Selenium,selenoproteins and cancer of the thyroid

Selenium is an essential mineral element with important biological functions for the whole body through incorporation into selenoproteins. This element is highly concentrated in the thyroid gland. Selenoproteins provide antioxidant protection for this tissue against the oxidative stress caused by free radicals and contribute, via iodothyronine deiodinases, to the metabolism of thyroid hormones. It is known that oxidative stress plays a major role in carcinogenesis and that in recent decades there has been an increase in the incidence of thyroid cancer. The anti-carcinogenic action of selenium, although not fully understood, is mainly attributable to selenoproteins antioxidant properties, and to the ability to modulate cell proliferation (cell cycle and apoptosis), energy metabolism, and cellular immune response, significantly altered during tumorigenesis. Researchers have suggested that different forms of selenium supplementation may be beneficial in the prevention and treatment of thyroid cancer; however, the studies have several methodological limitations. This review is a summary of the current knowledge on how selenium and selenoproteins related to thyroid cancer.     

Specific antioxidant selenoproteins are induced in the heart during hypertrophy

Selenium (Se) is thought to confer cardioprotective effects through the actions of antioxidant selenoprotein enzymes that directly limit levels of ROS such as hydrogen peroxide (H₂O₂) or that reverse oxidative damage to lipids and proteins. To determine how the selenoproteome responds to myocardial hypertrophy, two mouse models were employed: triidothyronine (T3)- or isoproterenol (ISO)-treatment. After 7 days of T3- and ISO-treatment, cardiac stress was demonstrated by increased H₂O₂ and caspase-3 activity. Neither treatment produced significant increases in phospholipid peroxidation or TUNEL-positive cells, suggesting that antioxidant systems were protecting the cardiomyocytes from damage. Many selenoprotein mRNAs were induced by T3- and ISO-treatment, with levels of methionine sulfoxide reductase 1 (MsrB1, also called SelR) mRNA showing the largest increases. MsrB enzymatic activity was also elevated in both models of cardiac stress, while glutathione peroxidase (GPx) activity and thioredoxin reductase (Trxrd) activity were moderately and nonsignificantly increased, respectively. Western blot assays revealed a marked increase in MsrB1 and moderate increases in GPx3, GPx4, and Trxrd1, particularly in T3-treated hearts. Thus, the main response of the selenoproteome during hypertrophy does not involve increased GPx1, but increased GPx3 for reducing extracellular H₂O₂ and increased GPx4, Trxrd1, and MsrB1 for minimizing intracellular oxidative damage.     

Biosynthesis of selenoproteins in cultured bovine mammary cells

The biosynthesis of selenoproteins was studied in relation to milk formation and mammary cell biology by incubating the bovine mammary cell line MAC-T with ((75)Se)selenite. Intracellular proteins and proteins secreted into the cell culture medium were separated by 2D electrophoresis, the selenoproteins were detected by autoradiography, and the proteins were identified by MALDI-TOF. Approximately 35 (75)Se-containing spots were found in the cell proteins from MAC-T cells. Among them, one-third showed high intensity. The strongest spot was identified as glutathione peroxidase 1. About 20 spots were observed in protein precipitated from cell culture medium, one-third of them being distinctly visible. In an attempt to study a perturbation of the system, the effect of retinoic acid (RA) on the formation of selenoproteins was investigated. The concentration of (75)Se in total cell protein was reduced by about 35% in cells cultured with RA compared with control cells, while the opposite effect was observed in protein precipitated from cell culture medium, which contained 60% more (75)Se in RA-treated samples than in controls. There were also indications that RA might affect different selenoproteins in different ways. The methods described provide a promising approach for further studies of the regulation of selenoprotein formation in the mammary gland.     

Biochemical analysis of selenoprotein expression in brain cell lines and in distinct brain regions

Selenium is present in various biologically important selenoproteins. The preferential incorporation of selenium into the brain indicates its significance for this organ, but so far knowledge concerning the cerebral selenoproteome is scarce. We therefore investigated the expression of selenoproteins in various regions of the rat brain, various subcellular fractions and several brain cell lines by (75)Se-labelling, gel electrophoretic separation and autoradiography, with the (75)Se tracer as the selenoprotein marker. Quantitative evaluation of the labelled proteins in selenium-deficient rats revealed information regarding preferentially supplied selenoproteins and their distribution; 21 selenoproteins could be distinguished, among them a novel or modified 15-kDa selenoprotein enriched in the cerebellum cytosol. The selenoproteins differed in the degree of their expression among the brain regions and within a region among the subcellular fractions. Some cell-type-specific selenium-containing proteins were found in the cell lines. Differences in the distribution patterns between mono-cultured and co-cultured endothelial cells and astrocytes showed that mediators produced by other cells could affect the selenoprotein expression of a specific cell-type. This effect might play a role in the uptake and distribution of selenium in the brain but could also be of significance in the selenium metabolism of other tissues.     

Characterization and tissue expression of twelve selenoproteins in yellow catfish Pelteobagrus fulvidraco fed diets varying in oxidized fish oil and selenium levels

BackgroundSelenium (Se) functions through selenoproteins and is essential to growth and metabolism of vertebrates. The present study was conducted to identify twelve selenoproteins genes (selenoe, selenof, selenoh, selneoi, selenom, selenok, selneon, selenoo, selenot, selenos, selenou and msrb1) from yellow catfish. Their mRNA expression patterns, as well as their response to dietary oxidized fish oils and Se addition were explored.MethodsWe use 3′and 5′ RACE PCR to clone full-length cDNA sequence of twelve selenoprotein genes from yellow catfish. Their mRNA expression patterns were assessed via quantitative real-time PCR. Yellow catfish were fed diet adequate Se+ fresh fish oil, adequate Se+ oxidized fish oil, high Se+ fresh fish oil and high Se+ oxidized fish oil, respectively, for 10 weeks. Their kidney, heart, brain and testis were used to assess the mRNA expression of twelve selenoprotein.ResultsTwelve selenoprotein genes had similar domains with mammals and the other fish. Their mRNAs were expressed widely in eleven tissues but varied with the tissues. Dietary oxidized fish oils and Se addition influenced their mRNA abundances of twelve selenoproteins in a tissue-dependent manner.ConclusionOur study demonstrated the characterization and expression of twelve selenoproteins, and elucidated their responses in yellow catfish fed diets varying in oxidized fish oils and Se addition, which increased our knowledge into the biological function and regulatory mechanism of Se and selenoproteins in fish.     

Identification of proteins regulated by interferon-alpha in resistant and sensitive malignant melanoma cell lines

Treatment of patients with malignant melanoma with interferon-alpha achieves a response in a small but significant subset of patients. Currently, although much is known about interferon biology, little is known about either the particular mechanisms of interferon-alpha activity that are crucial for response or why only some patients respond to interferon-alpha therapy. Two melanoma cell lines (MeWo and MM418) that are known to differ in their response to the antiproliferative activity of interferon-alpha, have been used as a model system to investigate interferon-alpha action. Using a proteomics approach based on two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, several proteins induced in response to interferon-alpha have been identified. These include a number of gene products previously known to be type I interferon responsive (tryptophanyl tRNA synthetase, leucine aminopeptidase, ubiquitin cross-reactive protein, gelsolin, FUSE binding protein 2 and hPNPase) as well as a number of proteins not previously reported to be induced by type I interferon (cathepsin B, proteasomal activator 28alpha and alpha-SNAP). Although the proteins upregulated by interferon-alpha were common between the cell lines when examined at the level of Western blotting, the disparity in the basal level of cathepsin B was striking, raising the possibility that the higher level in MM418 may contribute to the sensitivity of this cell line to interferon-alpha treatment.     

Markers of mitochondrial dysfunction during the diclofenac-induced apoptosis in melanoma cell lines

Melanoma is an aggressive cutaneous cancer, whose incidence is growing in recent years, especially in the younger population. The favorable therapy for this neoplasm consists in its early surgical excision; otherwise, in case of late diagnosis, melanoma becomes very refractory to any conventional therapy. Nevertheless, the acute inflammatory response occurring after excision of the primary melanoma can affect the activation and/or regulation of melanoma invasion and metastasis. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely employed in clinical therapy as cyclooxygenase inhibitors, also display a cytotoxic effect on some cancer cell lines; therefore, their possible usage in combination with conventional chemo- and radio-therapies of tumors is being considered. In particular, diclofenac, one of the most common NSAIDs, displays its anti-proliferative effect in many tumor lines, through an alteration of the cellular redox state. In this study, the possible anti-neoplastic potential of diclofenac on the human melanoma cell lines A2058 and SAN was investigated, and a comparison was made with the results obtained from the nonmalignant fibroblast cell line BJ-5ta. Either in A2058 or SAN, the diclofenac treatment caused typical apoptotic morphological changes, as well as an increase of the number of sub-diploid nuclei; conversely, the same treatment on BJ-5ta had only a marginal effect. The observed decrease of Bcl-2/Bax ratio and a parallel increase of caspase-3 activity confirmed the pro-apoptotic role exerted by diclofenac in melanoma cells; furthermore, the drug provoked an increase of the ROS levels, a decrease of mitochondrial superoxide dismutase (SOD2), the cytosolic translocation of both SOD2 and cytochrome c, and an increase of caspase-9 activity. Finally, the cytotoxic effect of diclofenac was amplified, in melanoma cells, by the silencing of SOD2. These data improve the knowledge on the effects of diclofenac and suggest that new anti-neoplastic treatments should be based on the central role of mitochondrion in cancer development; under this concern, the possible involvement of SOD2 as a novel target could be considered.     

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles’ heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.     

Establishment in monolayer culture and characterization of four human melanoma cell lines

Four new human melanoma cell lines were established in monolayer culture from xenograft lines originating from different patients. Several distinct characteristics of the source xenograft lines were retained in the cell lines, e.g., number of chromosomes, DNA-index, and cell ultrastructure. Cell volume was generally larger for the cell lines than for the corresponding xenograft lines, but the differences among the lines were similar in vitro and in vivo. The cell lines showed significant differences in growth pattern, i.e., cell motility and degree of intercellular contact. Cell cycle time (T_c) during exponential growth ranged from 15 to 21 h. The differences among the lines in T_c were mainly due to differences in the duration of S. Growth fraction was close to 100% and cell loss was negligible during exponential growth. Plating efficiency was 90–100% in the presence of feeder cells. The four cell lines represent a valuable supplement to the xenograft lines for future studies of the cell biology, pathophysiology, metastatic behavior, and treatment sensitivity of malignant melanoma.     

Dietary selenium levels modulates antioxidant,cytokine and immune response and selenoproteins mRNA expression in rats under heat stress condition

BackgroundHigher environmental temperature is a major abiotic stress factor for animals and human beings. The selenium (Se) is an important trace mineral having diverse health promoting effects under stress conditions. However, studies on dietary requirement of selenium under prolonged heat stress condition are lacking. Present study discern the effect of higher dietary Se levels on antioxidant, cytokine, haemato-biochemical profile, and immune response, and the selenoproteins mRNA expression in rats under prolonged heat stress (HS) condition.MethodsWeaned Wistar rats (4 wk age; 67.6 ± 1.53 g BW; n = 72) housed under thermoneutral (TN) or HS conditions and fed with purified diets containing three graded Se levels were divided in six experimental groups. The groups were 1) TN control with 138 ppb Se (TN_CON), 2) HS control with 138 ppb Se (HS_CON), 3) TN with higher Se @ 291 ppb (TN_Se1), 4) HS with higher Se @ 291 ppb (HS_Se1) 5) TN with higher Se @ 460 ppb (TN_Se2), 6) HS with higher Se @ 460 ppb (HS_Se2). Rats in all the six groups were maintained in TN environmental conditions (57.3 ± 0.22 temperature humidity index; THI) for initial 28 days period. Subsequently, rats of HS groups were exposed to 77.0 ± 0.11 THI for 6 h/d in a psychrometric chamber for last fourteen days.ResultsHigher dietary Se (291 and 460 ppb) significantly improved the blood hemoglobin concentration and reduced serum alanine aminotransferase activity of rats under HS conditions. The serum triiodothyronine and insulin levels were significantly higher in high dietary Se groups irrespective of the environmental conditions. Similarly, the serum reduced glutathione levels, and catalase and superoxide dismutase enzyme activity were increased and malondialdehyde levels were reduced in high dietary Se groups irrespective of stress conditions. The glutathione peroxidase (GPx) activity was significantly higher in 460 ppb dietary Se groups as compared to other groups. The serum pro-inflammatory cytokine interleukin (IL)− 1 was declined, whereas the anti-inflammatory cytokine IL-10 level was increased in high dietary Se fed rats under both HS and TN conditions with 460 ppb dietary Se groups showing pronounced effects. Further, there was heat stress- and dietary Se level dependent- up regulation in hepatic GPx and iodothyronine deiodinase-II mRNA expression and similar pattern was noticed in hepatic thioredoxin reductase mRNA expression. The selenoprotein-P mRNA expression was up regulated in 460 ppb Se fed HS group as compared to CON and Se1_C groups. High dietary Se improved the humoral immune response 7d after antigen inoculation under HS conditions whereas cell-mediated immune response was augmented in rats fed higher Se under TN condition.ConclusionIt is concluded that under prolonged heat stress conditions the dietary requirement of Se may be increased to 460 ppb for improving the antioxidant status and humoral immune response, cytokine levels, modulating the thyroid and insulin hormone, and the selenoproteins mRNA expression of rats.     

Characterization of HLA class I altered phenotypes in a panel of human melanoma cell lines

Background Altered HLA class I cell surface expression is one of the major mechanisms by which tumor cells escape from T lymphocytes. Immunohistochemistry-defined phenotypes of lost HLA class I expression have been described in human solid tumors, nut less information is available on melanoma cell lines. Objectives To describe the frequency and distribution of different types of HLA class I antigen alterations in 91 melanoma cell lines from the European Searchable Tumour Cell and Databank (ESTDAB). Methods The HLA class I expression was assessed by flow cytometry and HLA genotyping. Results We found various types of HLA class I cell surface alterations in about 67% of the melanoma cell lines. These alterations range from total to selective HLA class I loss due to loss of heterozygosity (LOH), haplotype loss, β2-microglobulin gene mutation, and/or total or selective down-regulation of HLA class I molecules. The most frequently observed phenotype is down-regulation of HLA-B locus that was reversible after treatment with IFN -γ. Conclusions In general, HLA class I alterations in the majority of the cells analyzed were of regulatory nature and could be restored by IFN-γ. Analysis of the frequency of distinct HLA class I altered phenotypes in these melanoma cell lines revealed specific differences compared to other types of tumors. Rosa Méndez and Teresa Rodríguez have equally contributed to this work and both should be considered as first authors.     

Assessing proteolytic events in bioinformatic reanalysis of public secretome data from melanoma cell lines

Autocrine and paracrine signals are of paramount importance in both normal and oncogenic events and the composition of such secreted molecular signals (i.e the secretome) designate the communication status of cells. In this context, the analysis of post-translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as proteolytic processing. In the present study, we have performed a bioinformatic reanalysis of public proteomics data on melanoma cell line secretomes, changing database searching parameters to allow for the identification of proteolytic events generated by active proteases. Such approach enabled the identification of proteolytic signatures which suggested active proteases and whose expression profiles might be targeted in patient tissues or liquid biopsies, as well as their cleaved substrates. Although N-terminomics approaches continue to be the method of choice for the evaluation of proteolytic signaling events in complex samples, the simple approach performed in this work resulted in the gain of biological insights derived from shotgun proteomics data.     

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.