Nicola Traverso

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.

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.

The increased activity of BACE1 correlates with oxidative stress in Alzheimer's disease.

We evaluated expression, protein levels and activity of the Beta-site cleaving enzyme (BACE1) as well as the amount of products of lipid peroxidation in frontal cortex of three groups of cases: sporadic Alzheimers disease (AD); control subjects (CTR); cognitively normal subjects with abundant amyloid plaques (NA). We found a significant increase of BACE1 activity and products of lipid peroxidation in brain tissue of AD cases, with normal gene expression, and non-significant elevation of protein levels. CTR and NA samples showed similar levels of BACE1 activity and oxidative products. BACE1 activity and the amount of oxidative products were significantly correlated in all cases.Moreover, both BACE1 activity and the level of 4-hydroxynonenal were correlated with the amount of Beta-amyloid pyroglutamated 3-42, the more toxic Beta-amyloid peptide that is characteristic of AD. These findings suggest that BACE1 activity reflects the type of ABeta species, rather than the Beta-amyloid plaques load. Hence, the increase of BACE1 activity occurring in sporadic AD is likely the effect, rather the cause, of ABeta accumulation and oxidative stress.

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.

MUTUAL INTERACTION BETWEEN GLYCATION AND OXIDATION DURING NON-ENZYMATIC PROTEIN MODIFICATION

Aging pathogenesis involves non-enzymatic modifications of proteins; protein oxidation, glycation and their interactions have aroused a particular interest. Possible interrelations between oxidation and glycation have been evaluated in vitro: bovine serum albumin was oxidized by gamma-irradiation and then exposed to in vitro glycation. Fluorescence modifications induced by radiolytic oxidation and glycation were similar and tended to be additive. Both non-enzymatic processes provoked a loss of free sulfhydryl groups and a strong increment of protein carbonyl content: this supports that glycation can act through oxidative mechanisms. The observed rearrangement of amino groups after irradiation could predispose proteins to glycation attacks. Protein peroxides generated during irradiation appear able to give birth to further protein modifications leading to the generation of carbonyl groups and to interact with monosaccharides, probably stimulating their autoxidation and in turn glycative protein damage. Glycation increases the oxidation-mediated structural damage revealed by SDS-PAGE. Therefore our data support the hypothesis of mutual enhancement between oxidation and glycation of proteins and suggest possible molecular mechanisms of interactions.

Levels of carbonyl groups in plasma proteins of type 2 diabetes mellitus subjects.

Abstract Carbonyl groups result from protein oxidation and their level in tissues and plasma is a relatively stable marker of oxidative damage. Carbonyl content of plasma proteins in 43 type 2 diabetic subjects, 30–87 years of age (25 males and 18 females) and in 20 age-matched healthy controls (31–89 years of age, 12 males and 8 females) was evaluated with 2,4-dinitro-phenyl-hydrazine method. In both groups, lipids, tocopherols (HPLC) and glycated hemoglobin (HPLC) were studied.Fasting blood glucose, glycated hemoglobin and lipids were significantly higher in the diabetic group; carbonyl content and α-tocopherol were slightly, but not significantly higher in the diabetic group (1.06 ± 0.03 vs. 0.97 ± 0.04 nmol/mg protein, 27.07 ± 2.82 vs. 31.55 ± 2.11 μmol/l, respectively). Significant relationships between age and lipids, α-tocopherol and proteins were found in controls, but not in diabetics. Alpha-tocopherol correlated with lipids in both groups; glycated hemoglobin, a marker of glycemic control, was related to lipids, α-tocopherol and protein carbonyl groups in diabetics, while only the correlation with carbonyls was found in controls.These results suggest that impaired glycemic control is connected to protein oxidation. Glycation cascade also releases free radicals, becoming responsible for further oxidative attacks. In conclusion, increased oxidative stress, if any, in the diabetic group, is doubtlessly induced by hyperglycemia, and the tocopherols are not seriously affected by a worsening of the metabolic control.

The Nrf2/HO-1 Axis in Cancer Cell Growth and Chemoresistance

The transcription factor, nuclear factor erythroid 2 p45-related factor 2 (Nrf2), acts as a sensor of oxidative or electrophilic stresses and plays a pivotal role in redox homeostasis. Oxidative or electrophilic agents cause a conformational change in the Nrf2 inhibitory protein Keap1 inducing the nuclear translocation of the transcription factor which, through its binding to the antioxidant/electrophilic response element (ARE/EpRE), regulates the expression of antioxidant and detoxifying genes such as heme oxygenase 1 (HO-1). Nrf2 and HO-1 are frequently upregulated in different types of tumours and correlate with tumour progression, aggressiveness, resistance to therapy, and poor prognosis. This review focuses on the Nrf2/HO-1 stress response mechanism as a promising target for anticancer treatment which is able to overcome resistance to therapies.

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.

Redox Homeostasis and Cellular Antioxidant Systems: Crucial Players in Cancer Growth and Therapy

Reactive oxygen species (ROS) and their products are components of cell signaling pathways and play important roles in cellular physiology and pathophysiology. Under physiological conditions, cells control ROS levels by the use of scavenging systems such as superoxide dismutases, peroxiredoxins, and glutathione that balance ROS generation and elimination. Under oxidative stress conditions, excessive ROS can damage cellular proteins, lipids, and DNA, leading to cell damage that may contribute to carcinogenesis. Several studies have shown that cancer cells display an adaptive response to oxidative stress by increasing expression of antioxidant enzymes and molecules. As a double-edged sword, ROS influence signaling pathways determining beneficial or detrimental outcomes in cancer therapy. In this review, we address the role of redox homeostasis in cancer growth and therapy and examine the current literature regarding the redox regulatory systems that become upregulated in cancer and their role in promoting tumor progression and resistance to chemotherapy.