fecal matter from six privately owned horses maintained on their standard grazing diet were collected. Three of the six horses received a probiotic supplement, SmartDigest, for several years prior to beginning the project. Supplementation ceased for one month, and samples were again obtained. The other three horses never received probiotics. Bacterial DNA was isolated from all fecal samples, the 16S rRNA gene amplified, tagged with index primers, and subsequently sequenced using the Illumina MiSeq. RESULTS: Dominant groups from non-supplemented horses residing on the same property included the phyla bacteriodetes, firmicutes, proteobacteria, and verrucomicrobia. Interestingly, Sphingobacterium bambusae was identified in all three horses, even though this species has previously been isolated from the soil of bamboo plantations. Currently, samples from horses with probiotics are being analyzed. We are also employing qPCR analysis to validate the NextGen data, and to further investigate relative abundance of specific bacterial groups relevant to equine intestinal health. CONCLUSIONS: Preliminary NextGen sequence analysis of the relative abundance of bacterial phyla suggests that, as expected, the horses residing on the same property and thus receiving the same diet possess a similar intestinal microbiome composition. Similarities between horses persist down to the genus level, and are now being compared to samples from horses on a probiotic-supplemented diet. FUNDING SOURCES: Financial support was provided by Towson University's Fisher College of Science and Mathematics, and Jess and Mildred Fisher Endowed Chair funds to P. Tsuji.

OBJECTIVES: Plant secondary metabolites, such as polyphenols, are found in many fruits, grains, and vegetables, and are thus a part of normal human diet. One such food is sorghum (Sorghum bicolor), a cereal grain that contains varying concentrations of polyphenols. Many polyphenols have been implicated in the regulation of colon cancer through modulating the antioxidant defense, which includes some of the major selenoproteins, e.g., thioredoxin reductases (TXNRD) and glutathione peroxidases (GPX). However, because such redox-active enzymes have been shown to be involved in both cancer prevention and promotion, the goal of our study is to assess the impact of high-polyphenol sorghum extracts on the expression of selenoproteins. METHODS: Human colon cancer cells (HT29, HCT116) were incubated with 1.25 mg high-polyphenol sorghum bran extract per mL medium for 48 h. RNA was extracted with Trizol/Chloroform, and reverse-transcribed to cDNA. mRNA expression of selenoproteins was quantitated using qPCR, normalized to GAPDH, and analyzed using GraphPad Prism. Protein lysates will be used for Western blotting and catalytic activity assays. RESULTS: Compared to solvent control, incubation of human colon cancer cells with high-polyphenol sorghum extracts for 48 h moderately impacted mRNA expression of investigated selenoproteins. One of the extracts resulted in a nearly doubled GPX1 mRNA expression in HCT116 cells (p = 0.08), whereas preliminary results suggest that TXNRD1 expression may be lowered. CONCLUSIONS: High-polyphenol varieties of sorghum may affect selenoprotein expression. Further investigations involving both shorter and longer-term incubation times, as well as effects on protein expression and activity will help elucidate the effects of these new sorghum varieties on selenoprotein expression important in prevention and promotion of colon cancer. FUNDING SOURCES: Financial support was provided by Towson University's Fisher College of Science and Mathematics (P. Tsuji) and the USDA (D. Smolensky).

Colorectal cancer is a major public health issue, accounting for nearly 150,000 cases annually. Because colorectal cancer is correlated with inflammation and oxidative cellular stress, it would be highly beneficial to exploit bioactive components in our diet that could potentially inhibit cancer promotion. Certain varieties of the dietary grain sorghum (S. bicolor) contain high amounts bioactive polyphenols, which may contribute to the prevention of colon cancer due to their antioxidant properties, anti-inflammatory, and anti-carcinogenesis effects, some of which may be mediated by epigenetic mechanisms. Tumor suppressor genes are often hyper-methylated in tumors. Therefore, DNA methylation and histone acetylation patterns may affect the expression of genes that play a role in the promotion or prevention of colon cancer. Our objective is to investigate how high-polyphenol sorghum varietals impact epigenetic mechanisms in human colon cancer cells, including quantitating global DNA methylation and histone acetylation. We treated HCT116 human colon cancer cells with high-polyphenol sorghum extracts or solvent control, or 5 µM epigallocatechin gallate as a positive control, as it's known to decrease DNA methylation. Total RNA, protein, and DNA were harvested after 24 and 48 hours. mRNA expression of DNA methyltransferases (DNMT) were quantitated with qPCR. Our preliminary results suggest that high polyphenol sorghum may decrease the mRNA expression of DNMT1, the key enzyme that upholds methylation patterns after DNA synthesis. Furthermore, mRNA expression of DNMT 3A, which is the enzyme responsible for de novo DNA methylation, also indicated a decrease. We further plan to assess protein expression and catalytic activity of DNMTs and histone acetyltransferases in addition to global DNA methylation and histone acetylation to investigate the impact of these high polyphenol sorghum varietals on relevant epigenetic mechanisms in both HCT116 and CACO-2 cells. Consumption of high-polyphenol sorghum as part of the human diet may lead to global or gene-specific hypo-methylation or hyper-acetylation, and this may be of importance in the prevention of colon cancer. Research supported by USDA ARS Kansas. Citation Format: Zeguela Kamagate, Dmitriy Smolensky, Petra Tsuji. The effect of sorghum on epigenetic modifications in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2088.

OBJECTIVES: Colorectal cancer (CRC) is a major public health issue, accounting for nearly 150,000 cases annually. Because CRC correlates with oxidative stress, exploiting the antioxidative nature of bioactive components in our diet that could potentially inhibit cancer promotion might be beneficial. New varietals of the grain Sorghum bicolor contain high amounts of polyphenols, which may contribute to CRC prevention due to their strong antioxidative and anti-carcinogenic properties. Because gene expression of enzymes may be mediated by epigenetic mechanisms, we are interested in DNA methylation and histone acetylation patterns of genes, including those enzymes that play a role in the promotion or prevention of CRC. Our objective is to examine how high-polyphenol sorghum varietals impact epigenetic mechanisms in human colon cancer cells. METHODS: We are currently assessing mRNA and protein expression, catalytic activity of DNA-methyltransferases (DNMTs) and histone acetyltransferases, in addition to global DNA methylation and histone acetylation in human HCT116 and CACO-2 colon cancer cells. We treated HCT116 cells with new high-polyphenol or commercially available S. bicolor extracts, solvent control, or 5 μM epigallocatechin gallate as a positive control for up to 48 hours. Total RNA, protein, and DNA were harvested, and mRNA expression of selected genes was quantitated with qPCR, and protein expression is being quantitated with Western blotting. RESULTS: Our preliminary results suggest that high-polyphenol S. bicolor may decrease mRNA expression of DNMT1, the key enzyme that upholds methylation patterns after DNA synthesis. Furthermore, mRNA expression of DNMT3A, the enzyme responsible for de novo DNA methylation, was also decreased. We will further investigate global and gene-specific DNA methylation, and the expression of genes involved in tumorigenesis and chemoprevention. CONCLUSIONS: Consumption of high-polyphenol S. bicolor as part of the human diet may lead to beneficial changes in chromatin methylation or acetylation. Subsequently, the expression of specific genes involved in the promotion or prevention of colorectal cancer may be modified, providing a dietary intervention to CRC. FUNDING SOURCES: Towson University. Bridges-to-Doctorate grant. USDA ARS Kansas.

Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogues, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyses the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1-knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by embryonic day (E)8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione transferase Omega 1 (GSTO1). To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma (EC) cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Furthermore, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homoeostasis and controlling cell growth.

The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4−/− mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4−/− livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4−/− pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4−/− mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation., Graphical abstract, Highlights • Conditional Gpx4 loss causes hepatocellular degeneration and early death of mice. • Dietary vitamin E supplementation rescues death of liver-specific Gpx4 null mice. • Nutritional vitamin E content in diet may severely impact experimental outcome.

Selenium is an essential element that is required to support a number of cellular functions and biochemical pathways. The objective of this study was to examine the effects of reduced dietary selenium levels on gene expression to assess changes in expression of non-selenoprotein genes that may contribute to the physiological consequences of selenium deficiency. Mice were fed diets that were either deficient in selenium or supplemented with selenium in the form of sodium selenite for six weeks. Differences in liver mRNA expression and translation were measured using a combination of ribosome profiling, RNA-Seq, microarrays, and qPCR. Expression levels and translation of mRNAs encoding stress-related selenoproteins were shown to be up-regulated by increased selenium status, as were genes involved in inflammation and response to interferon-γ. Changes in serum cytokine levels were measured which confirmed that interferon-γ, as well as IL-6, were increased in selenium adequate mice. Finally, microarray and qPCR analysis of lung tissue demonstrated that the selenium effects on immune function are not limited to liver. These data are consistent with previous reports indicating that adequate selenium levels can support beneficial immune responses, and further identify the IL-6 and interferon-γ pathways as being responsive to dietary selenium intake.

Selenocysteine-containing proteins (selenoproteins) have been implicated in the regulation of various cell signaling pathways, many of which are linked to colorectal malignancies. In this in-depth excurse into the selenoprotein literature, we review possible roles for human selenoproteins in colorectal cancer, focusing on the typical hallmarks of cancer cells and their tumor-enabling characteristics. Human genome studies of single nucleotide polymorphisms in various genes coding for selenoproteins have revealed potential involvement of glutathione peroxidases, thioredoxin reductases, and other proteins. Cell culture studies with targeted down-regulation of selenoproteins and studies utilizing knockout/transgenic animal models have helped elucidate the potential roles of individual selenoproteins in this malignancy. Those selenoproteins, for which strong links to development or progression of colorectal cancer have been described, may be potential future targets for clinical interventions.

The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility.

The selenocysteine (Sec) tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2’-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified post-transcriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA[Ser]Sec population by one or more procedures and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA[Ser]Sec and resolving the isoforms are used in only a few laboratories, these procedures will be discussed briefly and references provided for more detailed information, while the more recently developed procedures are discussed in detail. In addition, a novel technique that was developed in sequencing tRNA[Ser]Sec for identifying their occurrence in other organisms is also presented.

The selenocysteine (Sec) tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2'-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified posttranscriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA[Ser]Sec population by one or more procedures, and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA[Ser]Sec and resolving the isoforms are used in only a few laboratories, these procedures will be discussed briefly and references provided for more detailed information, while the more recently developed procedures are discussed in detail. In addition, a novel technique that was developed in sequencing tRNA[Ser]Sec for identifying their occurrence in other organisms is also presented.

OBJECTIVES: Polyphenols are plant secondary metabolites found in many fruits, grains, and vegetables, and are thus a part of normal human diet. One such food is sorghum (Sorghum bicolor), a cereal grain that contains varying concentrations of polyphenols. Many polyphenols have been implicated in the regulation of bioactivating (phase 1) and bioinactivating (phase 2) enzymes. The goal of this study is to assess the impact of sorghum polyphenol extracts on the expression of xenobiotic-activating and -inactivating enzymes important in cancer prevention and promotion. METHODS: Polyphenols were extracted from two types of sorghum bran. Human colon cancer cells (HT29, HCT116) were incubated with 1.25 mg extract per mL medium for 48 hours. RNA was extracted with Trizol/Chloroform, and reverse-transcribed to cDNA. mRNA expression of carcinogen-activating and -inactivating enzymes are quantitated using qPCR, normalized to GAPDH as internal control, and analyzed using GraphPad Prism. Protein extracts will be used for Western blotting to assess impact on protein expression and enzymatic activity. RESULTS: Compared to solvent control, mRNA expression of the arylhydrocarbon receptor (AHR) nuclear transferase (ARNT) increased in HT29 cells exposed to a novel high-polyphenol abstract (N = 4, p = 0.0017), whereas expression of AHR itself remained unaffected. Preliminary data suggest that expression of carcinogen-metabolizing enzymes, such as CYP1A1, may also increase in colon cancer cells upon exposure to the novel high-polyphenol extract. CONCLUSIONS: The balance of xenobiotic-bioactivating and –inactivating enzymes is important to consider, as we investigate carcinogenesis and its prevention using dietary micronutrients. We continue to elucidate the expression and activity of such phase I and II enzymes in colon cancer cell models exposed to polyphenolic extracts. FUNDING SOURCES: Financial support was provided by Towson University's Fisher College of Science and Mathematics (P. Tsuji), and the USDA (D. Smolensky).

OBJECTIVES: The isothiocyanate sulforaphane and the polyphenols chrysin, epigallocatechin-3-gallate, and resveratrol are dietary micronutrients that are thought to exhibit potential in cancer prevention. These compounds are commonly found in the human diet, and previous research suggests that they may play a role in cellular processes that are often labeled as the ‘hallmarks of cancer’. Our goal is to investigate the impact of these dietary nutrients on the expression of genes associated with apoptosis, metastasis, and invasion, and thus their potential role in regulating the expression of genes involved in colon cancer. METHODS: Colorectal cancer cells were incubated for up to 48 h. RNA was extracted with Trizol/Chloroform, and reverse-transcribed to cDNA. mRNA expression was quantitated using qPCR, normalized to Gapdh, and analyzed using GraphPad Prism. Total protein was extracted using lysis buffer for subsequent Western blotting and catalytic activity assays. RESULTS: Our preliminary results suggest that especially the isothiocyanate sulforaphane increases mRNA expression of apoptosis-regulatory genes, such as Cyclooxygenase 2 (p

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.

Selenium is essential to mammals and many other life forms, but its ingested levels are critical to health because too little or too much can have severe consequences on the well-being of an organism. Selenium may exhibit its effects through selenocompounds and/or selenoproteins; however, selenoproteins have received much more attention, and the data suggest that they are primarily responsible for the health benefits of this element. Selenium has been recognized for its role in preventing, and more recently in promoting, cancer. A combination of bioinformatics and experimental approaches and mouse models uncovered the roles of housekeeping and stress-related selenoproteins, and individual selenoproteins, in health and disease, including their contrasting roles in cancer. These and other topics on the role of selenium and selenoproteins in cancer are discussed.

International audience; The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

The discovery of multiple selenoproteins has raised tantalizing questions about their role in maintaining normal cellular function. Unfortunately, many of these remain inadequately investigated. While they have a role in maintaining redox balance, other functions are becoming increasingly recognized. As the roles of these selenoproteins are further characterized, a better understanding of the true physiological significance of this trace element will arise. This knowledge will be essential in defining optimum intakes to achieve cellular homeostasis in order to optimize health, including a reduction in cancer, for diverse populations. Human variation in the response to selenium likely reflects significant interactions between the type and amounts of selenium consumed with the genome and a host of environmental factors including the totality of the diet, as discussed in this review.

Selenium is an essential micronutrient for humans and animals, and is thought to provide protection against some forms of cancer. These protective effects appear to be mediated, at least in part, through selenium-containing proteins (selenoproteins). Recent studies in a mouse colon cancer cell line have shown that the 15 kDa selenoprotein (Sep15) may also play a role in promoting colon cancer. The current study investigated whether the effects of reversing the cancer phenotype observed when Sep15 was removed in mouse colon cancer cells, were recapitulated in HCT116 and HT29 human colorectal carcinoma cells. Targeted down-regulation of Sep15 using RNAi technology in these human colon cancer cell lines resulted in similarly decreased growth under anchorage-dependent and anchorage-independent conditions. However, the magnitude of reduction in cell growth was much less than in the mouse colon cancer cell line investigated previously. Furthermore, changes in cell cycle distribution were observed, indicating a delayed release of Sep15 deficient cells from the G(0)/G(1) phase after synchronization. The potential mechanism by which human colon cancer cells lacking Sep15 revert their cancer phenotype will need to be explored further.

Selenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic 75Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 ± 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G2-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle. Cancer Prev Res; 3(5); 630–9. ©2010 AACR.

Many flavonoids have been shown to possess prooxidant properties, capable of causing oxidative stress, especially at larger doses. Here, we examined the potential cell toxicity caused by exposure to the hydroxylated flavones chrysin, apigenin, luteolin and quercetin in comparison to the methylated flavones 5,7-dimethoxyflavone and 3',4'-dimethoxyflavone in normal Rainbow trout hepatocytes. The hydroxylated flavones, especially chrysin, demonstrated cell toxicity and inhibition of DNA synthesis at very low (2 microM) concentrations. The cytotoxicity of chrysin may partially be due to its metabolism by myeloperoxidase, which was shown to be present in these normal trout liver cells (164pmol/(min mg protein)). In contrast, methylated flavones showed no significant metabolism by myeloperoxidase and no signs of toxicity, even at much higher concentrations. These results may be useful for further investigations of cytotoxicity of dietary flavonoids.

When selenocysteine (Sec) tRNA[Ser]Sec was originally discovered, it was proposed to be the first nonsense suppressor tRNA found in mammalian and avian tissues, since it exclusively decoded the nonsense codon, UGA, which normally dictates the cessation of protein synthesis. This tRNA was subsequently shown to be Sec tRNA, which inserted Sec into protein as the 21st proteinogenic amino acid. Once it was established that this tRNA was aminoacylated with serine by seryl-tRNA synthetase and served as the scaffold for Sec synthesis, Sec tRNA was appropriately named Sec tRNA[Ser]Sec. The mammalian Sec-tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2′-O-methyl moiety on the uridine at position 34, designated Um34. The non-Um34 isoform is involved in the synthesis of a subclass of selenoproteins, called housekeeping selenoproteins, while the Um34 isoform supports synthesis of stress-related selenoproteins. These novel functions and other unique features of Sec tRNA are the subjects of this chapter, supporting the idea that this tRNA is the quintessential constituent in selenoprotein biosynthesis.

Malignant tumors are known to require robust antioxidant systems to sustain their rapidly dividing cells and protect them from oxidative damage. The dietary trace mineral selenium, through its incorporation into selenoproteins such as thioredoxin reductase 1 (TXNRD1), glutathione peroxidase (GPX) 2 and the 15 kDa selenoprotein (SEP15), has been shown to play important roles in redox-regulation. Given that the functions of these selenoenzymes protect both normal and malignant cells from oxidative stress, these very same redox-regulatory processes are thought to result in both anti- and pro-tumorigenic effects at a tissue-specific and cellular level; thus, these selenoproteins are often referred to as having a “Dr. Jekyll and Mr. Hyde personality”. Herein, we summarize the main findings with emphasis on TXNRD1 and SEP15, and their roles in the regulation of specific studies of lung, liver and colon cancers to illustrate the differences in the antioxidants involved, and the complexities of their interplay with other antioxidants or antioxidant systems. It should be noted that it remains to be established if any of the observed anti- and pro-tumorigenic effects of TXNRD1 and SEP15 are possibly tumor stage or grade-dependent.

The 15 kDa selenoprotein (SEP15) is one of more than 25 selenoproteins found in humans. This protein has been proposed to be involved in redox regulation in the endoplasmic reticulum (ER), although the biological function of SEP15 is still not completely understood. It appears to have strong tissue specificity, and has been shown to have a split personality in terms of cancer initiation and promotion. Polymorphisms in the human SEP15 gene have been linked to increases in several types of cancer. Thus, among the many selenoproteins, SEP15 continues to generate interest due to its potential implications in human health and disease.

Trace minerals are inorganic micronutrients, many of which are essential components of enzymes. Because trace minerals are involved in many cellular functions, deficiency of some of these dietary micronutrients may cause debility and disease and may possibly result in death. The dietary sources, human consumption, bioavailability, and health effects of the trace minerals chromium, copper, manganese, molybdenum, selenium, and zinc, as well as the trace element iodine, are described herein.

Selenium (Se) is an essential trace mineral found ubiquitously in the environment in geographically variable amounts. The bioavailability of Se compounds varies depending on the source and form of Se, with our primary source of Se intake through food, including grains, animal products and, increasingly recently, dietary supplements. Recommended dietary amounts vary slightly among countries due to differential average intakes and subsequent Se status across populations, and typically range from 40–45 μg/day for adults in Japan to 75 μg/day for adults in the United Kingdom. Controversies continue to prevail regarding adequate amounts of Se for health and disease prevention in the general population and for populations which are potentially more sensitive to changes in dietary Se. The biological response to dietary Se, much like that of other nutrients, likely follows a U-shaped curve, which may be modulated by a variety of factors, including gender, ethnicity, Se status, and background health.

The collection of diseases we call cancer accounts for about 20% of all deaths in industrialized countries. Dietary nutrients may interfere with the cancer process and thus may be employed as chemopreventive agents. Observational studies in humans, which frequently rely on food frequency questionnaires and extrapolation of nutrient content from databases, have indicated a variety of phytochemicals with great cancer-preventive potential. Most of the supporting data for chemoprevention are derived from preclinical cell culture or animal experiments, investigating a single nutrient in relation to selected gene products in the cancer process. In humans, randomized clinical trials with single nutrients or single foods have mostly shown conflicting results. This article presents early and recent findings of dietary food components that have been implicated as chemoprevention agents, because of either their antioxidative nature or their influence on molecular mechanisms important in the initiation and promotion of cancers.

A common characteristic of many cancer cells is that they suffer from oxidative stress. They, therefore, require effective redox regulatory systems to combat the higher levels of reactive oxygen species that accompany accelerated growth compared to the normal cells of origin. An elevated dependence on these systems in cancers suggests that targeting these systems may provide an avenue for retarding the malignancy process. Herein, we examined the redox regulatory systems in human liver and lung cancers by comparing human lung adenocarcinoma and liver carcinoma to their respective surrounding normal tissues. Significant differences were found in the two major redox systems, the thioredoxin and glutathione systems. Thioredoxin reductase 1 levels were elevated in both malignancies, but thioredoxin was highly upregulated in lung tumor and only slightly upregulated in liver tumor, while peroxiredoxin 1 was highly elevated in lung tumor, but downregulated in liver tumor. There were also major differences within the glutathione system between the malignancies and their normal tissues. The data suggest a greater dependence of liver on either the thioredoxin or glutathione system to drive the malignancy, while lung cancer appeared to depend primarily on the thioredoxin system.

Selenium (Se) exerts its mechanistic effects through incorporation into selenoproteins. Many selenoproteins, such as the 15kDa selenoprotein (Sep15), are involved in cancer prevention and promotion...

Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

Previously, systemic knockout (KO) of the ER-resident 15kDa selenoprotein (Sep15, SelenoF) protected mice against the formation of chemically-induced pre-neoplastic lesions and suggested modulation of inflammatory pathways. Our objective is to elucidate the potential role of SelenoF in the early steps of colon tumorigenesis by assaying gene expression of KO mice and littermate controls (WT) that were either treated with 2% dextran sulfate sodium (DSS) to induce colitis or given water (control). Based on previous results, we hypothesize that SelenoF expression is inversely correlated with a protective interferon (IFN)-γ pro-inflammatory response. Microarray analyses of colonic mucosa suggest that knockout of SelenoF expression itself caused more significant gene expression changes than DSS treatment (inflammation). Gene pathway analyses indicated galectin-6 was the highest up-regulated gene in DSS-treated (133-fold) and untreated (30-fold) KO compared to WT mice. The top significantly changed network included genes involved in the differentiation of T-lymphocytes. Colonic gene expression changes in SelenoF-KO mice, as validated with qPCR, included up-regulation of guanylate-binding protein 1 (p=0.004), and a down-regulation of T-box transcription factor 21 (p=0.08). Interestingly, a decrease in IFN-γ levels (p=0.026) was detected in the serum of KO compared to WT mice, whereas the opposite trend was shown in colonic mucosa. Thus, our preliminary analyses indicate that SelenoF may indeed influence the expression of inflammatory genes in the colon. Its contribution to the regulation of colitis will continue to be elucidated.

The 15-kDa selenoprotein (Sep15) has been implicated in etiology of some types of cancer. Herein, inducible RNAi cell lines were established and cell morphology and motility were analyzed. The majority of Sep15-deficient cells (>95%) formed membrane blebs in a dynamic manner. Blebbing cells transformed cell morphology from a normal flat spindle shape to a spherical morphology. In blebbing cells, actin fibers moved to the cell periphery, covering and obscuring visualization of α-tubulin. Bleb formation was suppressed by the inhibitors of Rho-associated protein kinase (ROCK), RhoA or myosin light chain (MLC), restoring blebbing cells to wild-type morphology. RhoA activation and phosphorylation of myosin phosphatase target subunit 1 was induced by Sep15 knockdown. Sep15-deficient cells were non-apoptotic, and displayed a distinct relative localization of F-actin and α-tubulin from typical apoptotic blebbing cells. Our data suggest that Sep15 in Chang liver cells regulates the pathway that antagonizes RhoA/ROCK/MLC-dependent non-apoptotic bleb formation.

The many biological and biomedical effects of selenium are relatively unknown outside the selenium field. This fascinating element, initially described as a toxin, was subsequently shown to be essential for health and development. By the mid-1990s selenium emerged as one of the most promising cancer chemopreventive agents, but subsequent human clinical trials yielded contradictory results. However, basic research on selenium continued to move at a rapid pace, elucidating its many roles in health, development, and in cancer prevention and promotion. Dietary selenium acts principally through selenoproteins, most of which are oxidoreductases involved in diverse cellular functions.