Response surface methodology optimizes selenium inhibition of prostate cancer PC-3 cell viability.

BACKGROUND: The rising incidence of prostate cancer in the U.S. necessitates innovative therapeutic approaches to this disease. Though extensive research has studied Selenium as an anticarcinogen against prostate cancer, results have varied due to overlooked experimental confounds. Recent studies have identified differential effects of various selenium compounds on prostate cancer cells. This study leverages Mixture Design Response Surface Methodology to characterize the ideal combination of select Se forms against the PC-3 prostate cancer cell line. METHODS: The PC-3 cell line was chosen as a model for its representation of advanced-stage malignancy. Three Se compounds-sodium selenite, methylseleninic acid, and nano-selenium-were selected for their promising antineoplastic potential. Nano-Se particles were synthesized and subsequently characterized by transmission electron microscopy. Cells were cultured, treated with Se compounds, and assessed for viability using an Alamar Blue Assay. IC50 values of individual Se compounds were determined, and treatment combinations evaluated. In collaboration with statical modeling experts, MDRSM was utilized to optimize Se compound combinations. RESULTS: Absolute IC50 values were identified for methylseleninic acid (5.01 µmol/L), sodium selenite (13.8 µmol/L), and nano-selenium (14.6 µmol/L). Combining methylseleninic acid and sodium selenite resulted in only 5% PC-3 cell viability, whereas individual treatments reduced viability by approximately 45%. Among the tested mixtures, the 50:50 combination of MSA and sodium selenite most effectively decreased PC-3 cell viability. Regression analysis indicated the special cubic model had a strong fit (multiple r² = 0.9853), predicting maximum cell viability reduction from the methylseleninic acid and selenite mixture. CONCLUSION: The specific form of Selenium plays a pivotal role in determining its physiological effects and therapeutic potential against prostate cancer. All three selenium compounds showed variable antineoplastic effects, with a 50:50 mixture of methylseleninic acid and selenite exhibiting optimal results. Nano-selenium, when combined with selenite, showed no additive effect, implying a shared mechanism of action. Our research underscores the critical need to consider Se compound forms as distinct entities in prostate cancer treatment and encourages further exploration of Se compounds against prostate cancer.

Application of Mixture Design Response Surface Methodology for Combination Chemotherapy in PC-3 Human Prostate Cancer Cells.

Combining chemotherapeutics to treat malignant tumors has been shown to be effective in preventing drug resistance, tumor recurrence, and reducing tumor size. We modeled combination drug therapy in PC-3 human prostate cancer cells using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize compositions that we applied in a novel manner to design combinations of chemotherapeutics. Conventional chemotherapeutics (mitoxantrone, cabazitaxel, and docetaxel) and natural bioactive compounds (resveratrol, piperlongumine, and flavopiridol) were used in 12 different combinations containing three drugs at varying concentrations. Cell viability and cell cycle data were collected and used to plot response surfaces in MDRSM that identified the most effective concentrations of each drug in combination. MDRSM allows for extrapolation of data from three or more compounds in variable ratio combinations, unlike the Chou-Talalay method. MDRSM combinations were compared with combination index data from the Chou-Talalay method and were found to coincide. We propose MDRSM as an effective tool in devising combination treatments that can improve treatment effectiveness and increase treatment personalization, because MDRSM measures effectiveness rather than synergism, potentiation, or antagonism.

Prenatal exposure to soy and selenium reduces prostate cancer risk factors in TRAMP mice more than exposure beginning at six weeks.

BACKGROUND: Diets high in soy and selenium (Se) decrease prostate cancer risk factors in healthy rats. The purpose of this study was to determine whether treatment with high levels of soy and/or supplemental Se would decrease prostate cancer risk factors in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, and whether timing of the introduction of these nutrients would affect risk reduction. METHODS: Male hemizygous [C57BL/6 × FVB]F1 TRAMP mice were exposed to stock diets high or devoid of soy, with or without a supplement of Se-methylselenocysteine (MSC) starting at conception (10 mg Se/L in drinking water of pregnant/nursing dams; daily bolus of 4 mg Se/kg body weight to pups after weaning) or at 6 weeks of age in a 2 × 2 factorial design. Mice were killed at 12 weeks (n per dietary treatment = 20-30). RESULTS: Liver and serum Se concentrations were increased by MSC supplementation (P < 0.001), high-soy diet (P < 0.05), and initiation of dietary treatments at conception (P < 0.05). MSC supplementation had greater effects in mice fed the zero-soy basal diet, compared to the high-soy formulation (Pinteraction < 0.01). These same three interventions, individually and interactively, decreased body weight and epididymal fat pad weights, and steady state levels of mRNA for Cyp19a1 (aromatase) and Srd5a1 (5α-reductase). In contrast, MSC was the only treatment that decreased urogenital tract weights (P < 0.001), serum IGF-1 levels (P < 0.002), and Gleason scores (P < 0.05). CONCLUSIONS: Supplemental MSC reduces risk of prostate cancer in TRAMP mice. Basal diet composition (zero- vs. high-soy) can modify MSC's chemopreventive effects. Initiation of dietary treatments from conception maximizes chemopreventive effects of MSC. Prenatal Se status may have long-lasting effects on development and progression of prostate cancer.

Selenium and prostate cancer prevention: what next--if anything?

Chemopreventive effects of the essential trace element selenium against prostate cancer have been shown in preclinical models and human observational studies, but results from clinical trials have been disappointing. It appears that there is a threshold selenium (Se) status below which improvement will decrease prostate cancer risk, but above which supplemental Se may be deleterious. Different forms of selenium have different effects, and genetic and other factors modify selenium's chemopreventive potential. Identification of men most likely to benefit from Se status improvement could have significant public health benefits.

A high isoflavone diet decreases 5' adenosine monophosphate-activated protein kinase activation and does not correct selenium-induced elevations in fasting blood glucose in mice.

Selenium (Se) has been implicated as a micronutrient that decreases adenosine monophosphate-activated protein kinase (AMPK) signaling and may increase diabetes risk by reducing insulin sensitivity. Soy isoflavones (IF) are estrogen-like compounds that have been shown to attenuate insulin resistance, hyperglycemia, adiposity, and increased AMPK activation. We hypothesized that a high IF (HIF) diet would prevent the poor metabolic profile associated with high Se intake. The purpose of this study was to examine changes in basal glucose metabolism and AMPK signaling in response to an HIF diet and/or supplemental Se in a mouse model. Male FVB mice were divided into groups receiving either a control diet with minimal IF (low IF) or an HIF diet. Each dietary group was further subdivided into groups receiving either water or Se at a dose of 3 mg Se/kg body weight daily, as Se-methylselenocysteine (SMSC). After 5 months, mice receiving SMSC had elevated fasting glucose (P < .05) and a tendency for glucose intolerance (P = .08). The increase in dietary IF did not result in improved fasting blood glucose. Interestingly, after 6 months, HIF-fed mice had decreased basal AMPK activation in liver and skeletal muscle tissue (P < .05). Basal glucose metabolism was changed by SMSC supplementation as evidenced by increased fasting blood glucose and glucose intolerance. High dietary IF levels did not protect against aberrant blood glucose. In FVB mice, decreased basal AMPK activation is not the mechanism through which Se exerts its effect. These results suggest that more research must be done to elucidate the role of Se and IF in glucose metabolism.

Combination effects of dietary soy and methylselenocysteine in a mouse model of prostate cancer.

BACKGROUND: High dietary intake of soy or selenium (Se) is associated with decreased risk of prostate cancer. Soy constituents and various chemical forms of Se have each been shown to downregulate expression of the androgen receptor (AR) and AR-regulated genes in the prostate. We hypothesized that downregulation of AR and AR-regulated genes by the combination of these dietary components would inhibit tumorigenesis in the TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse. METHODS: Male mice were exposed from conception to stock diets high or low in soy, with or without a supplement of Se-methylseleno-L-cysteine (MSC) in a 2 × 2 factorial design. Mice were sacrificed at 18 weeks. Prostate histopathology, urogenital tract (UGT) weight, hepatic activity of androgen-metabolizing enzymes, and expression of AR, AR-regulated, and AR-associated FOX family genes, in the dorsolateral prostate were examined. RESULTS: High soy intake decreased activity of hepatic aromatase and 5α-reductase, expression of AR, AR-regulated genes, FOXA1, UGT weight, and tumor progression, and upregulated protective FOXO3. Supplemental MSC upregulated AKR1C14, which reduces 5α-dihydrotestosterone. CONCLUSIONS: Soy is an effective pleiotropic dietary agent for prevention of prostate cancer. The finding of effects of soy on FOX family gene expression in animals is novel. Combination effects of supplemental MSC may depend upon the soy content of the basal diet to which it is added.

A retail market study of organic and conventional potatoes (Solanum tuberosum): mineral content and nutritional implications.

Whether or not all foods marketed to consumers as organic meet specified standards for use of that descriptor, or are nutritionally different from conventional foods, is uncertain. In a retail market study in a Western US metropolitan area, differences in mineral composition between conventional potatoes and those marketed as organic were analysed. Potatoes marketed as organic had more copper and magnesium (p < 0.0001), less iron (p < 0.0001) and sodium (p < 0.02), and the same concentration of calcium, potassium and zinc as conventional potatoes. Comparison of individual mineral concentrations between foodstuffs sold as organic or conventional is unlikely to establish a chemical fingerprint to objectively distinguish between organic and conventional produce, but more sophisticated chemometric analysis of multi-element fingerprints holds promise of doing so. Although statistically significant, these differences would only minimally affect total dietary intake of these minerals and be unlikely to result in measurable health benefits.

Soy content of basal diets determines the effects of supplemental selenium in male mice.

The effects of supplemental Se in rodent models may depend upon composition of the basal diet to which it is added. Wild-type male littermates of Transgenic Adenocarcinoma of Mouse Prostate mice were fed until 18 wk of age 1 of 2 Se-adequate stock diets high in soy (HS) or low in phytoestrogens (LP) or the same diets supplemented with 3.0 mg Se/kg diet as seleno-methylselenocysteine. Body and abdominal fat pad weights were lower (P < 0.01) in mice fed the HS diet. Supplemental Se reduced fat pad weights in mice receiving the LP diet but increased body and fat pad weights in mice consuming the HS formulation (P-interaction < 0.005). Serum free triiodothyronine concentrations were unaffected by supplemental Se in mice fed the LP diet but were decreased by Se supplementation of mice given the HS feed (P-interaction < 0.02). Free thyroxine concentrations were higher in mice consuming the HS diet regardless of Se intake (P < 0.001). Hepatic mRNA for iodothyronine deiodinase I was lower (P < 0.001) in mice fed the HS diet. Supplementation of Se increased this mRNA (P < 0.001) in both diet groups. Results from this study show a significant interaction between the composition of basal diets and the effects of supplemental Se with respect to body composition. These findings have important implications for future studies in rodent models of the effects of supplemental Se on heart disease, cancer, diabetes, and other conditions related to body weight and composition.

Diets high in selenium and isoflavones decrease androgen-regulated gene expression in healthy rat dorsolateral prostate.

BACKGROUND: High dietary intake of selenium or soybean isoflavones reduces prostate cancer risk. These components each affect androgen-regulated gene expression. The objective of this work was to determine the combined effects of selenium and isoflavones on androgen-regulated gene expression in rat prostate. METHODS: Male Noble rats were exposed from conception until 200 days of age to diets containing an adequate (0.33-0.45 mg/kg diet) or high (3.33-3.45 mg/kg) concentration of selenium as Se-methylselenocysteine and a low (10 mg/kg) or high (600 mg/kg) level of isoflavones in a 2 x 2 factorial design. Gene expression in the dorsolateral prostate was determined for the androgen receptor, for androgen-regulated genes, and for Akr1c9, whose product catalyzes the reduction of dihydrotestosterone to 5alpha-androstane-3alpha, 17beta-diol. Activity of hepatic glutathione peroxidise 1 and of prostatic 5alpha reductase were also assayed. RESULTS: There were no differences due to diet in activity of liver glutathione peroxidase activity. Total activity of 5alpha reductase in prostate was significantly lower (p = 0.007) in rats fed high selenium/high isoflavones than in rats consuming adequate selenium/low isoflavones. High selenium intake reduced expression of the androgen receptor, Dhcr24 (24-dehydrocholesterol reductase), and Abcc4 (ATP-binding cassette sub-family C member 4). High isoflavone intake decreased expression of Facl3 (fatty acid CoA ligase 3), Gucy1a3 (guanylate cyclase alpha 3), and Akr1c9. For Abcc4 the combination of high selenium/high isoflavones had a greater inhibitory effect than either treatment alone. The effects of selenium on gene expression were always in the direction of chemoprevention CONCLUSION: These results suggest that combined intake of high selenium and high isoflavones may achieve a greater chemopreventive effect than either compound supplemented individually.

Timing of supplementation of selenium and isoflavones determines prostate cancer risk factor reduction in rats.

BACKGROUND: High dietary intake of selenium or isoflavones reduces risk factors for prostate cancer. We tested whether combined supplementation of these two dietary components would reduce prostate cancer risk factors in rats more than supplementation of each component individually. METHODS: Male Noble rat pups were exposed from conception to diets containing an adequate (0.33-0.45 mg/kg diet) or high (3.33-3.45 mg/kg) concentration of selenium as Se-methylselenocysteine and a low (10 mg/kg) or high (600 mg/kg) level of isoflavones in a 2 x 2 factorial design. Pups consumed their respective diets until sacrifice at 35, 100, or 200 days. Male Noble rat breeders, whose exposure to the diets began after puberty, were sacrificed at 336 days. Rats were weighed biweekly. Blood was collected at the time of sacrifice and body fat and prostates were dissected and weighed. Serum levels of leptin, IGF-1, and testosterone were determined using ELISA kits. Serum levels of isoflavones were assayed by GC/MS. Liver activity of selenium-dependent glutathione peroxidase 1 was measured as an indicator of selenium status. RESULTS: Serum isoflavone concentrations were nearly 100-fold higher at 35 days of age (1187.1 vs. 14.4 ng/mL, mean +/- SD) in pups fed the high vs. low isoflavone diets, and remained so at 100 and 200 days, and in breeders. There were no dietary differences in liver glutathione peroxidase activity in pups or breeders. High isoflavone intake significantly (p = 0.001-0.047) reduced body weight in rat pups from 35 days onward, but not in breeders. Body fat and leptin were likewise significantly reduced by high isoflavones in pups while effects in breeders were less pronounced but still significant. High intake of Se and isoflavones each decreased serum IGF-1 in pups at 100 and 200 days, but not in breeders. No consistent dietary effects were observed on serum testosterone or relative weights of prostates. In pups, the combination of high isoflavones and high selenium produced the lowest weight gain, the lowest serum leptin, and the lowest serum IGF-1 concentrations of all four diets. CONCLUSION: Combined intake of high selenium and high isoflavones may achieve greater chemopreventive effects than either compound individually. The timing of supplementation may determine the significance of its effects.

High selenium reduces NF-kappaB-regulated gene expression in uninduced human prostate cancer cells.

Nuclear factor kappa B (NF-kappaB) induces expression of antiapoptotic and pro-inflammatory genes and is constitutively activated in prostate cancer. We tested the hypothesis that a biologically and physiologically relevant form and concentration of selenium (Se) may alter NF-kappa B activation in early prostate cancer cells in the absence of exogenously added inducers of the NF-kappaB pathway. LNCaP cells were cultured in medium without added tumor necrosis factor alpha or lipopolysaccharide but with methylseleninic acid added to provide final concentrations of Se of 30 nM-7.6 microM. Compared to 50 nM Se, treatment with 7.6 microM Se virtually eliminated NF-kappaB binding to its DNA response element and reduced transcription rates and mRNA levels by half for NF-kappaB-regulated genes. There were no differences due to Se in tyrosine phosphorylation, inhibitor of kappa B alpha (I kappa B alpha) levels, or NF-kappaB translocation from cytosol to nucleus. The observation in these basal, unstimulated cells of altered NF-kappaB binding to DNA in the absence of effects on the NF-kappaB activation pathway suggests an interaction of Se with the NF-kappaB protein or an effect on recruitment of NF-kappaB coactivators or corepressors. Inhibition of transcription factor binding and anti-apoptotic gene expression may be one mechanism for the chemopreventive effects of Se against prostate cancer.

Estrogen receptor activation and estrogen-regulated gene expression are unaffected by methylseleninic acid in LNCaP prostate cancer cells.

Prostate cancer is the most frequently diagnosed cancer and a leading cause of cancer deaths in American men. High dietary intake and status of the essential trace element selenium (Se) have been consistently correlated with reduced risk for prostate cancer. One molecular mechanism by which Se may reduce prostate cancer risk is by catalyzing disulfide bond formation or, otherwise, complexing with reactive sulfhydryl groups in transcription factors, thus altering their binding to DNA and regulation of gene expression. Estrogen plays a role in the etiology of prostate cancer. Estrogen receptors contain cysteines in zinc fingers that are susceptible to oxidation and internal disulfide bond formation, which can prevent DNA binding. We hypothesized that Se alteration of estrogen receptor (ER) binding to DNA and estrogen-regulated gene expression may be one mechanism by which it exerts its chemopreventive effects. LNCaP human prostate cancer cells were treated with 0.05 mumol/L (control) or 5.0 mumol/L (high) Se as methylseleninic acid (MSA). Electrophoretic mobility shift assays showed that binding of ER-beta to the estrogen response element was a nonsignificant 14% lower in cells treated with high MSA. Run-on transcription assays showed no significant changes in transcription rates for estrogen-regulated genes, and steady-state mRNA levels for those genes, assayed by reverse transcription-polymerase chair reaction, were likewise unaffected by MSA. These results suggest that the well-documented chemopreventive effects of Se against prostate cancer may be mediated by mechanisms other than inhibition by monomethylated Se compounds of ER-beta activation or estrogen-regulated gene expression.

High dietary intake of sodium selenite induces oxidative DNA damage in rat liver.

One mechanism for the cancer-chemopreventive effects of high selenium (Se) intake is hypothesized to be antioxidant protection of DNA. In this work we examine DNA oxidation in whole animals as a function of dietary Se intake and carcinogen administration. Weanling male Sprague-Dawley rats were fed a basal, Torula yeast-based, Se-deficient diet supplemented with 0, 0.15, or 2.0 ppm Se as sodium selenite for 10 wk. They were then injected with 0, 0.1, or 10 mg /kg body weight of the pro-oxidant carcinogen N-nitrosodiethylamine. High levels of carcinogen and high levels of selenite intake each increased concentration of 8-hydroxy-2'-deoxyguanosine in liver DNA. Se-dependent glutathione peroxidase I gene expression and enzyme activity were dramatically reduced by dietary Se deficiency but were unaffected by carcinogen administration. There were no significant main or interactive effects of Se or carcinogen on activity or gene expression of the DNA repair enzyme 8-oxoguanine glycosylase I. These results do not support the hypothesis that high Se intake may be cancer-preventive by inhibiting oxidative DNA damage. Rather than inhibiting oxidative DNA damage, these findings suggest that high dietary intake of inorganic Se may promote in vivo DNA oxidation.

Tissue specificity of selenoprotein gene expression in rats.

To investigate the tissue-specific effects of inadequate, adequate, and high selenium intake on selenoprotein gene expression and enzyme activity, weanling rats were fed a selenium-deficient diet or the same diet supplemented with 0.1 or 2.0 mg of selenium/kg of diet for 91 days. No significant differences in growth were observed. In liver, transcription of genes for cellular glutathione peroxidase, type I iodothyronine 5'-deiodinase, and selenoprotein P was unaffected by selenium intake. Steady-state levels of mRNA for glutathione peroxidase and selenoprotein P were higher in liver than in kidney. For iodothyronine 5' deiodinase in the opposite was true. In liver, selenium deficiency reduced glutathione peroxidase mRNA by 89% and virtually abolished enzyme activity. For iodothyronine 5' deiodinase, mRNA and enzyme activity were reduced 69 and 70%, respectively. In kidney, selenium deprivation decreased glutathione peroxidase mRNA by 91% and reduced enzyme activity to nearly zero. For iodothyronine 5'-deiodinase, decreases in mRNA and enzyme activity were 19 and 62%, respectively. Reductions in selenoprotein P mRNA were 50% in kidney but only 14% in liver. The only difference in the effects between the two supplements was in liver, where iodothyronine 5'-deiodinase activity was reduced by increasing the selenium supplement above a nutritionally adequate level. Hence, for these selenoproteins, mRNA turnover appears to be the pretranslational process most sensitive to selenium intake. In addition, selenoprotein mRNAs are stabilized differentially in selenium deficiency, depending upon the tissue examined. Percentage changes in the activity of selenoenzymes were not always the same as the changes in their mRNA levels. This suggests that other processes, including translation and protein turnover, may determine the ultimate level of enzyme activity attained in response to dietary selenium intake.