Raghu Sinha

Apoptosis is a Critical Cellular Event in Cancer Chemoprevention and Chemotherapy by Selenium Compounds

Epidemiological studies, preclinical investigations and clinical intervention trials support the role of selenium compounds as potent cancer chemopreventive agents; the dose and the form of selenium are critical factors in cancer prevention. Induction of apoptosis and inhibition of cell proliferation are considered important cellular events that can account for the cancer preventive effects of selenium. Toxicity should always be considered a determining factor in the selection of potential chemopreventive agents. Prior to induction of apoptosis, selenium compounds alter the expression and/or activities of a number of cell cycle regulatory proteins, signaling molecules, proteases, mitochondrial associated factors, transcriptional factors, tumor suppressor genes, polyamine and glutathione levels. Depending on the form, selenium compounds can target separate pathways but more efforts are needed to learn about disrupting different pathways converging to apoptosis. Numerous selenium compounds are known to inhibit carcinogenesis in several animal models but not all of these have been examined for their efficacy to induce apoptosis or vice versa in the corresponding target organ. Studies aimed at investigating the effects of selenium compounds on apoptosis in the target organ in vivo and in vitro are limited. On the basis of information provided in this review, we recommend that additional molecular markers should be added to those proposed in the Selenium and Vitamin E Cancer Prevention Trial (SELECT) on prostate cancer. Apart from the selenium compounds reviewed here, several novel synthetic organoselenium compounds need to be examined both in vitro and in vivo for their potential to induce apoptosis; such an investigation may provide better and mechanism-based cancer chemoprevention as well as chemotherapeutic agents.

Organic and inorganic selenium compounds inhibit mouse mammary cell growth in vitro by different cellular pathways.

Selenium, both organic and inorganic forms, inhibit mammary tumorigenesis in vivo and mammary cell growth in vitro. In the present study, sodium selenite was compared to methylselenocysteine (MSC) for their individual effects on cell growth, cdc2/cdk2 kinase activities and the levels of cyclins D1, E and A bound to cdk2 in a mouse mammary epithelial cell culture model. Selenite arrested the growth of cells in S-G2-M phase in contrast to MSC which arrested or delayed the cells in G1. In MSC-treated cells there was a 57% drop in the cdk2 kinase activity accompanied by a 73.5% decrease in cyclin E-cdk2 content as compared to the control cells. Selenite treatment increased the cdk2 kinase activity by 30% without any appreciable change in either of the cyclins D1, E or A bound to cdk2 when compared to the control cells. These data support the hypothesis that selenite and MSC have distinct modes of action in the inhibition of cell growth in vitro. Selenite has a strong genotoxic effect on the tumor cells; in contrast, MSC appears to inhibit cell growth via specific inhibition of cell cycle regulatory proteins.

α-Keto acid metabolites of naturally-occurring organoselenium compounds as inhibitors of histone deacetylase in human prostate cancer cells

Histone deacetylase (HDAC) inhibitors are gaining interest as cancer therapeutic agents. We tested the hypothesis that natural organoselenium compounds might be metabolized to HDAC inhibitors in human prostate cancer cells. Se-Methyl-l-selenocysteine (MSC) and selenomethionine are amino acid components of selenium-enriched yeast. In a cell-free system, glutamine transaminase K (GTK) and l-amino acid oxidase convert MSC to the corresponding α-keto acid, β-methylselenopyruvate (MSP), and l-amino acid oxidase converts selenomethionine to its corresponding α-keto acid, α-keto-γ-methylselenobutyrate (KMSB). Although methionine (sulfur analogue of selenomethionine) is an excellent substrate for GTK, selenomethionine is poorly metabolized. Structurally, MSP and KMSB resemble the known HDAC inhibitor butyrate. We examined androgen-responsive LNCaP cells and androgen-independent LNCaP C4-2, PC-3, and DU145 cells and found that these human prostate cancer cells exhibit endogenous GTK activities. In the corresponding cytosolic extracts, the metabolism of MSC was accompanied by the concomitant formation of MSP. In MSP-treated and KMSB-treated prostate cancer cell lines, acetylated histone 3 levels increased within 5 hours, and returned to essentially baseline levels by 24 hours, suggesting a rapid, transient induction of histone acetylation. In an in vitro HDAC activity assay, the selenoamino acids, MSC and selenomethionine, had no effect at concentrations up to 2.5 mmol/L, whereas MSP and KMSB both inhibited HDAC activity. We conclude that, in addition to targeting redox-sensitive signaling proteins and transcription factors, α-keto acid metabolites of MSC and selenomethionine can alter HDAC activity and histone acetylation status. These findings provide a potential new paradigm by which naturally occurring organoselenium might prevent the progression of human prostate cancer.

Cancer Chemoprevention by Garlic and Garlic-Containing Sulfur and Selenium Compounds

As early as 1550 B.C., Egyptians realized the benefits of garlic as a remedy for a variety of diseases. Many epidemiological studies support the protective role of garlic and related allium foods against the development of certain human cancers. Natural garlic and garlic cultivated with selenium fertilization have been shown in laboratory animals to have protective roles in cancer prevention. Certain organoselenium compounds and their sulfur analogs have been identified in plants. Organoselenium compounds synthesized in our laboratory were compared with their sulfur analogs for chemopreventive efficacy. Diallyl selenide was at least 300-fold more effective than diallyl sulfide in protecting against 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas in rats. In addition, benzyl selenocyanate inhibited the development of DMBA-induced mammary adenocarcinomas and azoxymethane-induced colon cancer in rats and benzo[a]pyrene-induced forestomach tumors in mice. The sulfur analog, benzyl thiocyanate, had no effect under the same experimental conditions. Furthermore, we showed that 1,4-phenylenebis(methylene)selenocyanate, but not its sulfur analog, significantly inhibited DMBA-DNA adduct formation and suppressed DMBA-induced mammary carcinogenesis. Collectively, these results indicate that structurally distinctive organoselenium compounds are superior to their corresponding sulfur analogs in cancer chemoprevention. Additionally, synthetic aromatic selenocyanates are more effective cancer chemopreventive agents than the naturally occurring selenoamino acids. Because plants are capable of utilizing selenium in a manner similar to that in sulfur assimilation pathways, future studies should aim at determining whether, under appropriate conditions, these potent cancer chemopreventive synthetic selenium compounds can be synthesized by garlic and related allium foods.

Methylseleninic acid, a potent growth inhibitor of synchronized mouse mammary epithelial tumor cells in vitro.

Selenium compounds have been shown to be effective chemopreventive agents in several animal models and in cultured cells in vitro. It has been proposed that compounds able to generate monomethyl Se have an increased potential to inhibit cell growth. To test this hypothesis, methylseleninic acid (MSeA) and other compounds that could generate methylselenol rapidly were compared with Se compounds that do not generate monomethyl Se, using a well-characterized synchronized TM6 mouse mammary epithelial tumor model in vitro. MSeA at a low micromolar concentration inhibited TM6 growth after 10- to 15-min treatment times. Cells resumed growth after 24 hr but remained sensitive to the fresh addition of monomethyl Se-generators. Dimethyl selenide (DMSe), a putative metabolite of methylselenol, was inactive. Cells treated with 5 microM MSeA were arrested in G1. The effects of 5 microM MSeA on gene expression were evaluated using the Atlas mouse cDNA expression array. A 10-min exposure with MSeA caused a 2- to 3-fold change in the expression of three genes: laminin receptor 1 (decreased), integrin beta (decreased), and Egr-1 (increased). The results provide experimental support for the hypothesis that monomethylated forms of Se are the critical effector molecules in Se-mediated growth inhibition in vitro.

Se-methylselenocysteine inhibits phosphatidylinositol 3-kinase activity of mouse mammary epithelial tumor cells in vitro

IntroductionSe-methylselenocysteine (MSC), a naturally occurring selenium compound, is a promising chemopreventive agent against in vivo and in vitro models of carcinogen-induced mouse and rat mammary tumorigenesis. We have demonstrated previously that MSC induces apoptosis after a cell growth arrest in S phase in a mouse mammary epithelial tumor cell model (TM6 cells) in vitro. The present study was designed to examine the involvement of the phosphatidylinositol 3-kinase (PI3-K) pathway in TM6 tumor model in vitro after treatment with MSC.MethodsSynchronized TM6 cells treated with MSC and collected at different time points were examined for PI3-K activity and Akt phosphorylation along with phosphorylations of Raf, MAP kinase/ERK kinase (MEK), extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The growth inhibition was determined with a [3H]thymidine incorporation assay. Immunoblotting and a kinase assay were used to examine the molecules of the survival pathway.ResultsPI3-K activity was inhibited by MSC followed by dephosphorylation of Akt. The phosphorylation of p38 MAPK was also downregulated after these cells were treated with MSC. In parallel experiments MSC inhibited the Raf–MEK–ERK signaling pathway.ConclusionThese studies suggest that MSC blocks multiple signaling pathways in mouse mammary tumor cells. MSC inhibits cell growth by inhibiting the activity of PI3-K and its downstream effector molecules in mouse mammary tumor cells in vitro.

Chemopreventive mechanisms of α-keto acid metabolites of naturally occurring organoselenium compounds

Previous studies on the chemopreventive mechanisms of dietary selenium have focused on its incorporation into antioxidative selenoproteins, such as glutathione peroxidase and thioredoxin reductase. Several studies, however, have revealed that dietary selenium in the form of l-selenomethionine and the 21st amino acid, selenocysteine, also have intrinsic anti-cancer properties. Biochemical mechanisms previously investigated to contribute to their anticancer effects involve β- and γ-lyase reactions. Some pyridoxal 5′-phosphate (PLP)-containing enzymes can catalyze a β-lyase reaction with Se-methyl-l-selenocysteine (MSC) generating pyruvate and ammonia. Other PLP-enzymes can catalyze a γ-lyase reaction with l-selenomethionine (SM) generating α-ketobutyrate and ammonia. In both cases, a purported third product is methylselenol (CH3SeH). Although not directly quantifiable, as a result of its extreme hydrophobicity and high vapor pressure, CH3SeH has been indirectly observed to act through the alteration of protein-sulfhydryl moieties on redox-responsive signal and transcription factors, thereby maintaining a non-proliferative intracellular environment. We have considered the possibility that α-keto acid analogues of MSC (i.e., methylselenopyruvate; MSP) and SM (i.e., α-keto-γ-methylselenobutyrate; KMSB), generated via a transamination and/or l-amino acid oxidase reaction may also be chemoprotective. Indeed, these compounds were shown to increase the level of histone-H3 acetylation in human prostate and colon cancer cells. MSP and KMSB structurally resemble butyrate, an inhibitor of several histone deacetylases. Thus, the seleno α-keto acid metabolites of MSC and SM, along with CH3SeH derived from β- and γ-lyase reactions, may be potential direct-acting metabolites of organoselenium that lead to de-repression of silenced tumor suppressor proteins and/or regulation of genes and signaling molecules.

Differential effects of naturally occurring and synthetic organoselenium compounds on biomarkers in androgen responsive and androgen independent human prostate carcinoma cells

Epidemiological studies and clinical trials show that selenium supplementation results in reduction of prostate cancer incidence; however, the form of selenium and mechanisms underlying protection remain largely unknown. Toward this end, we compared the effects of naturally occurring selenomethionine (SM) and Se‐methylselenocysteine (MSC) and synthetic 1,4‐phenylenebis(methylene)selenocyanate (p‐XSC) and p‐xylylbis(methylselenide) p‐XMS) organoselenium compounds in androgen responsive (AR) LNCaP and its androgen independent clone (AI) LNCaP C4‐2 human prostate carcinoma cells on cell growth, secretion of prostate specific antigen (PSA), intracellular redox status and genomic profiles with emphasis on identifying redox sensitive genes. Both p‐XSC and p‐XMS reduced cell number and total protein concentration compared to control‐treated AR and AI cells, while SM and MSC exhibited no effect on growth of AR and AI cells. SM, p‐XSC and p‐XMS but not MSC inhibited levels of secreted PSA in AR cells. SM, MSC and p‐XMS increased glutathione (GSH) levels in AI LNCaP cells. By contrast, in both cell types, only p‐XSC significantly decreased GSH concentrations to <50% of control suggesting either an increase in intracellular oxidative stress or a change in GSH/GSSG ratio. On the basis of RT‐PCR analysis, SM and p‐XSC increased p53 gene expression by 2‐fold in AR cells but not in AI cells and only SM enhanced epidermal growth factor receptor in AR cells. Depending on the structure, organoselenium compounds exhibit differential effects on growth, PSA secretion, oxidative stress and selective gene responses in human prostate cancer cells and suggest the potential of developing novel organoselenium compounds as chemopreventive agents in models of human prostate cancer.

Methionine restriction affects oxidative stress and glutathione-related redox pathways in the rat

Lifelong dietary methionine restriction (MR) is associated with increased longevity and decreased incidence of age-related disorders and diseases in rats and mice. A reduction in the levels of oxidative stress may be a contributing mechanistic factor for the beneficial effects of MR. To examine this, we determined the effects of an 80% dietary restriction of Met on different biomarkers of oxidative stress and antioxidant pathways in blood, liver, kidney and brain in the rat. Male F-344 rats were fed control (0.86% methionine) or MR (0.17% methionine) diets for up to six months. Blood and tissues were analyzed for glutathione (GSH) concentrations, related enzyme activities and biomarkers of oxidative stress. MR was associated with reductions in oxidative stress biomarkers including plasma 8-hydoxydeoxyguanosine (8-OHdG) and 8-isoprostane and erythrocyte protein-bound glutathione after one month with levels remaining low for at least six months (P < 0.05). Levels of free GSH in blood were increased after 1–6 months of MR feeding whereas liver GSH levels were reduced over this time (P < 0.05). In MR rats, GSH peroxidase activity was decreased in liver and increased in kidney compared with controls. No changes in the activities of GSH reductase in liver and kidney and superoxide dismutase in liver were observed as a result of MR feeding. Altogether, these findings indicate that oxidative stress is reduced by MR feeding in rats, but this effect cannot be explained by changes in the activity of antioxidant enzymes.

Osteopontin is a potential target gene in mouse mammary cancer chemoprevention by Se-methylselenocysteine

BackgroundSe-methylselenocysteine (MSC) is a naturally occurring organoselenium compound that inhibits mammary tumorigenesis in laboratory animals and in cell culture models. Previously we have documented that MSC inhibits DNA synthesis, total protein kinase C and cyclin-dependent kinase 2 kinase activities, leading to prolonged S-phase arrest and elevation of growth-arrested DNA damage genes, followed by caspase activation and apoptosis in a synchronized TM6 mouse mammary tumor model. The aim of the present study was to examine the efficacy of MSC against TM6 mouse mammary hyperplastic outgrowth (TM6-HOG) and to determine in vivo targets of MSC in this model system.MethodsTwenty mammary fat pads each from female Balb/c mice transplanted with TM6-HOG and fed with 0.1 ppm selenium and with 3 ppm selenium respectively, were evaluated at 4 and 12 weeks after transplantation for growth spread, proliferative index and caspase-3 activity. Thirteen mice transplanted with TM6-HOG in each selenium group were observed for tumor formation over 23 weeks. Tumors from mice in both groups were compared by cDNA array analysis and data were confirmed by reverse transcription–polymerase chain reaction. To determine the effect of MSC on the expression of the novel target gene and on cell migration, experiments were performed in triplicate.ResultsA dietary dose of 3 ppm selenium significantly reduced the growth spread and induced caspase-3 activity in mammary fat pads in comparison with mice fed with the basal diet (0.1 ppm selenium). The extended administration (23 weeks) of 3 ppm selenium in the diet resulted in a tumor incidence of 77% in comparison with 100% tumor incidence in 0.1 ppm selenium-fed animals. The size of TM6 tumors in the supplemented group was smaller (mean 0.69 cm2) than in the mice fed with the basal diet (mean 0.93 cm2). cDNA array analysis showed a reduced expression of osteopontin (OPN) in mammary tumors of mice fed with the 3 ppm selenium diet in comparison with OPN expression in tumors arising in 0.1 ppm selenium-fed mice. A 24-hour treatment of TM6 cells with MSC significantly inhibited their migration and also reduced their OPN expression in comparison with untreated cells.ConclusionsOPN is a potential target gene in the inhibition of mammary tumorigenesis by selenium.