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

Oxidative stress induces Wnt canonical/non-canonical pathways modulation in colon cancer cell models

BACKGROUND-AIM. Increased reactive oxygen species (ROS) levels play critical roles in chronic inflammation, and predispose to colon carcinogenesis. Wnt signaling is essential for gut morphogenesis, tissue homeostasis and self-renewal, but its aberrant activation may drive the colorectal cancer (CRC). The ROS production seems to induce the Wnt/β-Catenin pathways, but the molecular mechanisms involved in CRC progression are still undefined. To evaluate the molecular relationship among oxidative stress and canonical/non-canonical Wnt pathways, we analyzed the response to ROS exposure in CRC cell lines with different Wnt signaling behaviour. METHODS. HCT116 (MSI) and SW480 (MSS) cells were treated with H2O2 [2 mM and 10 mM] for 15’and 30’. We assayed cell viability by MTS and cell cycle by FACS. Gene expression was evaluated by SYBR Green qRT-PCR, and protein expression was analyzed by IHC. Statistical analysis was performed by T-test (p value<0.05). RESULTS. MTS revealed different inhibition rates of cell growth at H2O2 concentrations. Acute stress induced by H2O2 [2mM] up-regulated gene expression of canonical LRP6 and LEF1, and non canonical ROR2 and JUN/AP1 molecules in SW480, while reduced ROR2 and LRP6 expression in HCT116. Both pathways showed a dose dependent increase in SW480, at H2O2 [10mM]. In HCT116 down-regulated gene expression of APC, LRP6, LEF1, and p65-NFkB was dependent on treatment time, in opposition to non-canonical ROR2. MUTYH, OGG1, NRF2, COX2 and JUN/AP1 expression significantly increased. H2O2 treatment induced FZD6 protein expression in HCT116 cytoplasm and E-cadherin protein expression in SW480 cytoplasm, while beta-catenin increased in both cell lines. Intriguingly we relieved a de novo APC expression in both cell lines cytoplasm. FACS analysis of cell cycle showed time dependent changes: upon H2O2 [2mM] treatment at 15’, SW480 increased in G1 and G2 and decreased in S, whereas HCT116 increased in G1 and slightly reduced in G2; after 30’, SW480 enhanced in G1 and S, and reduced in G2 while HCT116 diminished in G1 and increased in S/G2. CONCLUSIONS. In MSI and MSS CRC cells, oxidative stress differently affects the WNT pathways at gene and protein expression levels. Our results could unravel a new scenario for innovative CRC therapeutic approaches

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 to its capability of developing biofilms as a response to environmental stress and changes. 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 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 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of bOMVs and pOMVs. The presence of eDNA in bOMVs and pOMVs was carried out using a DNase I-gold complex and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterized using the dynamic light scattering (DLS) analysis. The eDNA associated to OMVs was detected and quantified by using PicoGreen assay and spectrophotometer, while its extraction was performed through a DNA Kit. The TEM images showed that the eDNA was mainly isolated and identified on OMVs-membrane surface; while the PicoGreen staining showed a 4-fold increase of dsDNA in bOMVs compared to pOMVs. The eDNA extracted from OMVs was visualized by using gel electrophoresis. The DLS analysis demonstrated that H. pylori generate vesicles, both in its planktonic and biofilm phenotypes, with sizes in the nanometer scales and a broad size distribution. The DLS aggregation study of H. pylori OMVs demonstrated that eDNA may play a role in the OMVs aggregation, particularly for biofilm phenotype. The eDNA associated with vesicle membrane can affect the DNase I activity on H. pylori biofilms. OMVs derived from H. pylori ATCC 43629/NCTC 11639, particularly its biofilm phenotype, may play a structural role by preventing eDNA degradation by nucleases and provide a bridging function between eDNA strands

Emerging Role of Oxidative Stress on EGFR and OGG1-BER Cross-Regulation: Implications in Thyroid Physiopathology

Thyroid diseases have a complex and multifactorial aetiology. Despite the numerous studies on the signals referable to the malignant transition, the molecular mechanisms concerning the role of oxidative stress remain elusive. Based on its strong oxidative power, H2O2 could be responsible for the high level of oxidative DNA damage observed in cancerous thyroid tissue and hyperactivation of mitogen-activated protein kinase (MAPK) and PI3K/Akt, which mediate ErbB signaling. Increased levels of 8-oxoG DNA adducts have been detected in the early stages of thyroid cancer. These DNA lesions are efficiently recognized and removed by the base excision repair (BER) pathway initiated by 8-oxoG glycosylase1 (OGG1). This study investigated the relationships between the EGFR and OGG1-BER pathways and their mutual regulation following oxidative stress stimulus by H2O2 in human thyrocytes. We clarified the modulation of ErbB receptors and their downstream pathways (PI3K/Akt and MAPK/ERK) under oxidative stress (from H2O2) at the level of gene and protein expression, according to the mechanism defined in a human non-pathological cell system, Nthy-ori 3-1. Later, on the basis of the results obtained by gene expression cluster analysis in normal cells, we assessed the dysregulation of the relationships in a model of papillary thyroid cancer with RET/PTC rearrangement (TPC-1). Our observations demonstrated that a H2O2 stress may induce a physiological cross-regulation between ErbB and OGG1-BER pathways in normal thyroid cells (while this is dysregulated in the TPC-1 cells). Gene expression data also delineated that MUTYH gene could play a physiological role in crosstalk between ErbB and BER pathways and this function is instead lost in cancer cells. Overall, our data on OGG1 protein expression suggest that it was physiologically regulated in response to oxidative modulation of ErbB, and that these might be dysregulated in the signaling pathway involving AKT in the progression of thyroid malignancies with RET/PTC rearrangements

DOXORUBICIN REMEDY OR HARM? CARDIOTOXICITY AND NOT ONLY...

INTRODUCTION. Doxorubicin (Doxo) is a chemotherapeutic agent whose clinical use is hampered by the serious dose-dependent cardiotoxicity. The accumulation of Reactive Oxygen Species (ROS) is widely accepted as a key factor of cardiotoxic effects. Mitochondrial Connexin 43 (Cx43) conferred cardioprotection by reducing cytosolic and mitochondrial ROS production. Topic of this work was the identification of antioxidant enzymes and molecules involved in Doxo-induced damage, in absence and in presence of Radicicol (Rad), an inhibitor of Cx43 translocation to mitochondria. Due to increasing numbers of young cancer survivors and the raising concerns for their fertility state, elucidating the biological mechanisms of chemotherapy risk is highly relevant. Moreover, it is known that Doxo-induced ovarian toxicity is associated with apoptosis of mouse granulosa cells. MATERIALS AND METHODS. Rat cardiac cells H9c2 were treated with Doxo and Rad. Changes in expression level of SOD, iNOS, CAT and HO were evaluated by FACS and qReal Time-PCR. The expression of molecules involved in apoptosis and NFkB pathway was analysed by FACS and Western blot. Doxo effects were also investigated in Human Granulosa Cells (HGC). RESULTS: In H9c2 cells, FACS analysis showed that cotreatment with Doxo and Rad increased SOD, CAT, HO and the apoptotic response, as shown by hypodiploid nuclei. The induction of apoptosis was confirmed by Western blot analysis that showed how the combined treatment with Doxo and Rad increased caspase-9 expression and reduced procaspase-3 levels. Moreover, after 3h of Doxo treatment a significant increase in IKKα expression was observed, more evident after 6h of co-treatment with Doxo and Rad. These results suggested a rapid activation of pathway involved in inflammatory response mostly in the presence of Cx43 inhibitor. Our preliminary results obtained treating HGC with Doxo showed toxic effects, as determined by MTS assay. CONCLUSIONS: In the model of cardiomyocytes, the understanding of the basic mechanisms underlying the cellular insult induced by Doxo will result in developing new applications for preventing cardiotoxicity. Antioxidant enzymes and molecules involved in inflammatory pathways are mostly increased in presence of Rad. Therefore, our findings help to strengthen the role of Cx43 in cardioprotection mechanisms

Oxidative Distress Induces Wnt/&beta;-Catenin Pathway Modulation in Colorectal Cancer Cells: Perspectives on APC Retained Functions

Colorectal cancer (CRC) is a multistep process that arises in the colic tissue microenvironment. Oxidative stress plays a role in mediating CRC cell survival and progression, as well as promoting resistance to therapies. CRC progression is associated with Wnt/&beta;-Catenin signaling dysregulation and loss of proper APC functions. Cancer recurrence/relapse has been attributed to altered ROS levels, produced in a cancerous microenvironment. The effect of oxidative distress on Wnt/&beta;-Catenin signaling in the light of APC functions is unclear. This study evaluated the effect of H2O2-induced short-term oxidative stress in HCT116, SW480 and SW620 cells with different phenotypes of APC and &beta;-Catenin. The modulation and relationship of APC with characteristic molecules of Wnt/&beta;-Catenin were assessed in gene and protein expression. Results indicated that CRC cells, even when deprived of growth factors, under acute oxidative distress conditions by H2O2 promote &beta;-Catenin expression and modulate cytoplasmic APC protein. Furthermore, H2O2 induces differential gene expression depending on the cellular phenotype and leading to favor both Wnt/Catenin-dependent and -independent signaling. The exact mechanism by which oxidative distress can affect Wnt signaling functions will require further investigation to reveal new scenarios for the development of therapeutic approaches for CRC, in the light of the conserved functions of APC