This research explores anti-cancer activities of synthetic peptides that inhibit the CMG helicase, the major DNA replicative helicase. Targeting CMG helicases for cancer treatment provides a unique opportunity to develop a cancer treatment that effectively eradicates cancer cells without affecting normal healthy tissues. This approach exploits a natural difference between activation of CMG helicases in normal and cancer cells. Collective evidence shows that normal cells - compare to cancer cells - possess higher levels of CMG helicase reserves available for activation throughout the S phase. Cancer cells, on the other hand, activate the majority of their helicases at once, thereby exhausting their helicase reserves. Based on this, we hypothesized that our peptides - by targeting the CMG helicase - would lead to the death of cancer cells, while spare corresponding normal cells. In this study, we utilized two types of cancerous and normal cell lines: pancreatic cancerous Hs766T and normal hTERT-HPNE cell lines, as well as glioma M059J and normal glial SVGp12 cell lines. Cells were plated in 96 well plates at a density of 3,000 cells/well and treated with different concentrations of studied peptides. Viabilities of cells were scored by a colorimetric CCK-8 assay. According to our IC50 calculations, both cancerous cell lines were much more sensitive to the peptide treatments compare to their normal counterparts. Indeed, we observed that normal cell lines required almost 25 times higher concentrations of peptides to reach the same cytotoxic effect as the cancerous cells. Interestingly, we contrast these results with our studies with Gemcitabine, a therapeutic agent that is currently used for cancer treatment. Unlike our studied peptides, treatments with Gemcitabine led to an equal demise of both cancerous and normal cells.
Aberrant activation of the stress-response system in early life can alter neurodevelopment and cause long-term neurological changes. Activation of the hypothalamic–pituitary–adrenal axis releases glucocorticoids into the bloodstream, to help the organism adapt to the stressful stimulus. Elevated glucocorticoid levels can promote the accumulation of reactive oxygen species, and the brain is highly susceptible to oxidative stress. The essential trace element selenium is obtained through diet, is used to synthesize antioxidant selenoproteins, and can mitigate glucocorticoid-mediated oxidative damage. Glucocorticoids can impair antioxidant enzymes in the brain, and could potentially influence selenoprotein expression. We hypothesized that exposure to high levels of glucocorticoids would disrupt selenoprotein expression in the developing brain. C57 wild-type dams of recently birthed litters were fed either a moderate (0.25 ppm) or high (1 ppm) selenium diet and administered corticosterone (75 μg/ml) via drinking water during postnatal days 1 to 15, after which the brains of the offspring were collected for western blot analysis. Glutathione peroxidase 1 and 4 levels were increased by maternal corticosterone exposure within the prefrontal cortex, hippocampus, and hypothalamus of offspring. Additionally, levels of the glucocorticoid receptor were decreased in the hippocampus and selenoprotein W was elevated in the hypothalamus by corticosterone. Maternal consumption of a high selenium diet independently decreased glucocorticoid receptor levels in the hippocampus of offspring of both sexes, as well as in the prefrontal cortex of female offspring. This study demonstrates that early life exposure to excess glucocorticoid levels can alter selenoprotein levels in the developing brain.
Selenium, an essential trace element known mainly for its antioxidant properties, is critical for proper brain function and regulation of energy metabolism. Whole-body knockout of the selenium recycling enzyme, selenocysteine lyase (Scly), increases susceptibility to metabolic syndrome and diet-induced obesity in mice. Scly knockout mice also have decreased selenoprotein expression levels in the hypothalamus, a key regulator of energy homeostasis. This study investigated the role of selenium in whole-body metabolism regulation using a mouse model with hypothalamic knockout of Scly. Agouti-related peptide (Agrp) promoter-driven Scly knockout resulted in reduced weight gain and adiposity while on a high-fat diet (HFD). Scly-Agrp knockout mice had reduced Agrp expression in the hypothalamus, as measured by Western blot and immunohistochemistry (IHC). IHC also revealed that while control mice developed HFD-induced leptin resistance in the arcuate nucleus, Scly-Agrp knockout mice maintained leptin sensitivity. Brown adipose tissue from Scly-Agrp knockout mice had reduced lipid deposition and increased expression of the thermogenic marker uncoupled protein-1. This study sheds light on the important role of selenium utilization in energy homeostasis, provides new information on the interplay between the central nervous system and whole-body metabolism, and may help identify key targets of interest for therapeutic treatment of metabolic disorders.
The essential micronutrient selenium (Se) provides antioxidant defense and supports numerous biological functions. Obtained through dietary intake, Se is incorporated into selenoproteins via the amino acid, selenocysteine (Sec). Mice with genetic deletion of the Se carrier, selenoprotein P (SELENOP), and the Se recycling enzyme selenocysteine lyase (SCLY), suffer from sexually dimorphic neurological deficits and require Se supplementation for viability. These impairments are more pronounced in males and are exacerbated by dietary Se restriction. We report here that, by 10 weeks of age, female Selenop/Scly double knockout (DKO) mice supplemented with 1 mg/ml sodium selenite in drinking water develop signs of hyper-adiposity not seen in male DKO mice. Unexpectedly, this metabolic phenotype can be reversed by removing Se from the drinking water at post-natal day 22, just prior to puberty. Restricting access to Se at this age prevents excess body weight gain and restriction from either post-natal day 22 or 37 reduces gonadal fat deposits. These results provide new insight into the sex-dependent relationship between Se and metabolic homeostasis.
Selenoprotein P (SELENOP1) is a selenium-rich antioxidant protein involved in extracellular transport of selenium (Se). SELENOP1 also has metal binding properties. The trace element Zinc (Zn 2+ ) is a neuromodulator that can be released from synaptic terminals in the brain, primarily from a subset of glutamatergic terminals. Both Zn 2+ and Se are necessary for normal brain function. Although these ions can bind together with high affinity, the biological significance of an interaction of SELENOP1 with Zn 2+ has not been investigated. We examined changes in brain Zn 2+ in SELENOP1 knockout (KO) animals. Timm-Danscher and N-(6-methoxy-8-quinolyl)- p- toluenesulphonamide (TSQ) staining revealed increased levels of intracellular Zn 2+ in the SELENOP1 −/− hippocampus compared to wildtype (WT) mice. Mass spectrometry analysis of frozen whole brain samples demonstrated that total Zn 2+ was not increased in the SELENOP1 −/− mice, suggesting only local changes in Zn 2+ distribution. Unexpectedly, live Zn 2+ imaging of hippocampal slices with a selective extracellular fluorescent Zn 2+ indicator (FluoZin-3) showed that SELENOP1 −/− mice have impaired Zn 2+ release in response to KCl-induced neuron depolarization. The zinc/metal storage protein metallothionein 3 (MT-3) was increased in SELENOP1 −/− hippocampus relative to wildtype, possibly in response to an elevated Zn 2+ content. We found that depriving cultured cells of selenium resulted in increased intracellular Zn 2+ , as did inhibition of selenoprotein GPX4 but not GPX1, suggesting the increased Zn 2+ in SELENOP1 −/− mice is due to a downregulation of antioxidant selenoproteins and subsequent release of Zn 2+ from intracellular stores. Surprisingly, we found increased tau phosphorylation in the hippocampus of SELENOP1 −/− mice, possibly resulting from intracellular zinc changes. Our findings reveal important roles for SELENOP1 in the maintenance of synaptic Zn 2+ physiology and preventing tau hyperphosphorylation.
The role of the essential trace element selenium in hypothalamic physiology has begun to come to light over recent years. Selenium is used to synthesize a family of proteins participating in redox reactions called selenoproteins, which contain a selenocysteine residue in place of a cysteine. Past studies have shown that disrupted selenoprotein expression in the hypothalamus can adversely impact energy homeostasis. There is also evidence that selenium supports leptin signaling in the hypothalamus by maintaining proper redox balance. In this study, we generated mice with conditional knockout of the selenocysteine tRNA[Ser]Sec gene (Trsp) in an orexigenic cell population called agouti-related peptide (Agrp)-positive neurons. We found that female TrspAgrpKO mice gain less weight while on a high-fat diet, which occurs due to changes in adipose tissue activity. Female TrspAgrpKO mice also retained hypothalamic sensitivity to leptin administration. Male mice were unaffected, however, highlighting the sexually dimorphic influence of selenium on neurobiology and energy homeostasis. These findings provide novel insight into the role of selenoproteins within a small yet heavily influential population of hypothalamic neurons.
