Umberto M. Marinari

Role of Glutathione in Cancer Progression and Chemoresistance

Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and disturbances in GSH homeostasis are involved in the etiology and progression of many human diseases including cancer. While GSH deficiency, or a decrease in the GSH/glutathione disulphide (GSSG) ratio, leads to an increased susceptibility to oxidative stress implicated in the progression of cancer, elevated GSH levels increase the antioxidant capacity and the resistance to oxidative stress as observed in many cancer cells. The present review highlights the role of GSH and related cytoprotective effects in the susceptibility to carcinogenesis and in the sensitivity of tumors to the cytotoxic effects of anticancer agents.

Cytotoxicity, DNA fragmentation and sister-chromatid exchange in Chinese hamster ovary cells exposed to the lipid peroxidation product 4-hydroxynonenal and homologous aldehydes.

The cytotoxic and genotoxic activities of 4-hydroxypentenal (HPE), 4-hydroxyhexenal (HHE), 4-hydroxyoctenal (HOE), 4-hydroxynonenal (HNE) and 4-hydroxyundecenal (HUE) were investigated in Chinese hamster ovary (CHO) cells. All five 4-hydroxyalkenals reduced plating efficiency in a concentration (ranging from 7 to 170 microM) lower than that producing a parallel reduction of trypan blue-excluding cells, but with both methods the increase in molarity needed to obtain a lethal effect was constantly rather small. With all five 4-hydroxyalkenals a significant amount of DNA fragmentation, as revealed either by the alkaline elution assay or by alkaline denaturation followed by chromatographic partition of single- and double-stranded DNA, was detected only after cell exposure to a cytotoxic concentration. HPE, HHE and HOE induced a clear-cut increase of sister-chromatid exchange (SCE) frequency, while that displayed by cells treated with HNE and HUE was minimal, even if dose-dependent and statistically significant. Since 4-hydroxyalkenals have been shown to originate from biomembrane lipids peroxidation, these findings should be taken into consideration in the assessment of the genotoxic role of lipoperoxidation in humans.

GEBR-7b, a novel PDE4D selective inhibitor that improves memory in rodents at non-emetic doses.

BACKGROUND AND PURPOSE Strategies designed to enhance cerebral cAMP have been proposed as symptomatic treatments to counteract cognitive deficits. However, pharmacological therapies aimed at reducing PDE4, the main class of cAMP catabolizing enzymes in the brain, produce severe emetic side effects. We have recently synthesized a 3‐cyclopentyloxy‐4‐methoxybenzaldehyde derivative, structurally related to rolipram, and endowed with selective PDE4D inhibitory activity. The aim of the present study was to investigate the effect of the new drug, namely GEBR‐7b, on memory performance, nausea, hippocampal cAMP and amyloid‐β (Aβ) levels.

Microarray Analysis in Alzheimer's Disease and Normal Aging

The purpose of this study was to investigate gene expression in Alzheimers disease (AD), the most common form of senile dementia. We utilized the microarray technology to simultaneously compare the expression profile of 12,000 human genes in cerebral cortex of AD and normal aging. To identify gene expression related to neurodegeneration, beside the presence of amyloid deposition, we used control brains with abundant amyloid plaques, derived from cognitively normal elderly subjects. The microarray analysis indicated that 314 genes were differentially expressed in AD cerebral cortex, with differences greater than 5 folds in 25 genes. RT‐PCR performed on a selected group of genes confirmed the increased expression of the interferon‐induced protein 3 in AD brain. This protein, which is highly inducible by both type I and type II interferons, was not previously associated with the neurodegenerative disease. IUBMB Life, 56: 349‐354, 2004

Role of peroxisome proliferator-activated receptor γ in amyloid precursor protein processing and amyloid β-mediated cell death

Recent data indicate that PPARgamma (peroxisome proliferator-activated receptor gamma) could be involved in the modulation of the amyloid cascade causing Alzheimers disease. In the present study we show that PPARgamma overexpression in cultured cells dramatically reduced Abeta (amyloid-beta) secretion, affecting the expression of the APP (Abeta precursor protein) at a post-transcriptional level. APP down-regulation did not involve the pathway of the secretases and correlated with a significant induction of APP ubiquitination. Additionally, we demonstrate that PPARgamma was able to protect the cells from H(2)O(2)-induced necrosis by decreasing Abeta secretion. Taken together, our results indicate a novel mechanism at the basis of the neuroprotection shown by PPARgamma agonists and an additional pathogenic role for Abeta accumulation.

Immunological evidence for increased oxidative stress in diabetic rats

Summary The role of oxidative stress in aging and diabetes mellitus is currently under discussion. We previously showed age-dependent accumulations of fluorescent protein adducts with lipoperoxidative aldehydes, (malondialdehyde (MDA), and hydroxynonenal (HNE)) in rat skin collagen with diabetic BB rats exhibiting faster accumulation. Modified proteins have been shown to be immunogenic: antibody titres against rat serum albumin modified by MDA and HNE (MDA-RSA and HNE-RSA) or oxidized by reactive oxygen species were measured by ELISA as markers of oxidative damage in BB diabetic and non-diabetic rats. Each tested antibody titre was significantly higher in the diabetic than in the non-diabetic rats. A significant correlation existed between anti-MDA-RSA and anti-HNE-RSA antibody titers. Only the anti-HNE-RSA antibody titre increased significantly with age (p = 0.052) in diabetic animals, while no titres increased significantly in non-diabetic animals. A major factor which correlated with the development of these antibodies was diabetes duration: this was significant (p = 0.032) for anti-HNE-RSA antibody titre and slightly significant (p = 0.05) for anti-MDA-RSA antibody titre. Thus, chronic hyperglycaemia is probably fundamental in the increase of oxidative stress. There is correlation between anti-aldehyde-RSA antibody titres and the corresponding aldehyde-related collagen-linked fluorescence: modified collagen may play a part in the observed immune response. Our data indicate a stronger immune response of diabetic rats against proteins modified by lipoperoxidative aldehydes and oxygen free radicals, and they support the hypothesis of increased oxidative damage in diabetes. [Diabetologia (1998) 41: 265–270]

Diabetes impairs the enzymatic disposal of 4-hydroxynonenal in rat liver.

This study assesses whether the HNE accumulation we formerly observed in liver microsomes and mitochondria of BB/Wor diabetic rats depends on an increased rate of lipoperoxidation or on impairment of enzymatic removal. There are three main HNE metabolizing enzymes: glutathione-S-transferase (GST), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH). In this study we show that GST and ALDH activities are reduced in liver microsomes and mitochondria of diabetic rats; in contrast, ADH activity remains unchanged. The role of each enzyme in HNE removal was evaluated by using enzymatic inhibitors. The roles of both GST and ALDH were markedly reduced in diabetic rats, while ADH-mediated consumption was significantly increased. However, the higher level of lipohydroperoxides in diabetic liver indicated more marked lipoperoxidation. We therefore think that HNE accumulation in diabetic liver may depend on both mechanisms: increased lipoperoxidation and decreased enzymatic removal. We suggest that glycoxidation and/or hyperglycemic pseudohypoxia may be involved in the enzymatic impairment observed. Moreover, since HNE exerts toxic effects on enzymes, HNE accumulation, deficiency of HNE removal, and production of reactive oxygen species can generate vicious circles able to amplify the damage.

Protein oxidation in hemodialysis and kidney transplantation

Oxidative damage of plasma proteins determined with the markers carbonyl group (CG) content and thiobarbituric acid-reactive substances (TBARS) was studied in 13 hemodialyzed and eight kidney-transplanted patients. The level of CGs was 38% higher in hemodialysis (HD) patients (1.49 +/- 0.05 nmol/mg protein) than in the healthy subjects (1.08 +/- 0.03 nmol/mg protein); the TBARS level was also higher in HD patients than in the control group (2.64 +/- 0.15 v 1.81 +/- 0.09 nmol/mL, P < .001). These data confirm that in end-stage renal failure, an increased oxidative stress is present and is able to induce protein damage. After transplantation, the CG content in protein was reduced (1.34 +/- 0.08 nmol/mg protein), but it was not significantly different from the level in the HD group. The failure to return to the normal range suggests that an impaired redox status is maintained, resulting in a sustained elevation of CG. Conversely, the level of TBARS in transplanted patients (1.99 +/- 0.22 nmol/mL) was not significantly different from that in the control group (1.81 +/- 0.09), suggesting that lipoperoxidation may be inhibited. These results may be explained by the different turnover rates of the molecules and by the distinct origin of the two markers, resulting from the damage of proteins or lipids. Thus, lipoperoxidation would produce rapidly removable molecules, whereas protein oxidation damage would tend to accumulate. However, the significant correlation found between CGs and TBARS indicates that a common cause (oxidative stress) binds the two markers of damage.

Glutathione depletion induces apoptosis of rat hepatocytes through activation of protein kinase C novel isoforms and dependent increase in AP-1 nuclear binding

Treatment of isolated rat hepatocytes with the glutathione depleting agents L-buthionine-S,R-sulfoximine or diethylmaleate reproduced various cellular conditions of glutathione depletion, from moderate to severe, similar to those occurring in a wide spectrum of human liver diseases. To evaluate molecular changes and possible cellular dysfunction and damage consequent to a pathophysiologic level of GSH depletion, the effects of this condition on protein kinase C (PKC) isoforms were investigated, since these are involved in the intracellular specific regulatory processes and are potentially sensitive to redox changes. Moreover, a moderate perturbation of cellular redox state was found to activate novel PKC isoforms, and a clear relationship was shown between novel kinase activation and nuclear binding of the redox-sensitive transcription factor, activator protein-1 (AP-1). Apoptotic death of a significant number of cells, confirmed in terms of internucleosomal DNA fragmentation was a possible effect of these molecular reactions, and was triggered by a condition of glutathione depletion usually detected in human liver diseases. Finally, the inhibition of novel PKC enzymatic activity in cells co-treated with rottlerin, a selective novel kinase inhibitor, prevented glutathione-dependent novel PKC up-regulation, markedly moderated AP-1 activation, and protected cells against apoptotic death. Taken together, these findings indicate the existence of an apoptotic pathway dependent on glutathione depletion, which occurs through the up-regulation of novel PKCs and AP-1.

PKC delta and NADPH oxidase in AGE-induced neuronal death.

Advanced glycation end product (AGE) accumulation in brain is believed to contribute to neuronal death in several neurodegenerative diseases. Neurons exposed to AGEs undergo oxidative stress, but the molecular mechanisms able to induce ROS generation and cell death are not yet clear. In this work, we exposed SH-SY5Y neuroblastoma cells to glycated albumin, as a model of AGE-modified protein, and we observed that cells differentiated by retinoic acid died after AGE exposure, through anion superoxide and peroxide generation, while undifferentiated cells resulted resistant. Retinoic acid induced marked increase in p47phox expression and in catalytic activity of PKC delta: the upregulation of a pathway involving NADPH oxidase and PKC delta is likely to be responsible for neuronal susceptibility to AGE. This hypothesis is confirmed by the fact that pre-treatments of differentiated cells with DPI, an inhibitor of NADPH oxidase, or with rottlerin, an inhibitor of PKC delta, were able to prevent AGE-induced neuronal death.