Global and region-specific post-transcriptional and post-translational modifications of bisphenol A in human prostate cancer cells.

Bisphenol A (BPA), as synthetic monomer used in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Epigenetic alterations could act an important role in BPA-induced toxicity, but its mechanism has not been fully understood. We investigated the effects of BPA on gene expression of chromatin modifying enzymes, promoter methylation of tumor suppressor genes and histone modifications in human prostate carcinoma cells (PC-3). IC50 value of BPA was determined as 217 and 190 µM in PC-3 cells by MTT and NRU tests, respectively. We revealed an increase in global levels of 5-methylcytocine and 5-hydroxymethylcytocine at 10 µM of BPA for 96 h. We observed a significant increase on promoter DNA methylation and decrease on gene expression of p16 gene while no change was observed for Cyclin D2 and Rassf1. Significant changes were observed in global histone modifications (H3K9ac, H3K9me3, H3K27me3, and H4K20me3) in PC-3 cells. According to these results, we investigated wide-range epigenetic modifications using PCR arrays. After 96 h BPA exposure, chromatin modifying enzymes including KDM5B and NSD1 were significantly downregulated. Also, promoter methylation of tumor suppressor genes including BCR, GSTP1, LOX, MGMT, NEUROG1, PDLIM4, PTGS2, PYCARD, TIMP3, TSC2 and ZMYDN10 altered significantly. ChIP results showed that H3K9ac, H3K9me3 and H3K27me3 modifications on p16 gene showed significant increases after 1 and 10 µM of BPA exposure. In conclusion, epigenetic signatures such as DNA methylation and histone modifications could be proposed as molecular biomarkers of BPA-induced prostate cancer progression.

Tub and ß-catenin play a key role in insulin and leptin resistance-induced pancreatic beta-cell differentiation.

The aim of this study was to investigate the molecular mechanism of pancreatic islet-derived mesenchymal stem cell (PID-MSC) differentiation into beta-cells in the presence of insulin and leptin resistance stimulators. We determined that beta-cell differentiation was stimulated by glucose, insulin, and leptin. Co-administration of insulin and leptin resulted in greater, at a further stage of differentiation but non-functional beta-cell formation. The levels of p-AKT(Ser473) did not change; SOCS3, PTP1B, p-IRS1(Ser307), PTEN levels increased and p-IRS1(Try) levels decreased due to insulin and leptin co-administration. These findings suggest that co-administration of insulin and leptin to PID-MSCs results in the development of both insulin and leptin resistance together. We showed that this differentiation signaling is mainly mediated by AKT/GSK-3ß/ß-catenin and Tub. Moreover, ß-catenin and Tub were linked to each other in the nucleus under this condition. Furthermore, we found that Tub and ß-catenin contributes to insulin production by increasing the expression of transcription factors by binding to the promoter regions of ins1, ins2, and pdx1 genes. In addition, Tub is also bound to the promoter region of the MafA gene. These findings demonstrate that when insulin and leptin resistance develop together in rat PID-MSCs beta-cell differentiation increases markedly via ß-catenin and Tub. New therapeutic agents that inhibit AKT/GSK-3ß/ß-catenin and in particular Tub may help prevent the development or retard the progression of type 2 diabetes.

The effect of plant lectins on the survival and malignant behaviors of thyroid cancer cells.

Altered or aberrant glycosylation is a common phenomenon in cancer cells and it originates from changes in the expression of the enzymes, glycosyltransferase, and glycosidase which up-regulate in response to some oncogenes in the glycan synthesis pathway. In this present study, it has been aimed to determine the alteration of sialic acid and fucose expressions in the cell surface of human thyroid carcinoma cells and investigate the changes in tumorigenic and malignant characters after treating them with specific plant lectins. Our study showed that the cell surface glycan chains of anaplastic 8305C, follicular FTC-133, and papillary K1 thyroid carcinoma cells were rich in α-2,6, α-2,3, sialic acid, and α-1,6 fucose residues. When the cells were treated with specific doses of Maackia amurensis lectin II (MAL), Sambucus nigra agglutinin (SNA), and Aleuria aurantia lectin (AAL) which have specific binding capacity for the detected glycan residues, respectively their cancerous traits changed dramatically. Remarkable findings obtained from MAL treatment leading to necrosis in 8505C cells without any toxicity for normal thyroid epithelial cells but it had proliferative effect on K1 and FCT-133 cells. Besides, MAL and SNA treatment decreased the mobility of 8505C and K1 cells. MAL and SNA lectins dramatically reduced the endothelial affinity of the cells and AAL significantly attenuated that of 8050C and K1 cells but not FTC-133. These results suggest that altered cell surface glycosylation in thyroid cancer seems to be a strong candidate for developing new therapeutic strategies.

The immune system as a chronotoxicity target of the anticancer mTOR inhibitor everolimus.

The circadian timing system controls many biological functions in mammals including xenobiotic metabolism, detoxification, cell proliferation, apoptosis and immune functions. Everolimus is a mammalian target of rapamycin inhibitor, whose immunosuppressant properties are both desired in transplant patients and unwanted in cancer patients, where it is indicated for its antiproliferative efficacy. Here we sought whether everolimus circadian timing would predictably modify its immunosuppressive effects so as to optimize this drug through timing. C57BL/6J mice were synchronized with light-dark 12h:12h, with L onset at Zeitgeber Time (ZT) 0. Everolimus was administered orally to male (5 mg/kg/day) and female mice (15 mg/kg/day) at ZT1, during early rest span or at ZT13, during early activity span for 4 weeks. Body weight loss, as well as hematological, immunological and biochemical toxicities, were determined. Spleen and thymus were examined histologically. Everolimus toxicity was less severe following dosing at ZT13, as compared to ZT1, as shown with least body weight inhibition in both genders; least reductions in thymus weight both in males (p < 0.01) and females (p < 0.001), least reduction in female spleen weight (p < 0.05), and less severe thymic medullar atrophy both in males (p < 0.001) and females (p < 0.001). The mean circulating counts in total leukocytes, total lymphocytes, T-helper and B lymphocytes displayed minor and non-significant changes following dosing at ZT13, while they were decreased by 56.9% (p < 0.01), 45.5% (p < 0.01), 43.1% (p < 0.05) and 48.7% (p < 0.01) after everolimus at ZT1, respectively, in only male mice. Chronotherapy of everolimus is an effective way to increase the general tolerability and decrease toxicity on the immune system.

Exendin-4 partly ameliorates - hyperglycemia-mediated tissue damage in lungs of streptozotocin-induced diabetic mice.

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion, - plays anti-inflammatory role in atherosclerosis, and has surfactant-releasing effects in lungs. GLP-1 analogues are used in diabetes therapy. This is the first study to investigate the effects of exendin-4, a GLP-1 receptor agonist, on lung injury in diabetic mice. BALB/c male mice were divided into four groups. The first group was given only citrate buffer, the second group was given only exendin-4, the third group was given only streptozotocin (STZ), and the fourth group was given both exendin-4 and STZ. Exendin-4 (3µg/kg) was administered daily by subcutaneous injection for 30days after mice were rendered diabetic with a single dose of STZ (200mg/kg). Structural alterations, oxidative stress, apoptosis, insulin signaling and expressions of prosurfactant-C, alpha-smooth muscle actin, collagen-I and fibronectin were evaluated in lung tissue. Diabetic mice lungs were characterized by induced oxidative stress, apoptosis, edema, and cell proliferation. They had honeycomb-like alveoli, thicker alveolar walls, and hypertrophic pneumocytes. Although exendin-4 treatment improved pulmonary edema, apoptosis, oxidative stress, and lung injury, it led to the disrupted insulin signaling and interstitial collagen accumulation in the lungs of diabetic mice. Exendin-4 ameliorates hyperglycemia-mediated lung damage by reducing glucose, -oxidative stress and stimulating cell proliferation. However, exendin-4 led to increased lung injury partly by reducing insulin signaling - and collagen accumulation around pulmonary vasculature in diabetic mice.

Involvement of dying beta cell originated messenger molecules in differentiation of pancreatic mesenchymal stem cells under glucotoxic and glucolipotoxic conditions.

Beta cell mass regulation represents a critical issue for understanding and treatment of diabetes. The most important process in the development of diabetes is beta cell death, generally induced by glucotoxicity or glucolipotoxicity, and the regeneration mechanism of new beta cells that will replace dead beta cells is still not fully understood. The aim of this study was to investigate the generation mechanism of new beta cells by considering the compensation phase of type 2 diabetes mellitus. In this study, pancreatic islet derived mesenchymal stem cells (PI-MSCs) were isolated from adult rats and characterized. Then, beta cells isolated from rats were co-cultured with PI-MSCs and they were exposed to glucotoxicity, lipotoxicity and glucolipotoxicity conditions for 72 hr. As the results apoptotic and necrotic cell death were increased in both PI-MSCs and beta cells especially by the exposure of glucotoxic and glucolipotoxic conditions to the co-culture systems. Glucotoxicity induced-differentiated beta cells were functional due to their capability of insulin secretion in response to rising glucose concentrations. Moreover, beta cell proliferation was induced in the glucotoxicity-treated co-culture system whereas suppressed in lipotoxicity or glucolipotoxicity-treated co-culture systems. In addition, 11 novel proteins, that may release from dead beta cells and have the ability to stimulate PI-MSCs in the direction of differentiation, were determined in media of glucotoxicity or glucolipotoxicity-treated co-culture systems. In conclusion, these molecules were considered as important for understanding cellular mechanism of beta cell differentiation and diabetes. Thus, they may be potential targets for diagnosis and cellular or therapeutic treatment of diabetes.

Runt-Related Transcription Factor 2 (Runx2) Is Responsible for Galectin-3 Overexpression in Human Thyroid Carcinoma.

Runx2 promotes metastatic ability of cancer cells by directly activating some of the mediators regarding malignancy. Galectin-3 (Gal-3) extensively expressed in normal and transformed cells and it is responsible for many cellular processes. In this study, we aimed to investigate whether there is any relationship between runx2 transcription factor and regulation of galectin-3 expression in different human thyroid carcinoma cell lines. To show effects of runx2 transcription factor on gal-3 expression, we developed runx2 knockdown model in the thyroid carcinoma cell lines; anaplastic 8505C and 8305C and, papillary TPC-1 and follicular FTC-133 by using siRNA transfection. We analyzed the protein expressions and mRNA levels of gal-3 and MMP2/9 in the runx2-silenced cell lines using Western blotting, qPCR, and fluorescent microscopy. Our results showed that mRNA expression levels of gal-3 and MMP2/9 were downregulated in runx2-silenced cell lines. In this investigation, we revealed that regulation of gal-3 expression was strongly correlated with runx2 transcription factor in human thyroid carcinoma. Considering the contribution of human gal-3 in collaboration with MMP2/9 to the malignant characters of many cancers, regulation of their expressions through runx2 seems like one of the key regulatory mechanism for malignant potential of human thyroid carcinoma. Accordingly, runx2 transcription factor inhibitors can be a potential target in order to prevent gal-3 mediated malignancy of human thyroid carcinoma. J. Cell. Biochem. 118: 3911-3919, 2017. © 2017 Wiley Periodicals, Inc.

Exendin-4 attenuates renal tubular injury by decreasing oxidative stress and inflammation in streptozotocin-induced diabetic mice.

In this study, we aimed to research the restorative effects of exendin-4, a GLP-1 analog, on renal tubular injury in streptozotocin-induced diabetes model. BALB/c male mice were divided into four groups: non-diabetic, non-diabetic + exendin-4 (3 µg/kg), diabetic and diabetic + exendin-4. In our diabetic model, we observed renal injury mainly in tubular area rather than glomeruli and exendin-4 decreased tubular injury with its glucose lowering effect. Besides, PCNA positive tubular cells, activities of LDH and Na(+)-K(+)-ATPase were also significantly declined by the administration of exendin-4. Furthermore, exendin-4 attenuated the levels of ROS, MDA, 8-OHdG, proinflammatory cytokines (TNF-α, IL-1ß), chemokine MCP-1, ICAM-1, and fibrosis-related molecules (transforming growth factor ß1 and fibronectin). In consistent with reducing tubular injury, macrophage infiltration and both MCP-1 and ICAM-1 production in tubular cells were decreased. These results indicate that exendin-4 may decrease renal tubular injury seen in the beginning of diabetic nephropathy by decreasing ROS production and inflammation.

Lectin binding properties of liver, small intestine and tail of metamorphosing marsh frog (Pelophylax ridibundus Pallas 1771).

In this present study, localization and variations of specific sugar moieties in the terminal carbohydrate chains of glycoconjugates in the small intestine, liver and tail have been investigated during the metamorphosis of Pelophylax ridibundus larvae. For this purpose, four lectins were used: wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA-I), Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), in different larval stages of the frog. Some cells stained specifically in the intestinal mucosa and in tail epidermal cells with the lectins and their affinity changed during metamorphic transformation. For the most part, they decreased in the climax and postmetamorphic periods. It was also found that WGA, DBA and UEA-I lectins exhibited strong affinity to white blood cells in the liver and their binding affinities were the highest in prometamorphosis and they gradually decreased until the end of metamorphosis. These results suggest that the changes of lectin binding in metamorphosis may be an indication of some cellular events occurring in larval metamorphosis such as cell differentiation and damage of cell adhesion between death and differentiating cells. They also can be useful markers for detection of white blood cells in amphibian hematopoietic organs.

Distribution of prolactin receptor in frog (Rana ridibunda) dorsal skin during hibernation.

The role of prolactin in the regulation of frog skin functions is still unclear particularly during environmental changes. In this study, prolactin receptor (PRLR) was detected in active and hibernating frog dorsal skin using immunohistochemical method. PRLR immunoreactivity in active frogs was observed in the epidermis, in the secretory epithelium of granular glands and the secretory channel cells of the glands. Myoepithelial cells of granular glands that started accumulating secretory material or those with a full lumen were PRLR immunoreactive, while some myoepithelial cells of empty granular glands were negative for PRLR. In hibernating frogs, this immunoreactivity was observed in the same regions; however, immunoreactivity was more intense than that in active frogs. PCNA was employed for detection of proliferative activity of PRL in the dorsal skin, and immunoreactivity was detected in the nuclei of a few epidermis cells and in the duct of glands of active frogs. The number of immunoreactive nuclei in these regions increased in hibernating and in prolactin injected groups. We conclude that prolactin provides morphological and functional integrity of skin stimulating the proliferation and regulating the function of granular glands and plays an important role in the adaptation of amphibians to the long winter period.