High concentration calcitriol induces endoplasmic reticulum stress related gene profile in breast cancer cells

WOS: 000399165600038PubMed ID: 28177777Calcitriol, the active form of vitamin D, is known for its anticancer properties including induction of apoptosis as well as the inhibition of angiogenesis and metastasis. Understanding the mechanisms of action for calcitriol will help with the development of novel treatment strategies. Since vitamin D exerts its cellular actions via binding to its receptor and by altering expressions of a set of genes, we aimed to evaluate the effect of calcitriol on transcriptomic profile of breast cancer cells. We previously demonstrated that calcitriol alters endoplasmic reticulum (ER) stress markers, therefore in this study we have focused on ER-stress-related genes to reveal calcitriols action on these genes in particular. We have treated breast cancer cell lines MCF-7 and MDA-MB-231 with previously determined IC50 concentrations of calcitriol and evaluated the transcriptomic alterations via microarray. During analysis, only genes altered by at least 2-fold with a P value < 0.05 were taken into consideration. Our findings revealed an ER-stress-associated transcriptomic profile induced by calcitriol. Induced genes include genes with a pro-survival function (NUPR1, DNAJB9, HMOX1, LCN2, and LAMP3) and with a pro-death function (CHOP (DDIT3), DDIT4, NDGR1, NOXA, and CLGN). These results suggest that calcitriol induces an ER-stress-like response inducing both pro-survival and pro-death transcripts in the process.Ege UniversityEge University [2011-TIP-038]This work is supported by Ege University Scientific Research Project 2011-TIP-038

The effect of calcitriol on endoplasmic reticulum stress response

WOS: 000355225900013PubMed ID: 25916601Calcitriol, the active form of vitamin D, is known for its anticancer properties including induction of apoptosis, inhibition of angiogenesis, and metastasis. Calcitriol also increases intracellular calcium triggering apoptosis in a calpain-dependent manner. Since the main storage unit for cellular calcium is endoplasmic reticulum (ER) and a decrease in ER calcium levels might induce ER stress associated cell death, we hypothesized that the cellular actions of calcitriol occur via ER stress. We have evaluated induction of ER stress by assessing BIP expression and XBP-1 splicing in breast cancer cell lines (MCF-7 and MDA-MB-231) and mammary epithelial cell line MCF10A. Our results suggest that cytotoxic concentrations of calcitriol induce an ER stress related response indicated as increased BIP levels and XBP-1 splicing not only in breast cancer cells but also in mammary epithelial cell line. However, vehicle treatment also induced a similar response de-emphasizing the importance of such effect. Calcitriol also failed to activate calpains, further weakening the idea of ER stress as the main mechanism for apoptotic effects of calcitriol. Taken together our results suggest an association between ER stress and vitamin D signaling. However present data indicates that ER stress by itself is not sufficient to explain anticancer properties of calcitriol.Ege UniversityEge University [2012-TIP-072]This project is supported by Ege University Scientific Research Project Grant No. 2012-TIP-072

Practitioners&rsquo; Perspectives towards Requirements Engineering: A Survey

In this paper, we discuss the results of our survey among 84 practitioners in order to understand practitioners&rsquo; perspectives towards requirements engineering. We asked 28 questions to learn the practitioners&rsquo; motivations, the techniques and technologies used for different activities, practitioners&rsquo; experiences with customer involvement, and any challenges encountered. Some important results are as follows: the practitioners&rsquo; top motivations are the precise communication of requirements and analyzing the requirements to detect issues. Most practitioners (i) insist on using natural languages, (ii) specify requirements as the use case and scenario descriptions, (iii) neglect using/transforming requirements for making high-level decisions and reasoning about requirements, (iv) neglect the specifications of quality requirements and their reasoning while considering quality requirements important, and (v) neglect any technologies for facilitating requirements engineering (e.g., meta-modeling technologies, formal verification tools, and advanced tools). Practitioners are challenged by the cost and effort spent in specifying requirements, the omissions of errors, misinterpretations of requirements and their incorrect (manual) transformations, and customers&rsquo; lack of technical knowledge. With the survey results, practitioners can gain an awareness on the general perspectives, academics can trigger new research addressing the observed issues, and tool vendors can improve their tools with regard to the weaknesses determined

Mitochondrial DNA polymorphisms associated with longevity in the Turkish population

WOS: 000341072700002PubMed ID: 24792352The accumulation of mutations in mitochondrial DNA is a widely recognized mechanism for aging and age related diseases. However, studies indicate that some mutations could be beneficial to longevity by slowing down the function of the electron transport chain, reducing free radical production. In this study, we re-sequenced the entire mitochondrial DNA from 50 individuals and examined aging-related variations in the Turkish population. We evaluated sequence data by comparing whole SNP frequencies, individual SNP frequencies, the effect of SNPs, SNP accumulation in certain mtDNA regions and haplotype profiles between elderly and control groups. The frequency of total mitochondrial SNPs was significantly higher in nonagenarians than controls (p = 0.0094). Furthermore, non-coding, synonymous and tRNA mutations were more prevalent in the 90 + group compared to controls (p = 0.0001, p < 0.001, p = 0.0096, respectively). A73G and C152T polymorphisms were significantly associated with longevity in the Turkish population (p = 0.0086 and p = 0.004, respectively). Additionally, C150T was specific to the 90 + group, but the difference failed to reach statistical significance (p = 0.053). We also detected a novel transversion in the ATPase6 gene (C8899A) that was negatively associated with longevity (p = 0.0016). Examining the distribution of SNPs among genes and functionally associated gene regions revealed a significant accumulation of mutations in the D-loop region and genes encoding Complex I subunits (ND1-6) (p < 0.0001, p = 0.0302, respectively). Moreover, there was an increase in the non-synonymous mutation frequency of Complex I genes in aged subjects (p < 0.0001). Haplotype H was also significantly increased in the control group (p = 0.0405). Overall, our findings support a role for mitochondrial genome variations and the functionality of oxidative phosphorylation in longevity. In this report, we sequenced the whole mtDNA of the Turkish population for the first time. (C) 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.Ege University Research FundsEge University [2010 TIP 013]This project was supported by the Ege University Research Funds (Project No. 2010 TIP 013)

Design and development of a self-microemulsifying drug delivery system of olmesartan medoxomil for enhanced bioavailability

###EgeUn###Olmesartan medoxomil (OM) is a hydrophobic antihypertensive drug with low bioavailability (26%) and is known to have adverse effects such as celiac disease and enteropathy. The purpose of this study was to develop SMEDDS to increase bioavailability and decrease potential side effects of OM. Hydrophilic lipophilic balance was calculated by testing solubility of OM in different oils, surfactants, and cosurfactants to obtain the most suitable combination of SMEDDS. Pseudoternary phase diagram was used to select the better oil/water formulation of SMEDDS. After a test for 3-month stability, dissolution tests and parallel artificial membrane permeability assay (PAMPA) were conducted to investigate drug solubility and permeability. Biodistribution of fluorescent marked SMEDDS was observed by using in vivo imaging system. The pharmacodynamics of the drug were determined by measuring blood pressure from tails of rats. At the end of the experiment, intestines were examined for adverse effects of OM. Compared with tablet formulation according to the dissolution study, SMEDDS formulation showed 1.67 times improvement in solubility of OM. PAMPA studies suggested a much faster permeability rate for OM SMEDDS compared to the suspension form. Labeled SMEDDS gave 3.96 times stronger fluorescent emission than control dye administered mice in in vivo imaging system (IVIS®) studies, indicating an increased bioavailability. Treating effect of SMEDDS was 3.1 times more efficient compared to suspension in hypertensive rats. It caused neither celiac-like enteropathy nor diarrhea, during 21-day noninvasive blood pressure system (NIBP) assay. Our results suggest that SMEEDS formulation improves dissolution and oral bioavailability of OM while reducing its adverse effects. © 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group.British Association for Psychopharmacology: 15-ECZ-014The authors express deep gratitude to Aliye Uster Foundation for their generous funding of this research. This work was also supported by the Ege University Scientific Research Project Commission (BAP), Izmir-Turkey, Grant Number 15-ECZ-014. -

Y Design and development of a self-microemulsifying drug delivery system of olmesartan medoxomil for enhanced bioavailability

###EgeUn###Olmesartan medoxomil (OM) is a hydrophobic antihypertensive drug with low bioavailability (26%) and is known to have adverse effects such as celiac disease and enteropathy. The purpose of this study was to develop SMEDDS to increase bioavailability and decrease potential side effects of OM. Hydrophilic lipophilic balance was calculated by testing solubility of OM in different oils, surfactants, and cosurfactants to obtain the most suitable combination of SMEDDS. Pseudoternary phase diagram was used to select the better oil/water formulation of SMEDDS. After a test for 3-month stability, dissolution tests and parallel artificial membrane permeability assay (PAMPA) were conducted to investigate drug solubility and permeability. Biodistribution of fluorescent marked SMEDDS was observed by using in vivo imaging system. The pharmacodynamics of the drug were determined by measuring blood pressure from tails of rats. At the end of the experiment, intestines were examined for adverse effects of OM. Compared with tablet formulation according to the dissolution study, SMEDDS formulation showed 1.67 times improvement in solubility of OM. PAMPA studies suggested a much faster permeability rate for OM SMEDDS compared to the suspension form. Labeled SMEDDS gave 3.96 times stronger fluorescent emission than control dye administered mice in in vivo imaging system (IVISA (R)) studies, indicating an increased bioavailability. Treating effect of SMEDDS was 3.1 times more efficient compared to suspension in hypertensive rats. It caused neither celiac-like enteropathy nor diarrhea, during 21-day noninvasive blood pressure system (NIBP) assay. Our results suggest that SMEEDS formulation improves dissolution and oral bioavailability of OM while reducing its adverse effects.Aliye Uster Foundation; Ege University Scientific Research Project Commission (BAP), Izmir-TurkeyEge University [15-ECZ-014]The authors express deep gratitude to Aliye Uster Foundation for their generous funding of this research. This work was also supported by the Ege University Scientific Research Project Commission (BAP), Izmir-Turkey, Grant Number 15-ECZ-014

Acrylamide-encapsulated glucose oxidase inhibits breast cancer cell viability

Geyik, Oyku Gonul/0000-0003-3014-1253; KILINC, ALI/0000-0002-0470-4297; Yuce, Zeynep/0000-0002-2762-0942WOS:000603517600022Objectives: Cancer cells modulate metabolic pathways to ensure continuity of energy, macromolecules and redoxhomeostasis. Although these vulnerabilities are often targeted individually, targeting all with an enzyme may prove a novel approach. However, therapeutic enzymes are prone to proteolytic degradation and neutralizing antibodies leading to a reduced half-life and effectiveness. We hypothesized that glucose oxidase (GOX) enzyme that catalyzes oxidation of glucose and production of hydrogen peroxide, may hit all these targets by depleting glucose; crippling anabolic pathways and producing reactive oxygen species (ROS); unbalancing redox homeostasis. Methods: We encapsulated GOX in an acrylamide layer and then performed activity assays in denaturizing settings to determine protection provided by encapsulation. Afterwards, we tested the effects of encapsulated (enGOX) and free (fGOX) enzyme on MCF-7 breast cancer cells. Results: GOX preserved 70% of its activity following encapsulation. When fGOX and enGOX treated with guanidinium chloride, fGOX lost approximately 72% of its activity, while enGOX only lost 30%. Both forms demonstrated remarkable resilience against degradation by proteinase K and inhibited viability of MCF-7 cells in an activity-dependent manner. Conclusions: Encapsulation provided protection to GOX against denaturation without reducing its activity, which would prolong half-life of the enzyme when administered intravenously

Effects of Ganoderma lucidum (Higher Basidiomycetes) Extracts on the miRNA Profile and Telomerase Activity of the MCF-7 Breast Cancer Cell Line

WOS: 000354909700003PubMed ID: 25954907Ganoderma lucidum is a medicinal higher Basidiomycetes mushroom that exerts anticancer effects through several different mechanisms. This study investigated the effects of Ganoderma lucidum on the telomerase activity and microRNA (miRNA) profiles of MCF-7 cells. According to the cytotoxicity results, the Ganoderma lucidum ether extract exhibits the highest cytotoxic potency; therefore it was chosen for the subsequent telomerase activity assay and miRNA profiling. The telomerase activity observed in the cells treated with a half-maximal inhibitory concentration of Ganoderma lucidum ether extract (100 mu g/mL in dimethyl sulfmdde) was 32.2% lower than that of the control cells treated with 1% dimethyl sulfoxide. Among 1066 miRNAs, the most downregulated miRNA was hsa-miR-27a* (4.469-fold), and the most upregulated miRNA was hsa-miR-1285 (10.462-fold). A database search revealed the predicted miRNAs that target the catalytic subunit of the telomerase enzyme telomerase reverse transcriptase, and only miR-3687 (upregulated 2.153-fold) and miR-1207-5p (upregulated 2.895-fold) were changed by at least 2-fold. The miRNA profile changes demonstrated in this study provide a data set regarding their effects on the pathways that regulate telomerase activity in MCF-7 breast cancer cells treated with Ganoderma lucidum. These data should aid the development of novel cancer treatment strategies