Large-scale screening for factor V Leiden (G1691A), prothrombin (G20210A), and MTHFR (C677T) mutations in Greek population.

Background and aims: To provide a fair estimate of the prevalence of factor V Leiden (FVL) (G1691A), prothrombin (G20210A), and MTHFR (C677T) mutations in the Greek population. Methods: We genotyped a representative sample of 974 apparently healthy Greek adults by the method of real-time PCR and we calculated the allele frequencies of factor V Leiden (FVL) (G1691A), prothrombin (G20210A), and MTHFR (C677T) mutations. In addition, we determined the frequency of co-occurrence of FVL (1691A) and prothrombin (20210A), FVL (1691A) and MTHFR (677T), prothrombin (20210A) and MTHFR (677T) mutations. Results: Τhe career frequencies of FVL (1691A), prothrombin (20210A), and MTHFR (677T) alleles were 7.5%, 4.5%, and 49.3% while the allele frequencies were 4%, 2.25%, and 39.5%, respectively. The coexistence of the allele frequencies combinations of two, FVL (1691A) and Prothrombin (20210A), FVL (1691A) and MTHFR (677T), prothrombin (20210A) and MTHFR (677T) was found in 1 (0.9%), 29 (3.5%), and 22 (3%) samples, respectively. Triple heterozygous carriers were not found. Conclusion: Allele frequencies of the two (FVL and MTHFR) mutations are higher compared with published data. The large sample size of our study enhances the validity of our results and suggests a biological affinity of Greek population with Southern Italian populations.

Paving the way for successful islet encapsulation.

Type 1 diabetes mellitus (T1DM) is a disorder that decimates pancreatic ß-cells which produce insulin. Direct pancreatic islet transplantation cannot serve as a widespread therapeutic modality owing to the need for lifelong immunosuppression and donor shortage. Therefore, several encapsulation techniques have been developed to enclose the islets in semipermeable vehicles that will allow oxygen and nutrient input as well as insulin, other metabolites and waste output, while accomplishing immunoisolation. Although encapsulation technology continues to face significant obstacles, recent advances in material science, stem cell biology and immunology potentially serve as pathways to success. This review summarizes the accomplishments of the past 5 years.

Greek nurses' knowledge on the prevention of surgical site infection: an investigation.

OBJECTIVE: The aim of the study was to investigate nurses' knowledge regarding the prevention of surgical site infection (SSI), and to examine the relationship between nurses' demographic characteristics and educational level and their level of knowledge in prevention of SSIs. A further aim was to examine the differences in nurses' knowledge with respect to selected variables and to identify the most significant predictors of nurses' knowledge regarding the prevention of SSIs, to support the provision of high-quality nursing care. METHOD: A prospective, observational study of a convenience sample of nurses and assistant nurses working in surgical departments, in a public general hospital for adults in Attica, during May to August 2016. For data collection, an anonymous self-completion questionnaire was developed and tested for comprehension and acceptability. RESULTS: Data was collected from 148 nurses and assistant nurses, 121 (81.8%) were female, 73.6% were aged 36-50 years and 43.9% had 11-20 years of experience. With regards to educational level, 66.2% had a degree from a technological educational institute and 10.1% had a Master's degree. Furthermore, 18.2% had a surgical specialty and 59.5% had received special training on surgical infections. The majority of respondents did not chose the correct definition of the time of occurrence of SSIs. Several statistically significant correlations were observed between knowledge on safer hair removal and respndent age (p=0.037), educational level (p=0.003), professional experience (p=0.048), and training in SSIs (p=0.009). CONCLUSION: The results of this study revealed that the majority respondents had a high level of knowledge regarding the prevention of SSIs, which contrasted with a low level of knowledge regarding their full definition of the time of occurrence.

Advanced glycation end products interfere in luteinizing hormone and follicle stimulating hormone signaling in human granulosa KGN cells.

Advanced glycation end products accumulate in the ovarian granulosa-cell layer of women with polycystic ovarian syndrome. Taken that the MAPK/ERK-pathway is a key regulator of oocyte maturation and function, consisting the main pathway used by the gonadotrophic hormones (luteinizing hormone, follicle stimulating hormone) to control ovulation, the present study aims to assess advanced glycation end products' interference into luteinizing hormone-and follicle stimulating hormone-signaling via the MAPK/ERK-pathway in the human granulosa KGN cell line. KGN cells were treated with luteinizing hormone or follicle stimulating hormone in the absence or presence of human glycated albumin. The specific activation of the main components of the MAPK/ERK1/2-pathway (namely c-Raf, MEK and ERK1/2) was assessed. Treatment of KGN cells with an MEK1/2-inhibitor or a blocking anti-RAGE-antibody was also performed to shed further light on the mechanism of the involvement of advanced glycation end products in luteinizing hormone and/or follicle stimulating hormone-related signaling pathways. Luteinizing hormone treatment increased p-ERK1/2 levels in human granulosa cells, while the combined treatment of luteinizing hormone and human glycated albumin provoked a decrease of p-ERK1/2 levels. A similar reducing effect was also observed for the upstream molecule phospho-cRaf upon combined treatment, while treatment with an MEK-inhibitor confirmed that the phenomenon is MAPK/ERK-pathway-dependent. Similarly, follicle stimulating hormone treatment increased p-ERK1/2 and p-MEK1/2 levels, while the combined treatment of follicle stimulating hormone and human glycated albumin downregulated their levels. Advanced glycation end products reduce the luteinizing hormone- and follicle stimulating hormone-induced ERK1/2 activation that is critical for granulosa cell mitogenesis and proliferation. Inappropriate activation of ERK1/2 in granulosa cells may block the granulosa cell differentiation pathway and/or impair follicular responses to hormones, potentially leading to ovulation failure that characterizes polycystic ovarian syndrome.

The COOH-terminus of the IGF-1Ec Isoform Enhances the Proliferation and Migration of Human MCF-7 Breast Cancer Cells.

BACKGROUND/AIM: The insulin-like growth factor-1 (IGF-1) signaling is well implicated in cancer biology, however the potential roles of the distinct IGF-1 isoforms in human malignancies are largely unknown. Recently, the carboxyl-terminal of the IGF-1Ec variant (hEc; 24aa) has been associated with osteosarcoma and prostate cancer. Herein, we investigated the potential role of hEc in breast cancer. MATERIALS AND METHODS: Synthetic hEc peptide was administrated to MCF-7 and MDA-MB-231 breast cancer cells. In addition MCF-7 cells were engineered to overexpress hEc. The proliferation and migratory capacities in response to hEc were analyzed using MTT, trypan blue and wound healing/scratch assays, while the activation of the ERK/AKT signaling pathways were investigated using phospho western blotting. RESULTS: We found that exogenous administration of hEc stimulated the proliferation of estrogen-responsive MCF-7, but not that of hormone-resistant MDA-MB-231 cells. In addition, MCF-7 cells stably-overexpressing hEc acquired an increased proliferation rate and migratory capacity, as well as, enhanced ERK1/2 phosphorylation, compared to mock and wild-type cells. CONCLUSION: hEc stimulates the proliferation and migration of MCF-7 breast cancer cells and enhances the intracellular ERK1/2 signaling.

The human Ec peptide: the active core of a progression growth factor with species-specific mode of action.

OBJECTIVE: Preferential IGF-1Ec expression has been firmly associated with skeletal muscle repair mechanisms, post-infarction remodeling of the myocardium, the pathophysiology of endometriosis and prostate cancer biology. Therefore, we have studied the possible biological significance of synthetic Ec peptide, a putative cleavage product of IGF-1Ec in PC-3 cells and C2C12 myoblasts. DESIGN: We had previously designed and synthesized commercially peptides corresponding to the human Ec and its mouse igf1 counterpart as well as synthetic peptides that correspond to parts of the hEc. Using proliferation and mitogenic signaling assays, we tested their effect on PC-3 cells and C2C12 myoblasts at different doses and in different culture conditions. RESULTS: Human Ec, hEc, was documented as exerting progression but not competence growth factor actions, activating ERK1/2 without affecting Akt phosphorylation in PC-3 cells. A narrow concentration range of hEc (5-50nM) stimulated the growth of PC-3 cells grown in culture media supplemented with 10% FBS. hEc did not stimulate the growth of PC-3 cells cultured with media containing 0.5% FBS or in mouse C2C12 myoblasts under any culture conditions. The activity of hEc was blocked by a neutralizing anti-human IGF-1Ec antibody but not by a neutralizing anti-human IGF-1 receptor antibody. The synthetic mouse Ec was inactive in human PC-3 cells; however, it stimulated significantly the proliferation of mouse C2C12. By analyzing the bioactivity of synthetic hEc fragments, we documented that hEc's active core is located in the last 4aa of its C-terminal end. CONCLUSION: The hEc peptide is an important progression factor for human PC-3 prostate cancer cells.

Advanced glycation end-products and insulin signaling in granulosa cells.

Advanced glycation end-products (AGEs) may interfere with insulin intracellular signaling and glucose transport in human granulosa cells, potentially affecting ovarian function, follicular growth, linked with diminished fertility. The potential interaction of AGEs with insulin signaling pathways and glucose transport was investigated in human granulosa KGN cells. KGN cells were cultured with variable concentrations of human glycated albumin (HGA, 50-200 µg/mL) or insulin (100 ng/mL). Combined treatments of KGN cells with insulin (100 ng/mL) and HGA (200 µg/mL) were also performed. p-AKT levels and glucose transporter type 4 (Glut-4) translocation analysis were performed by Western blot. Phosphatidylinositol-3-kinase (PI3K)-specific signaling was checked by using the PI3K-inhibitor, LY294002. p-AKT levels were significantly increased following insulin treatment compared to basal levels or HGA exposure. This insulin-mediated AKT-phosphorylation was PI3K-specific and it was inhibited after combined treatment of insulin and HGA. Furthermore, Glut-4 translocation from the cytoplasm to the membrane compartments of KGN cells was remarkably reduced after the combined treatment of insulin and HGA. The present findings support that AGEs interfere with insulin signaling in granulosa cells and prevent Glut-4 membrane translocation suggesting that intra ovarian AGEs accumulation, from endogenous or exogenous sources, may contribute to the pathophysiology of states characterized with anovulation and insulin resistance such as polycystic ovary syndrome.

PCK3145 inhibits proliferation and induces apoptosis in breast and colon cancer cells.

AIM: To explore the effects of PCK3145 beyond prostate cancer. MATERIALS AND METHODS: Using Trypan blue, MTT proliferation assays, cell cycle and apoptosis analysis, we assessed the effects of PCK3145 on prostate (PC-3), breast (MCF-7) and colon (HT-29) human cancer cell lines and in osteosarcoma (MG-63) cells; any synergistic effects with docetaxel and oxaliplatin were also explored. RESULTS: PCK3145 inhibited proliferation and induced apoptosis of PC-3, MCF-7 and HT-29 cells in a dose- and time-dependent manner but not in the MG-63 cell line, consistent with the low expression of the laminin receptor (LR) in the latter cell line. PCK3145 produced rapid (within 5 min) and transient (up to 60 min) activation of MEK and ERK1/2. Synergistic effects were observed with docetaxel and oxaliplatin. CONCLUSION: PCK3145 can exert anticancer activity not only on prostate but also on breast and colon cancer cells, possibly through LR-mediated activation of MEK and ERK1/2 phosphorylation.

Oncogenic Role of the Ec Peptide of the IGF-1Ec Isoform in Prostate Cancer.

IGF-1 is one of the key molecules in cancer biology; however, little is known about the role of the preferential expression of the premature IGF-1 isoforms in prostate cancer. We have examined the role of the cleaved COO- terminal peptide (PEc) of the third IGF-1 isoform, IGF-1Ec, in prostate cancer. Our evidence suggests that endogenously produced PEc induces cellular proliferation in the human prostate cancer cells (PC-3) in vitro and in vivo, by activating the ERK1/2 pathway in an autocrine/paracrine manner. PEc overexpressing cells and tumors presented evidence of epithelial to mesenchymal transition, whereas the orthotopic injection of PEc-overexpressing, normal prostate epithelium cells (HPrEC) in SCID mice was associated with increased metastatic rate. In humans, the IGF-1Ec expression was detected in prostate cancer biopsies, where its expression correlates with tumor stage. Our data describes the action of PEc in prostate cancer biology and defines its potential role in tumor growth, progression and metastasis.

Insulinlike growth factor-1Ec (MGF) expression in eutopic and ectopic endometrium: characterization of the MGF E-peptide actions in vitro.

The transcription of the insulinlike growth factor 1 (igf-1) gene generates three mRNA isoforms, namely IGF-1Ea, IGF-1Eb and IGF-1Ec (or MGF [mechano growth factor]). Herein, we analyzed the expression of IGF-1 isoforms in eutopic and ectopic endometrium (red lesions and endometriotic cysts) of women with endometriosis, and we characterized the actions of a synthetic MGF E-peptide on KLE cells. Our data documented that all three igf-1 gene transcripts are expressed in the stromal cells of the eutopic and ectopic endometrium; however, endometriotic cysts contained significantly lower IGF-1 isoform expression, both at the mRNA and protein level, as was shown using semiquantitative PCR and immunohistochemical methods. In addition, the glandular cells of the eutopic endometrium did not express any of the IGF-1 isoforms; however, the glandular cells of the ectopic endometrium (red lesions) did express the IGF-1Ec at mRNA and protein level. Furthermore, synthetic MGF E-peptide, which comprised the last 24 amino acids of the MGF, stimulated the growth of the KLE cells. Experimental silencing of the type 1 IGF receptor (IGF-1R) and insulin receptor expression of KLE cells (siRNA knock-out methods) did not alter the mitogenic action of the synthetic MGF E-peptide, revealing that MGF E-peptide stimulates the growth of KLE cells via an IGF-1R-independent and insulin receptor-independent mechanism. These data suggest that the IGF-1Ec transcript might generate, apart from mature IGF-1 peptide, another posttranslational bioactive product that may have an important role in endometriosis pathophysiology.

IL-6 and PPARgamma signalling in human PC-3 prostate cancer cells.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands and interleukin (IL)-6 are key factors for controlling prostate cancer cell proliferation and survival. MATERIALS AND METHODS: Herein we used the natural PPARgamma ligand, 15deoxy Delta12-14 PGJ(2) (15dPGJ(2)), and IL-6 to define their interactions on proliferation and signal transduction in PC-3 cells. Cytotoxic and trypan blue exclusion assays, Western blot analysis of mitogen-activated protein kinases (MAPK) and Janus kinase/Signal transducer and activator of transcription (JAK/Stat) and real-time polymerase chain reaction (PCR) were methods employed as investigation tools. RESULTS: 15dPGJ(2) reduced PC-3 cell proliferation, while IL-6 increased it. IL-6 induced PPARgamma expression but did not affect the PPARgamma ligand-mediated effects on the proliferation of PC-3 cells. However, 15dPGJ(2) inhibited the IL-6-mediated increase of PC-3 cell proliferation. 15dPGJ(2) activated Erk1/2 phosphorylation without affecting Akt phosphorylation and reduced phosphorylated and unphosphorylated Stat3 in PC-3 cells. IL-6 suppressed endogenous activation of Stat3 without affecting Erk1/2 and Akt phosphorylation and suppressed the 15dPGJ(2)-mediated activation of Erk1/2 phosphorylation in PC-3 cells. CONCLUSION: The interplay between PPARgamma ligands and IL-6 signalling could be important in controlling the growth of androgen independent prostate cancer cells as exemplified by PC-3 cells.

The glutamatergic system expression in human PC-3 and LNCaP prostate cancer cells.

BACKGROUND: The glutamatergic system (Glu system) comprises the Glu receptors (GluRs), the Glu transporters (GluTs) and glutamine synthetase (GS). MATERIALS AND METHODS: Using PCR-based detection and Western blot analysis, the expression of Glu system components was assessed in human androgen-independent PC-3 and androgen-dependent LNCaP prostate cancer cells. RESULTS: iGluRs, such as NR1, NR2A, NR2C, NR2D and NR3B; mGLuRs such as mGluR1, mGluR2, mGluR3, mGluR4 and mGluR5; GluTs such as EAAT1, EAAT2, EAAT3 and EAATS; and GS mRNA were steadily expressed in both cell lines. In addition, NR3A, mGluR6, mGluR8 and EAAT4 mRNA were differentially expressed in PC-3 and LNCaP cells, mGluR7 and EAAT4 mRNA expression was induced and mGluR8 was silenced by dihydrotestosterone (DHT) treatment in LNCaP cells. GS, EAAT1 and mGLuR5 were also detected at the protein level in both PC-3 and LNCAP cells. CONCLUSION: These data suggest that the Glu system could be an important regulator of prostate cancer cell biology.

IGF-1 expression in infarcted myocardium and MGF E peptide actions in rat cardiomyocytes in vitro.

Insulinlike growth factor-1 (IGF-1) expression is implicated in myocardial pathophysiology, and two IGF-1 mRNA splice variants have been detected in rodents, IGF-1Ea and mechano-growth factor (MGF). We investigated the expression pattern of IGF-1 gene transcripts in rat myocardium from 1 h up to 8 wks after myocardial infarction induced by left anterior descending coronary artery ligation. In addition, we characterized IGF-1 and MGF E peptide action and their respective signaling in H9C2 myocardial-like cells in vitro. IGF-1Ea and MGF expression were significantly increased, both at transcriptional and translational levels, during the late postinfarction period (4 and 8 wks) in infarcted rat myocardium. Measurements of serum IGF-1 levels in infarcted rats were initially decreased (24 h up to 1 wk) but remained unaltered throughout the late experimental phase (4 to 8 wks) compared with sham-operated rats. Furthermore, specific anti-IGF-1R neutralizing antibody failed to block the synthetic MGF E peptide action, whereas it completely blocked IGF-1 action on the proliferation of H9C2 cells. Moreover, this synthetic MGF E peptide did not activate Akt phosphorylation, whereas it activated ERK1/2 in H9C2 rat myocardial cells. These data support the role of IGF-1 expression in the myocardial repair process and suggest that synthetic MGF E peptide actions may be mediated via an IGF-1R independent pathway in rat myocardial cells, as suggested by our in vitro experiments.

Rosiglitazone attenuates insulin-like growth factor 1 receptor survival signaling in PC-3 cells.

PPARgamma, a member of the peroxisome proliferator-activated receptor family, is overexpressed in prostate cancer. Natural and synthetic ligands of PPARgamma via genomic and nongenomic actions promote cell cycle arrest and apoptosis of several prostate cancer cells, in vitro. Insulin-like growth factor 1 (IGF-1) inhibits the adriamycin-induced apoptosis of PC-3 human prostate cancer cells. Therefore, we have analyzed the ability of two PPARgamma ligands,15dPGJ2 and rosiglitazone, a natural and a synthetic PPARgamma ligand, respectively, to increase the adriamycin-induced cytotoxicity of PC-3 cells and to suppress the IGF-1 survival effect on adriamycin-induced apoptosis of PC-3 cells. Our data revealed that both the PPARgamma ligands increased the adriamycin-induced cytostasis of PC-3 cells, however, only rosiglitazone added to the adriamycin-induced apoptosis of PC-3 cells. In addition, rosiglitazone attenuated the type I IGF receptor (IGF-1R) survival signaling on adriamycin-induced apoptosis of PC-3 cells via its nongenomic action on ERK1/2 and AKT phosphorylation. Because the IGF-1R signaling is probably the most important host tissue (bone) metastasis microenvironment-related survival signaling for prostate cancer cells, we conclude that rosiglitazone effects on IGF-1R-mediated activation of ERK1/2 and AKT could have clinical implications for the management of androgen ablation-refractory and chemotherapy-resistant advanced prostate cancer with bone metastasis.

The non-genomic crosstalk between PPAR-gamma ligands and ERK1/2 in cancer cell lines.

Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily acting as transcription factors. PPAR-gamma, one of the three PPAR subtypes, is expressed in many malignant and non-malignant cells and tissues. PPAR-gamma ligands influence cancer biology via both genomic as well as non-genomic events. The non-genomic action of PPAR-gamma ligands, including the activation of MAPK signaling pathways, is under intense investigation. In the presence of PPAR-gamma ligands, a rapid phosphorylation of ERK1/2 is observed in many cancer cell lines. Activated ERK1/2 elicits rapid, non-genomic cellular effects and can directly repress PPAR-gamma transcriptional activity by phosphorylation. This paper reviews the interrelation of PPAR-gamma ligands and activated ERK1/2, in relation to their antineoplastic actions in cancer cell lines, which may offer the potential for improved anticancer therapies.