Identification of the zinc finger 216 (ZNF216) in human carcinoma cells. A potential regulator of EGFR activity

Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling

EGF and TGF-β1 Effects on Thyroid Function

Normal epithelial thyroid cells in culture are inhibited by TGF-β1. Instead, transformed thyroid cell lines are frequently resistant to its growth inhibitory effect. Loss of TGF-β responsiveness could be due to a reduced expression of TGF-β receptors, as shown in transformed rat thyroid cell lines and in human thyroid tumors, or to alterations of other genes controlling TGF-β signal transduction pathway. However, in thyroid neoplasia, a complex pattern of alterations occurring during transformation and progression has been identified. Functionally, TGF-β1 acts as a tumor suppressor in the early stage of transformation or as a tumor promoter in advanced cancer. This peculiar pleiotropic behaviour of TGF-β may result from cross-talk with signalling pathways mediated by other growth factors, among which EGF-like ligands play an important role. This paper reports evidences on TGF-β1 and EGF systems in thyroid tumors and on the cross-talk between these growth factors in thyroid cancer

Detection and Physicochemical Characterization of Membrane Vesicles (MVs) of Lactobacillus reuteri DSM 17938

Membrane vesicles (MVs) are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs), and planktonic (pMVs) phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM) and Dynamic Light Scattering (DLS). Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM) analysis. eDNA (extracellular DNA) was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (eMVsDNA). DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the eMVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems

DOXORUBICIN-INDUCED HEARTH FAILURE IS DEPENDENT ON BOTH OXIDATIVE- AND NITROSATIVE STRESS.

INTRODUCTION. Due to increasing numbers of young cancer survivors, the understanding of the biological mechanisms underlying the chemotherapy side effects, is highly relevant. Doxorubicin is a chemotherapeutic agent whose clinical use is hampered by the serious dose-dependent cardiotoxicity which ultimately results in left ventricular dysfunction, and, in the worst cases, congestive heart failure. Doxorubicin-induced cardiomyopathy is a lethal disease and, when congestive heart failure develops, mortality is approximately 50%. Unfortunately, no effective treatment is at present available. There is a large evidence that oxidative stress and inflammation are implicated in the pathogenesis of congestive heart failure. The sustained inflammatory/oxidative environment leads to cell damage becoming stuck in a vicious circle of impaired pathways. The accumulation of Reactive Oxygen Species is widely accepted as a key factor of cardiotoxic effects, but evidence indicates that also nitrosative stress is involved. Oxidative and nitrosative stress are strictly linked in Doxorubicin-induced heart failure. Mitochondrial Connexin 43 conferred cardioprotection by reducing mitochondrial ROS production in Doxorubicin-induced cardiotoxicity. This study aimed to evaluate the involvement of Mitochondrial Connexin 43 in Doxorubicin-induced nitrosative stress and heart failure. In fact, Doxorubicin stimulates mitochondrial superoxide over production and consequently the generation of other ROS able to trigger the activation of NF-κB, thus leading to enhanced iNOS expression and NO production. It is reported that an increase in NO production in the myocardium may cause nitration of actin and other cytoskeletal proteins altering their structure and causing harmful effects on the contractile function of myofilaments. MATERIALS AND METHODS. Rat cardiomyocytes H9c2 were treated with Doxorubicin and in absence or in presence of Radicicol, an inhibitor of Connexin 43 translocation to mitochondria. Fluorescence-activated cell sorting (FACS) analysis and quantitative Real Time-PCR showed that Doxorubicin increased SOD and CAT gene and protein expression. Moreover, the expression of molecules involved in apoptosis and NF-kB pathway was analysed by FACS and Western blot. It is known that increased expression of iNOS results in NO overproduction that quickly reacts with hydrogen peroxide or superoxide, generated by mitochondria, forming highly reactive and harmful peroxynitrite. RESULTS: In H9c2 cells, FACS analysis showed that co-treatment with Doxorubicin and Radicicol increased SOD and CAT expression and the apoptotic response, as shown by hypodiploid nuclei. In H9c2 cells low iNOS gene expression levels have been observed in both untreated and treated cells, while it increased after 6 hours of Doxorubicin-Radicicol co-treatment. Western blot analysis showed a significant increase of iNOS expression in Doxorubicin-treated H9c2 cells and a further increase in Radicicol-pretreated cells after 6 hours of treatment. Since in presence of high levels of O2-, NO quickly reacted to form peroxynitrite thus inducing nitration of the aromatic side-chains of tyrosine in proteins, we evaluated the levels of nytrotirosine in H9c2 cells treated as described. The cytofluorimetric analysis showed a significant increase of nitrotyrosine levels in Doxorubicin-treated cells. Inhibition of mitochondrial Connexin43 translocation by Radicicol further enhanced nitrotyrosine levels in particular after 6 hours of treatment. The induction of apoptosis was confirmed by Western blot analysis that showed how the combined treatment with Doxorubicin and Radicicol increased caspase 9 expression and reduced procaspase 3 levels. Moreover, after 3 hours of Doxorubicin treatment a significant increase in IKKα expression was observed, more evident after 6 hours of co-treatment with Doxorubicin and Radicicol. These results suggested a rapid activation of pathway involved in inflammatory response mostly in the presence of Connexin 43 inhibitor. CONCLUSIONS: In our models of rat cardiomyocytes, the understanding of the mechanisms underlying the cellular insult induced by Doxorubicin will result in developing new applications for preventing cardiotoxicity and consequently the heart failure. Antioxidant enzymes are mostly increased in presence of Radicicol. Therefore, our findings help to strengthen the role of Connexin 43 in cardioprotection mechanisms. Moreover, this study was aimed to explore the molecular basis of cardiomyocytes damage for tailoring medical treatment to the specific Doxorubicin-induced intracellular alterations not only to prevent heart failure, but also in an attempt to stop the progression of end-stage heart failure

Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from biofilm and planktonic phase associated with extracellular DNA (eDNA)

Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation