Generation of hydrogen peroxide-resistant murine neuroblastoma cells: a target discovery platform for novel neuroprotective genes

Oxidative stress has been suggested to play an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer’s disease (AD). Hydrogen peroxide (H2O2), one of the main reactive oxygen species, is converted into the highly toxic ·OH radical in the presence of redox-active transition metals, which then oxidises nucleic acids, lipids and proteins, leading to neurodegeneration and cell death. There is an urgent need to gain more knowledge about relevant therapeutic targets to combat oxidative stress and it neurotoxic effects, and how this knowledge can be utilized to develop novel neuroprotective therapies for AD. One way to identify new mechanisms combating oxidative stress was via the creation of H2O2-resistant cell lines and identification of the mechanisms responsible for their resistance. However, in most cases catalase overexpression or increased glutathione content was identified as the primary mode of H2O2 resistance in these cell lines. In this study, we have generated six different resistant neuronal cell lines or populations (from the same original murine Neuro2a neuroblastoma line) by exposing cells to increasing concentrations of H2O2 and performing continuous selection for survivors over a period of several months, which appear to have acquired H2O2 resistance based on other, novel mechanisms. These six populations showed a significant, but differential resistance against H2O2 when compared with the parental cell line. Using combinations of catalase-, glutathione synthesis- and glutathione peroxidase-inhibitors it was shown that the increased resistance of Neuro2a-HR cells is not solely based on an increased activity of catalase or the glutathione system, suggesting that their resistance might be based on yet unknown, novel defence mechanisms

Novel neuroprotective therapies for Alzheimer's and Parkinsons's disease

One of the major age-related damaging agents are reactive oxygen species (ROS). The brain is more vulnerable to oxidative stress than other organs as concomitant low activity and capacity of antioxidative protection systems allow for increased exposure of target molecules to ROS. Since neurons are postmitotic cells, they have to live with cellular damage accumulated over many decades. Increased levels of ROS (also termed "oxidative stress"), produced by normal mitochondrial activity, inflammation and excess glutamate levels, are proposed to accelerate neurodegenerative processes characteristic for Alzheimer's disease and Parkinson's disease. This review presents evidence for the importance of oxidative stress in the pathogenesis of these diseases and explains the nature of different types of ROS mediating neuronal damage. Furthermore, the potential beneficial effects of neuroprotective treatments, including synthetic and plant deroved antioxidants, energy supplements and anti - glutamatergic drugs are discussed

Chronic inflammation alters production and release of glutathione and related thiols in human U373 astroglial cells

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD

A versatile high throughput screening system for the simultaneous identification of anti-inflammatory and neuroprotective compounds

In many chronic neurodegenerative diseases including Frontotemporal Dementia and Alzheimer's disease (AD), microglial activation is suggested to be involved in pathogenesis or disease progression. Activated microglia secrete a variety of cytokines, including interleukin-1β, interleukin-6, and tumor necrosis factor as well as reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS contribute to alterations in neuronal glucose uptake, inhibition of mitochondrial enzymes, a decrease in mitochondrial membrane potential, impaired axonal transport, and synaptic signaling. In addition, ROS act as signaling molecules in pro-inflammatory redox-active signal transduction pathways. To establish a high throughput screening system for anti-inflammatory and neuroprotective compounds, we have constructed an "Enhanced Green Fluorescent protein" (EGFP) expressing neuronal cell line and set up a murine microglia/neuron co-culture system with these EGFP expressing neuronal cells. We show that microglia activation leads to neuronal cell death, which can be conveniently measured by loss of neuronal EGFP fluorescence. Moreover, we used this system to test selected polyphenolic compounds for their ability to downregulate inflammatory markers and to protect neurons against microglial insult. We suggest that this system might allow accelerated drug discovery for the treatment of inflammation-mediated neurodegenerative diseases

Novel Aspects of the Liver Microenvironment in Hepatocellular Carcinoma Pathogenesis and Development

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer that is derived from hepatocytes and is characterised by high mortality rate and poor prognosis. While HCC is driven by cumulative changes in the hepatocyte genome, it is increasingly recognised that the liver microenvironment plays a pivotal role in HCC propensity, progression and treatment response. The microenvironmental stimuli that have been recognised as being involved in HCC pathogenesis are diverse and include intrahepatic cell subpopulations, such as immune and stellate cells, pathogens, such as hepatitis viruses, and non-cellular factors, such as abnormal extracellular matrix (ECM) and tissue hypoxia. Recently, a number of novel environmental influences have been shown to have an equally dramatic, but previously unrecognized, role in HCC progression. Novel aspects, including diet, gastrointestinal tract (GIT) microflora and circulating microvesicles, are now being recognized as increasingly important in HCC pathogenesis. This review will outline aspects of the HCC microenvironment, including the potential role of GIT microflora and microvesicles, in providing new insights into tumourigenesis and identifying potential novel targets in the treatment of HCC

Circulating CD147 predicts mortality in advanced hepatocellular carcinoma

Background and Aim: The glycoprotein CD147 has a role in tumor progression, is readily detectable in the circulation, and is abundantly expressed in hepatocellular carcinoma (HCC). Advanced HCC patients are a heterogeneous group with some individuals having dismal survival. The aim of this study was to examine circulating soluble CD147 levels as a prognostic marker in HCC patients. Methods: CD147 was measured in 277 patients (110 HCC, 115 chronic liver disease, and 52 non‐liver disease). Clinical data included etiology, tumor progression, Barcelona Clinic Liver Cancer (BCLC) stage, and treatment response. Patients with HCC were stratified into two groups based upon the 75th percentile of CD147 levels (24 ng/mL). Results: CD147 in HCC correlated inversely with poor survival (P = 0.031). Increased CD147 predicted poor survival in BCLC stages C and D (P = 0.045), and CD147 levels >24 ng/mL predicted a significantly diminished 90‐day and 180‐day survival time (hazard ratio [HR] = 6.1; 95% confidence interval [CI]: 2.1–63.2; P = 0.0045 and HR = 2.8; 95% CI: 1.2–12.6; P = 0.028, respectively). In BCLC stage C, CD147 predicted prognosis; levels >24 ng/mL were associated with a median survival of 1.5 months compared with 6.5 months with CD147 levels ≤24 ng/mL (P = 0.03). CD147 also identified patients with a poor prognosis independent from treatment frequency, modality, and tumor size. Conclusions: Circulating CD147 is an independent marker of survival in advanced HCC. CD147 requires further evaluation as a potential new prognostic measure in HCC to identify patients with advanced disease who have a poor prognosis

Hepatocyte Produced Matrix Metalloproteinases Are Regulated by CD147 in Liver Fibrogenesis

<div><p>Background</p><p>The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090571#pone.0090571-Shackel1" target="_blank">[1]</a>. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.</p><p>Methods</p><p>Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by <i>in-situ</i> zymography. The role of CD147 was studied <i>in-vitro</i> with siRNA to CD147 in hepatocytes and <i>in-vivo</i> in mice with CCl₄ induced liver injury using <b>ã</b>CD147 antibody intervention.</p><p>Results</p><p>In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant <i>in-vitro</i> regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, <i>in-vivo</i> α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl₄ treated mice compared to controls.</p><p>Conclusion</p><p>We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both <i>in-vitro</i> and <i>in-vivo</i>, with the net result being reduced fibrotic matrix turnover <i>in-vivo</i>. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.</p></div

Expression of MMP mRNA in human liver.

<p>Quantitative PCR on whole liver tissue, non-diseased and HCV cirrhosis, of MMP-1, MMP-2, MMP-9 and MMP-14 mRNA (n = 4 per group). The expression of MMPs measured was significantly increased in cirrhosis compared to non-diseased controls, *p<0.05.</p

MMP expression in CCl₄ induced liver injury with α-CD147 antibody intervention.

<p>Cirrhosis was induced in a mouse model of liver injury (<i>Balb/c</i>) with CCl₄. An antibody targeting CD147 (RL73.2) was given twice weekly for 4 weeks, mice given an equal amount of IgG were used as control. There were no appreciable changes in MMP expression with either α-CD147 antibody or IgG control treatment in the absence of CCl₄ (not shown). Tissue hydroxyproline was significantly increased with α-CD147 antibody treatment (Panel A). Compared with control MMP expression is significantly increased in the CCl₄ injury with IgG group and reversed by intervention with α-CD147 antibody (Panels B–E). TIMP-1 expression was upregulated in injury but unaffected by intervention with α-CD147 antibody (Panel F) (n = 5−7 per group data expressed as mean and SEM. *p<0.05, **p<0.01 and ***p<0.001).</p