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Structural characterization of recombinant humangastrokine-1: biochemical properties of the purified protein
No full textGastric cancer (GC) is one of the most leading cause of cancer death. The
high mortality is due to late diagnosis, although the gastric infection by
Helicobacter pylori represents one of the most significant risk factors for
this type of cancer. Gastrokine-1 (GKN1) is expressed in normal gastric
tissue, while it is scarcely present in samples from patients infected by H.
pylori, and completely absent in GC tissues and cell lines. Therefore,
GKN1 could be a potential marker for GC diagnosis and a possible tumor
suppressor. In order to get more insights into the molecular mechanisms
on the pathology of GC, this study was directed to better understand the
structural features and the biological activity of this protein. In details,
this study was directed to: 1) synthesis of recombinant mature GKN1 by
cloning, expression and purification; 2) characterization of structural and
biochemical properties of recombinant GKN1 by circular dichroism,
fluorescence spectroscopy, and limited proteolysis; 3) evaluation of the
effect of recombinant GKN1 on gastric cancer cell lines growth; 4)
construction of a 3D structural model of the protein. The first protocol for
biosynthesis and purification of native human GKN1 in the homologous
expression system of Pichia pastoris was settled. The use of biochemical
analytical methods such as limited proteolysis led to the identification of
exposed amino acid residues on the protein surface. The resistance of
GKN1 to the action of proteolytic enzymes was somehow explanatory of
its stability in the harsh stomach environment. Spectroscopic studies
(fluorescence and circular dichroism) showed that the protein is endowed
by a non-proper globular structure due probably to the presence of two
domains. These domains showed a different behaviour toward chemical
and physical denaturant. The results well correlate with predicted GKN1
secondary structure and 3D structure model. Finally, the recombinant
protein showed anti-proliferative properties on gastric cancer cell lines.
Our findings contribute to a preliminary clarification of the role of GKN1
in the pathogenesis of gastric cancer
Role of Gastrokine 1 in Gastric Cancer
Full text linkGastric cancer (GC) has high incidence (> 1.000.000 new cases/year) and mortality rate in several countries and is still one of the most frequent and lethal (> 600.000 dead/year) neoplasia with an average surviving of five years (less than 20%) (Pisani et al., 1990; Lands et al., 1998). It is already well known that infection of gastric antrum mucosal with the bacterium Helicobacter pylori is the cause of the chronic inflammation that leads to intestinal-type gastric cancer in the majority of the cases. The H. pylori infection is widespread but only a small number of the total population of infected individuals might eventually develop adenocarcinoma (around 3/10,000 individuals per year or 2.1% for lifetime infection) 27 (Correa & Piazuelo, 2008). Risk factors influencing the outcome of H. pylori–associated pathology include bacterial cytotoxic heterogeneity, diet, and geographic differences. The phenomenon of decreased gastric cancer incidence in Africa compared with other regions where H. pylori is endemic 28 (Holcombe, 1992) is probably due to the different diet of these populations compared to the western countries. This discrepancy has been partially attributed to helminth co-infection that likely modifies the characteristic proinflammatory type 1 T-helper 1 cell response, to a T-helper 2–predominant response 29 (Whary et al., 2005), typified by the release of non-inflammatory cytokines and reduced incidence of H. pylori–associated glandular atrophy, an early marker of cancer development. The identification of novel genes regulated by H. pylori in vivo, particularly those contributing to these early stages of gastric cancer, would facilitate improved understanding of the differential susceptibility to this pathogen. The different susceptibility among individuals to H. pylori infection is still not yet defined. Some works, however, suggested that the polymorphisms in host genetic factors like the proinflammatory cytokines interleukin-1, interleukin-8, and tumor necrosis factor may play a relevant role 30 (El-Omar et al., 2003).
The evolution of intestinal tumours is characterized by a progression of several sequential steps that starts with gastritis and then progresses to mucosal atrophy (atrophic gastritis), intestinal metaplasia, dysplasia and carcinoma with subsequent metastatic dissemination (Correa, 1992, 1995). The diffuse-type has instead a poorer prognosis and develops through unknown genetic and morphological events from normal gastric epithelium. No preceding steps have been identi_ed in the pathogenesis of diffuse carcinoma other than the chronic gastritis. The pathogenesis of gastric cancer remains poorly understood although it is evident that several environmental factors, such as H. pylori infection can be one of the causes leading to this disease. In fact, the risk to develop gastric cancer is increased in patients with H. pylori infections probably as the result of a combination of genetic and environmental factors in which the infection by H. pylori is of particular relevance, especially when the inflammation involves the gastric body region with respect to the antrum (Correa, 1995; Goldstone et al., 1996; Nabewera & Logan, 1999). Generally, this condition is associated to different degrees of atrophy and alterations of the secretor function that, in the long term, became associated to gastric carcinoma (Forman et al., 1991; Parsonnet et al., 1997; Watanabe et al., 1998).
Diffuse adenocarcinoma shows an increased propensity for intra and transmural spread and is therefore associated with a poorer prognosis. Unfortunately, the histological classification of an individual gastric adenocarcinoma is not clear-cut with a tumour often comprising a mixture of intestinal and diffuse tissue types. Under these considerations, we think that there is an urgent necessity to dispose of an efficient tool for the detection of early stage gastric cancer like the identification of highly sensitive and specific biomarkers that will aid disease diagnosis and ensure early clinical intervention, thereby preventing mortality and reducing morbidity (Boussioutas & Taupin, 2001). Since most of GC (around 73%) is developed at antrum/pylorus, proteins secreted by antrum/pylorus mucosa might play a critical role in maintaining normal gastric mucosa structure and function
Gastric Cancer: Molecular Pathology State
No full textDespite the progressive decrease observed in the past fifty years, gastric cancer (GC) is the
fourth of the world rankings incidence of various types of cancer and is the second as a cause
of cancer-related death. There is distinct geographical variation in gastric cancer incidence with
the highest rates reported from Japan, Korea and Eastern Asia. Other high incidence areas are
Eastern Europe and parts of Latin America, while Western Europe, Africa, Australia and the
US generally have low incidence rates. In the last decade there has been a downward trend in
the incidence and mortality from this cancer. The reasons are to be found in the improvement
of food both as regards its preservation procedures and the variability in the diet and for the
decrease of infection by Helicobacter pylori (H. pylori). H. pylori infection is strongly associated
with risk for stomach cancer. Likely, this association is supported by the strong link between
this bacterium infections and precancerous lesions, including chronic atrophic gastritis and
dysplasia. The development of gastric cancer is characterized by multistage process in which
several alterations of genetic and epigenetic nature accumulate. These alterations are mainly
related to abnormalities of growth factors and receptors, DNA mismatch repair genes,
angiogenic factors, transcription factors, adaptor proteins, cell cycle regulators, and many
other macromolecular cell components. All these abnormalities identify from one side the
molecular and biological aspect of gastric cancer cells and from the other might suggest
possible strategies for therapeutic intervention
An overview on factors underlying gastric cancer; strategies for its management with particular reference to diet
No full textThe incidence of stomach cancer and the number of victims of this disease have decreased dramatically over the
last 60 years. However, gastric cancer still remains a very serious disease that requires further studies to enlarge
knowledge on its causes and to prevention methods. To date, the causes of gastric cancer are still not yet well
known but it is clear that some people are more prone than others to develop this disease. Gastric cancer affects
mostly adults aged 55 and over and men in percentage double than women. Stomach ulcer apparently does not
increase the risk of gastric cancer however, Helicobacter pylori, usually due to inflammation and gastric ulcers, may
be an important risk factor for this disease. Moreover, patients who have undergone stomach surgery or suffering
from pernicious anemia, achlorhydria or atrophic gastritis (that typically produce a reduction in the amount of acid)
are subject to a higher risk of gastric cancer. Exposure in workplaces to certain agents such as dust or fumes is
linked to a higher risk than average of developing stomach cancer. Smoking also contributes to increase this risk.
Moreover, epidemiological studies and animal models, conducted for years, have shown that some eating habits can
increase the risk of cancer. Other studies instead report that fresh foods (especially fruits and vegetables) play a
protective function against gastric cancer. For this reason, this paper provides an overview of the possible causes of
gastric cancer and the different therapeutic approaches, focusing in particular on the effects of diet
Overexpression of gastrokine 1 in gastric cancer cells induces Fas-mediated apoptosis.
No full textGastrokine 1 (GKN1) is involved in the replenishment of the surface lumen epithelial cell layer, in maintaining the mucosal integrity, and could play a role in cell proliferation and differentiation. In fact, after injury of the gastric mucosa, restoration may occur very rapidly in the presence of GKN1. In contrast, if the protein is downregulated, the repair process may be hampered; however, application of GKN1 to gastrointestinal cells promoted epithelial restoration. Because GKN1 possesses some mitogenic effects on intestinal epithelial cells (IEC- 6) whereas this protein was also capable of inhibiting proliferation in gastric cancer cells (MKN28), we decided to study its involvement in apoptosis to understand the role of GKN1 in the modulation of inflammatory damage or tumorigenesis in gastric mucosa. We found by cytofluorimetry, Western blot and RT-PCR that the overexpression of GKN1 in gastric cancer cell lines (AGS and MKN28) stimulated the expression of Fas receptor. Moreover, compared to control cells, a significant increase of apoptosis, evaluated by TUNEL, was observed when GKN1 transfected cells were treated with a monoclonal antibody (IgM) anti-Fas. The activation of Fas expression was also observed by the overexpression of GKN1 in other cancer cell lines. Moreover, in GKN1-overexpressing gastric cancer cells exposed to FasL, the activation of caspase-3 was also observed by Western blot and fluorescence assays. Our data represent the first report for GKN1 as modulator of apoptotic signals and suggest that GKN1 might play an important role for tissue repair during the early stages of neoplastic transformation
Raf kinases in signal transduction and interaction with translation machinery
No full textIn recent years, a large amount of evidence has given a central role to translational control in diseases such as cancer, tissue hypertrophy and neurodegeneration. Its deregulation can directly modulate cell cycling, transformation and survival response. The aim of this review is to describe the interaction between Raf activation and the main characters of the translational machinery, such as the elongation factor 1A (eEF1A), which has been recognized in recent years as one of the most interesting putative oncogenes. A particular emphasis is given to an intriguing non-canonical role that eEF1A can play in the relationship between the Ras???Raf-1???MEK1???ERK-1/2 and PI3K???Akt signaling pathways. Recently, our group has described a C-Raf kinase-mediated phosphorylation of eEF1A triggered by a survival pathway induced upon interferon alpha (IFN??) treatment in the human epidermoid cancer cell line (H1355). This phosphorylation seems to be the center of the survival pathway that counteracts the well-known pro-apoptotic function of IFN??. Furthermore, we have identified two new phosphorylation sites on eEF1A (Ser21 and Thr88) that are substrates for Raf kinases in vitro and, likely, in vivo as well. These residues seem to have a significant functional role in the control of cellular processes, such as cell proliferation and survival. In fact, overexpression of eEF1A2 in gemcitabine-treated cancer cells caused the upregulation of phosphoAkt and an increase in cell viability, thereby suggesting that eEF1A2 could exert its oncogenic behavior by participating in the regulation of PI3K pathway
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
New insights on the interaction between the isoforms 1 and 2 of human translation elongation factor 1A
No full textThe eukaryotic translation elongation factor 1A (eEF1A) is a moonlighting protein that besides to its canonical role in protein synthesis is also involved in many other cellular processes such as cell survival and apoptosis. In a previous work, we identified eEF1A Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and apoptosis of human cancer cells. We proposed that the phosphorylation of eEF1A by C-Raf required the presence of both eEF1A isoforms thus suggesting the formation of a potential eEF1A heterodimer owning regulatory properties. This study aimed at investigating the cellular localization and interaction between two eEF1A isoforms. To this end, we developed chimera proteins by adding at the N-terminal end of both eEF1A1 and eEF1A2 cyan fluorescence protein (mCerulean) and yellow fluorescence protein (mVenus), respectively. The fluorescent eEF1A1 and eEF1A2 chimeras were both addressed to COS-7 cells and found co-localized in the cytoplasm at the level of cellular membranes. We highlighted FRET between the labeled N-termini of eEF1A isoforms. The intra-molecular FRET of this chimera was about 17%. Our results provide novel information on the intracellular distribution and interaction of eEF1A isoforms
Role of human GKN1 on APP processing in gastric cancer
No full textGastrokine 1 (GKN1) is highly expressed in gastric tissue and is secreted into the stomach but is not expressed in gastric cancer. GKN1 belongs to the BRICHOS domain family and plays a major role in maintaining gastric mucosa integrity. We previously demonstrated that a recombinant human GKN1 protein was able to interact with the amyloid precursor protein (APP) and was endowed with an anti-amyloidogenic property because it inhibited polymerization of the Aβ(1-40) peptide released from APP upon its partial hydrolysis. Here, we report that GKN1 can act as a physiological suppressor of Aβ production in gastric cancer cells. GKN1 blocked the access of γ-secretase to APP, thereby facilitating the cleavage of APP by α- and β-secretases. GKN1 directly interacted with APP C-terminal fragments, C83 and C99. In addition, it did not affect γ-secretase activity in gastric cancer cells because it did not alter Notch1 processing. GKN1-mediated inhibition of APP processing might represent a new approach for the prevention and therapy of Alzheimer's disease (AD)
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