Howard E. Ganther

GLUTATHIONE PEROXIDASE ACTIVITY OF GLUTATHIONE S-TRANSFERASES PURIFIED FROM RAT LIVER

Abstract Gel filtration chromatography demonstrated the presence of two peaks of glutathione peroxidase activity assayed with cumene hydroperoxide in the soluble fraction of rat liver, brain, kidney, and testis. The peak with an approximate molecular weight of 45,000 (GSH-Px II) was purified from rat liver labeled in vivo with Na275SeO3. Chromatography on DEAE-cellulose, Sephadex G-150, DEAE-cellulose, and CM-cellulose resulted in the co-purification of glutathione-S-transferase activity measured with 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity assayed with cumene hydroperoxide, and in the removal of all detectable 75Se. Studies on GSH-Px II indicated that the apparent Km for both cumene and t-butyl hydroperoxides was considerably higher than that for purified seleno-glutathione peroxidase. The Vmax estimated with cumene hydroperoxide was only 1 300 of that determined for the selenoenzyme at pH 7.5 and with 1 mM GSH.

Selenium-induced inhibition of angiogenesis in mammary cancer at chemopreventive levels of intake

The trace element nutrient selenium (Se) has been shown to possess cancer‐preventive activity in both animal models and humans, but the mechanisms by which this occurs remain to be elucidated. Because angiogenesis is obligatory for the genesis and growth of solid cancers, we investigated, in the study presented here, the hypothesis that Se may exert its cancer‐preventive activity, at least in part, by inhibiting cancer‐associated angiogenesis. The effects of chemopreventive levels of Se on the intra‐tumoral microvessel density and the expression of vascular endothelial growth factor in 1‐methyl‐1‐nitrosourea–induced rat mammary carcinomas and on the proliferation and survival and matrix metalloproteinase activity of human umbilical vein endothelial cells in vitro were examined. Increased Se intake as Se‐enriched garlic, sodium selenite, or Se‐methylselenocysteine led to a significant reduction of intra‐tumoral microvessel density in mammary carcinomas, irrespective of the manner by which Se was provided: continuous exposure (7‐wk feeding) with a chemoprevention protocol or acute bolus exposure (3 d) after carcinomas had established. Compared with the untreated controls, significantly lower levels of vascular endothelial growth factor expression were observed in a sizeable proportion of the Se‐treated carcinomas. In contrast to the mammary carcinomas, the microvessel density of the uninvolved mammary glands was not altered by Se treatment. In cell culture, direct exposure of human umbilical vein endothelial cells to Se induced cell death predominantly through apoptosis, decreased the gelatinolytic activities of matrix metalloproteinase‐2, or both. These results indicate a potential for Se metabolites to inhibit key attributes (proliferation, survival, and matrix degradation) of endothelial cells critical for angiogenic sprouting. Therefore, inhibition of angiogenesis associated with cancer may be a novel mechanism for the anticancer activity of Se in vivo, and multiple mechanisms are probably involved in mediating the anti‐angiogenic activity. Mol. Carcinog. 26:213–226, 1999.

New concepts in selenium chemoprevention.

This article highlights some recent advances in selenium cancer chemoprevention research. It has been well documented that the chemical transformation of selenium to a monomethylated metabolite is an important step in achieving cancer prevention. Studies with the rat mammary carcinogenesis model suggested that methylselenocysteine (MSC), a good precursor for generating methylselenol endogenously, is able to block clonal expansion of premalignant lesions in the mammary gland. This finding supports the notion that selenium intervenes at an early stage of carcinogenesis. In addition to decreasing cell proliferation of the transformed colonies in vivo, MSC also enhances apoptosis. These same cellular responses are replicated with human premalignant breast cells grown in culture. cDNA microarray analysis indicated that selenium affects a multitude of molecular targets. Based on this information, a number of signaling pathways are proposed that could potentially provide insight into how selenium might block cell cycle progression and induce cell death.

Dissociation of the genotoxic and growth inhibitory effects of selenium

The effects of forms of selenium compounds that enter the cellular selenium metabolic pathway at different points were investigated in a mouse mammary carcinoma cell line. The goal of these experiments was to determine if the genotoxicity of selenium, defined as its ability to induce DNA single-strand breaks, could be dissociated from activities proposed to account for its cancer inhibitory activity. The results demonstrated that growth inhibition, measured as inhibition of cell proliferation and induction of cell death, was induced by all the forms of selenium evaluated. However, sodium selenite and sodium selenide, which are metabolized predominantly to hydrogen selenide, caused the rapid induction of DNA single-strand breaks as an early event that preceded growth inhibition. Interestingly methylselenocyanate and Se-methylselenocysteine, which are initially metabolized predominantly to methylselenol, induced growth inhibition in the absence of DNA single-strand breakage. Differences in the time course of selenium retention, in the occurrence of membrane damage, and in the induction of morphological changes by selenite versus methylselenocyanate were noted. Collectively, these data indicate that different pathways affecting cell proliferation and cell death are induced depending on whether selenium undergoes metabolism predominantly to hydrogen selenide or to methylselenol.

Se-methylselenocysteine: a new compound for chemoprevention of breast cancer.

Selenium compounds have attracted renewed interest as chemopreventive agents for human cancer on the basis of the pioneering intervention study by Clark and co-workers. The rodent mammary gland has been used extensively as a model for examining the chemopreventive activities of inorganic and organic selenium compounds. This review summarizes the rationale and results for use of a new organic selenium compound, Se-methylselenocysteine, which exhibits greater efficacy as a chemopreventive agent than several previously used selenium compounds in experimental models of breast cancer and has potential for use in human populations.

Monomethyl selenium--specific inhibition of MMP-2 and VEGF expression: implications for angiogenic switch regulation.

Previous work suggested that antiangiogenic activity may be a novel mechanism contributing to the cancer chemopreventive activity of selenium (Se). Because methylselenol has been implicated as an in vivo active chemopreventive Se metabolite, experiments were conducted to test the hypothesis that this metabolite pool might inhibit the expression of matrix metalloproteinase‐2 (MMP‐2) by vascular endothelial cells and of vascular endothelial growth factor (VEGF) by cancer epithelial cells, two proteins critical for angiogenesis and its regulation. In human umbilical vein endothelial cells (HUVECs), zymographic analyses showed that short‐term exposure to methylseleninic acid (MSeA) and methylselenocyanate (MSeCN), both immediate methylselenol precursors, decreased the MMP‐2 gelatinolytic activity in a concentration‐dependent manner. In contrast, Se forms that enter the hydrogen selenide pool lacked any inhibitory effect. The methyl Se inhibitory effect on MMP‐2 was cell dependent because direct incubation with Se compounds in the test tube did not result in its inactivation. Immunoblot and enzyme‐linked immunosorbent assay analyses showed that a decrease of the MMP‐2 protein level largely accounted for the methyl Se–induced reduction of gelatinolytic activity. The effect of MSeA on MMP‐2 expression occurred within 0.5 h of exposure and preceded MSeA‐induced reduction of the phosphorylation level of mitogen‐activated protein kinases (MAPKs) 1 and 2 (∼3 h) and endothelial apoptosis (∼25 h). In addition to these biochemical effects in monolayer culture, MSeA and MSeCN exposure decreased HUVEC viability and cell retraction in a three‐dimensional context of capillary tubes formed on Matrigel, whereas comparable or higher concentrations of selenite failed to exert such effects. In human prostate cancer (DU145) and breast cancer (MCF‐7 and MDA‐MB‐468) cell lines, exposure to MSeA but not to selenite led to a rapid and sustained decrease of cellular (lysate) and secreted (conditioned medium) VEGF protein levels irrespective of the serum level (serum‐free medium vs. 10% fetal bovine serum) in which Se treatments were carried out. The concentration of MSeA required for suppressing VEGF expression was much lower than that needed for apoptosis induction. Taken together, the data support the hypothesis that the monomethyl Se pool is a proximal Se for inhibiting the expression of MMP‐2 and VEGF and of angiogenesis. The data also indicate that the methyl Se–specific inhibitory effects on these proteins are rapid and primary actions, preceding or independent of inhibitory effects on mitogenic signaling at the level of MAPK1/2 and on cell growth and survival. Mol. Carcinog. 29:236–250, 2000.

INTERACTIONS OF VITAMIN E AND SELENIUM WITH MERCURY AND SILVER

It is well established that the toxicity of silver and methylmercury is suppressed by the presence of low levels of vitamin E or selenium in the diet, but little is known of the mechanisms involved. Silver induces a conditioned deficiency of selenium in rats, as shown by its effects on tissue levels of selenium and glutathione peroxidase (a selenoprotein), but methylmercury does not. Supplements of selenium do not decrease mercury levels in tissues of animals given methylmercury, and animals given selenium plus methylmercury may accumulate high levels of mercury without signs of toxicity. Although an equimolar accumulation of selenium and mercury in tissues sometimes occurs and could lead to mutual detoxification, such a coaccumulation is not always linked to protection. The only known functions of vitamin E and selenium are related to the prevention of oxidative damage. It is possible that their protective effects against heavy metals may involve such functions, thus accounting for the protection afforded by low levels of the nutrients against high levels of the metal toxicants.

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems.

A pathway for the synthesis of dimethyl selenide from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30,000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO2 (93% inhibition by 10(-6) M arsenite in the presence of a 20,000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.

Differential induction of growth arrest inducible genes by selenium compounds

The effects of two types of selenium compounds on the expression levels of growth arrest and DNA damage-inducible (gadd) genes and on selected cell death genes were examined in mouse mammary MOD cells to test the hypothesis that the diversity of selenium-induced cellular responses to these compounds could be distinguished by unique gene expression patterns. Whereas the expression patterns of known cell death-related genes (bcl-2 and bax) were not informative with respect to the cellular response patterns upon exposure to selenium compounds, time-dependent and selenium species-specific induction patterns were observed for gadd34, gadd45 and gadd153 genes. It was also observed that the MOD cells expressed a truncated p53 transcript but no detectable immunoreactive P53 protein, indicating a null p53 phenotype. The fact that selenium compounds induced growth arrest and death of these cells and that these compounds induced specific patterns of expression of gadd genes indicates that these genes may mediate some selenium-induced cellular responses. The findings further imply that selenium compounds may be effective chemopreventive agents for human breast carcinogenesis, in which p53 mutations are frequent.

CHEMICAL TRANSFORMATIONS OF SELENIUM IN LIVING ORGANISMS. IMPROVED FORMS OF SELENIUM FOR CANCER PREVENTION

Abstract Compounds having cancer-preventing activity are developed during the metabolism of selenium in plants and animals. Monomethylated forms of selenium appear to be one class of chemopreventive metabolites. Synthetic organoselenium compounds have been used to explore determinants of activity and differentiation from other biological effects of selenium. Triphenyl selenonium chloride, a new type of chemopreventive selenium compound, has been synthesized in radioactive form for use as a tracer to facilitate studies of its mode of action.