Experimental therapeutics: targeting the redox Achilles heel of cancer

Reactive oxygen species (ROS) have recently emerged as promising targets for anticancer drug discovery. Constitutively elevated levels of cellular oxidative stress and dependence on mitogenic and anti-apoptotic ROS signaling represent a specific vulnerability of malignant cells that can be selectively targeted by novel pro- and antioxidant redox chemotherapeutics. This review discusses small-molecule anticancer redox drugs currently in various phases of preclinical and clinical development that are characterized by their unique mechanism of action, including small-molecule superoxide dismutase and catalase mimetics, bioreductively activated pro-oxidant redox catalysts, metal-based pro-oxidants, hypoxia-selective free radical precursors, and specific antagonists of the cancer cell antioxidant glutathione or thioredoxin redox systems. Based on ongoing redox biomarker discovery and validation, future redox phenotyping and genotyping may guide the selection of novel redox chemotherapeutics that efficiently target the redox Achilles heel of the individual tumor.     

Animal models of human disease in drug safety assessment

Animal models of human disease have been widely used in drug discovery, but they are rarely utilized in toxicological research and screening (except for transgenic models in carcinogenicity testing). Although genetic and/or acquired pathophysiological alterations associated with a particular disease may greatly exacerbate toxic responses to drugs in certain patient subsets, these pre-existing pathological conditions are usually not considered in preclinical safety assessment. Examples of disease-related determinants of susceptibility include disruption of the cytokine network in pro-inflammatory conditions, mitochondrial alterations and oxidative stress in certain neurodegenerative diseases, altered antioxidant defense in certain viral infections, and altered gene expression and mitochondrial dysfunction in type 2 diabetes. Hence, if cellular stress caused by drugs or metabolites and the disease-related effects are superimposed, then an individual can become sensitized to potential drug toxicity. Animal models of modest inflammation indeed can potentiate the toxicity of certain drugs. Similarly, rodent models of type 2 diabetes predispose the animals to hepatotoxic effects of thiazolidinediones antidiabetics. In conclusion, it is suggested that tailor-made and simplified models be adopted and increasingly used, in spite of clear limitations, as optimal substrates for satellite toxicity studies to facilitate candidate selection, help predict rare and unexpected toxicity, and identify new biomarkers.     

The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.     

The interplay of glutathione-related processes in antioxidant defense

This review summarizes current knowledge on glutathione (GSH) associated cellular processes that play a central role in defense against oxidative stress. GSH itself is a critical factor in maintaining the cellular redox balance and has been demonstrated to be involved in regulation of cell signalling and repair pathways. Enhanced expression of various enzymes involved in GSH metabolism, including glutathione peroxidases, γ-glutamyl cysteinyl synthetase (γ-GCS), glutathione S-transferases (GST) and membrane proteins belonging to the ATP-binding cassette family, such as the multidrug resistance associated protein, have all been demonstrated to play a prominent role in cellular resistance towards oxidative stress. This review stresses the fact that a co-ordinate interplay between these systems is essential for efficient protection against oxidative stress.     

Effects of ozone oxidative preconditioning on different hepatic biomarkers of oxidative stress in endotoxic shock in mice

In endotoxic shock, variations are known to occur in different biochemical parameters of oxidative stress. Ozone oxidative preconditioning (OOP) is a good candidate to restore the redox balance on different tissue. This investigation examined the effect of OOP on different biomarkers of oxidative stress in hepatic tissue of mice treated with lipopolysaccharide (LPS). LPS doses of 30?mg/kg were administered intraperitoneally (i.p.) and pretreatment with ozone/oxygen mixture (OOM) was applied i.p. at 0.2, 0.4, and 1.2?mg/kg once daily during 5 days before LPS injection. The mice were euthanized under ether atmosphere at different times, 1 and 24?h after LPS injection. Hepatic tissue from all animals was taken for biochemical determinations of oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) content and activity of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST). The results demonstrated that OOP reduces levels of TBARS content and increases the activity of GPx in hepatic tissue. In conclusion, OOP was able to recover the redox balance and in this way to protect the animals against the oxidative damage induced by endotoxemia.     

Oxidative Stress and Aging - Second International Conference. Technologies for assessment and intervention strategies. 2-5 April 2001, Maui, USA

The study of oxidative contributions to aging has reached sufficient maturity to support the development of interventional strategies designed to forestall or reverse protein cross-linking, oxidation of DNA and lipids, and mitochondrial senescence associated with chronic pathology and aging. Catalytic antioxidants, including combined superoxide dismutase (SOD) and catalase mimics, extend the lifespan of oxidatively compromised animals such as Mn-SOD knockout mice, and will be entering the clinic for radiation-induced dermatitis. Substituted phenacylthiozolium compounds that slow the formation and break extant protein cross-linkages formed via Maillard reactions, restoring vascular compliance in aged animals, and are showing efficacy in clinical trials. Non-feminizing estrogen analogs (eg, 17- alpha estradiol) block cytotoxicity in a host of oxidative stress models and moderate neuronal loss in MCAO stroke models. Efficacy of the polycyclic phenols is synergistically amplified by glutathione supplementation, suggesting that the two function as a redox couple analogous to vitamin E and ascorbate. Finally, discovery of novel small molecules designed to stabilize mitochondrial function during Ca(2+)-induced oxidative stress, such as that occurring during stroke or myocardial ischemia reperfusion, will be accelerated by a proprietary fluorescence resonance energy transfer assay developed at MitoKor. Maintaining mitochondrial function under these circumstances will improve cellular bioenergetic and oxidative status, and hence moderate secondary necrosis and apoptosis.     

Effects of ozone oxidative preconditioning on different hepatic biomarkers of oxidative stress in endotoxic shock in mice

In endotoxic shock, variations are known to occur in different biochemical parameters of oxidative stress. Ozone oxidative preconditioning (OOP) is a good candidate to restore the redox balance on different tissue. This investigation examined the effect of OOP on different biomarkers of oxidative stress in hepatic tissue of mice treated with lipopolysaccharide (LPS). LPS doses of 30?mg/kg were administered intraperitoneally (i.p.) and pretreatment with ozone/oxygen mixture (OOM) was applied i.p. at 0.2, 0.4, and 1.2?mg/kg once daily during 5 days before LPS injection. The mice were euthanized under ether atmosphere at different times, 1 and 24?h after LPS injection. Hepatic tissue from all animals was taken for biochemical determinations of oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) content and activity of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST). The results demonstrated that OOP reduces levels of TBARS content and increases the activity of GPx in hepatic tissue. In conclusion, OOP was able to recover the redox balance and in this way to protect the animals against the oxidative damage induced by endotoxemia.     

Oxidative stress,redox signalling and endothelial dysfunction in ageing-related neurodegenerative diseases: a role of NADPH oxidase 2

Chronic oxidative stress and oxidative damage of the cerebral microvasculature and brain cells has become one of the most convincing theories in neurodegenerative pathology. Controlled oxidative metabolism and redox signalling in the central nervous system are crucial for maintaining brain function; however, excessive production of reactive oxygen species and enhanced redox signalling damage neurons. While several enzymes and metabolic processes can generate intracellular reactive oxygen species in the brain, recently an O₂⁻-generating enzyme, NADPH oxidase 2 (Nox2), has emerged as a major source of oxidative stress in ageing-related vascular endothelial dysfunction and neurodegenerative diseases. The currently available inhibitors of Nox2 are not specific, and general antioxidant therapy is not effective in the clinic; therefore, insights into the mechanism of Nox2 activation and its signalling pathways are needed for the discovery of novel drug targets to prevent or treat these neurodegenerative diseases. This review summarizes the recent developments in understanding the mechanisms of Nox2 activation and redox-sensitive signalling pathways and biomarkers involved in the pathophysiology of the most common neurodegenerative diseases, such as ageing-related mild cognitive impairment, Alzheimer’s disease and Parkinson’s disease.     

Thiols and the chemoprevention of cancer

Thiols such as glutathione interfere with the complex carcinogenic process. Under stress conditions, they scavenge harmful molecules: Glutathione conjugation of electrophilic carcinogens may prevent tumor initiation, and reduced thiols may defend against oxidative stress. Thus, associated chemopreventive strategies involve enhancement of antioxidant or conjugating capacity by increasing the levels of, particularly, glutathione through precursor application or synthesis stimulation and by inducing the corresponding enzymes. The antioxidant potential of thiols is, however, a part of a more general capacity to regulate redox status even in the absence of unequivocal stress conditions. Redox status controls the activities of various cellular signalling proteins through oxidation or reduction of particular sensor structures that are also mostly thiols. The development of feasible chemotherapeutic strategies on the basis of this complex system of redox-sensitive messenger proteins is a goal in ongoing and future research.     

Cellular redox pathways as a therapeutic target in the treatment of cancer

The vulnerability of some cancer cells to oxidative signals is a therapeutic target for the rational design of new anticancer agents. In addition to their well characterized effects on cell division, many cytotoxic anticancer agents can induce oxidative stress by modulating levels of reactive oxygen species (ROS) such as the superoxide anion radical, hydrogen peroxide and hydroxyl radicals. Tumour cells are particularly sensitive to oxidative stress as they typically have persistently higher levels of ROS than normal cells due to the dysregulation of redox balance that develops in cancer cells in response to increased intracellular production of ROS or depletion of antioxidant proteins. In addition, excess ROS levels potentially contribute to oncogenesis by the mediation of oxidative DNA damage. There are several anticancer agents in development that target cellular redox regulation. The overall cellular redox state is regulated by three systems that modulate cellular redox status by counteracting free radicals and ROS, or by reversing the formation of disulfides; two of these are dependent on glutathione and the third on thioredoxin. Drugs targeting S-glutathionylation have direct anticancer effects via cell signalling pathways and inhibition of DNA repair, and have an impact on a wide range of signalling pathways. Of these agents, NOV-002 and canfosfamide have been assessed in phase III trials, while a number of others are undergoing evaluation in early phase clinical trials. Alternatively, agents including PX-12, dimesna and motexafin gadolinium are being developed to target thioredoxin, which is overexpressed in many human tumours, and this overexpression is associated with aggressive tumour growth and poorer clinical outcomes. Finally, arsenic derivatives have demonstrated antitumour activity including antiproliferative and apoptogenic effects on cancer cells by pro-oxidant mechanisms, and the induction of high levels of oxidative stress and apoptosis by an as yet undefined mechanism. In this article we review anticancer drugs currently in development that target cellular redox activity to treat cancer.     

Antioxidant pharmacological therapies for COPD

Increased oxidative stress occurs in the lungs and systemically in COPD, which plays a role in many of the pathogenic mechanisms in COPD. Hence, targeting local lung and systemic oxidative stress with agents that modulate the antioxidants/redox system or boost endogenous antioxidants would be a useful therapeutic approach in COPD. Thiol antioxidants (N-acetyl-l-cysteine [NAC] and N-acystelyn, carbocysteine, erdosteine, and fudosteine) have been used to increase lung thiol content. Modulation of cigarette smoke (CS) induced oxidative stress and its consequent cellular changes have also been reported to be effected by synthetic molecules, such as spin traps (α-phenyl-N-tert-butyl nitrone), catalytic antioxidants (superoxide dismutase [ECSOD] mimetics), porphyrins, and lipid peroxidation and protein carbonylation blockers/inhibitors (edaravone and lazaroids/tirilazad). Preclinical and clinical trials have shown that these antioxidants can reduce oxidative stress, affect redox and glutathione biosynthesis genes, and proinflammatory gene expression. In this review the approaches to enhance lung antioxidants in COPD and the potential beneficial effects of antioxidant therapy on the course of the disease are discussed.     

Ariadne's ChemEffect and Pathway Studio knowledge base

Importance of the field: Drug discovery and development is a very complex and costly process. Understanding the detailed molecular mechanisms of a disease and drug actions can make it more efficient not only for new target discovery but also for lead prioritization, drug repositioning and development of biomarkers for drug efficacy and safety. Access to formalized knowledge about functions of proteins and small molecules is crucial for rationalization of the drug development process, and scientific publications are the main source of this knowledge. Protein knowledge networks capturing protein functions, protein–protein relations and organization of proteins in complex cellular sub-systems are making their way into modern drug discovery. Chemical networks representing multiple aspects of chemical functional information integrated into a protein systems biology network is even more advanced and promising paradigm.

Areas covered in this review: This review describes utilization of literature-derived protein and chemical functional knowledge bases in drug development.

What the reader will gain: Readers will gain an understanding of how integrated protein and chemical knowledge networks can be used for understanding and building the models of cellular events, disease mechanisms, and drug actions, finding biomarkers of drug efficacy and safety, as well as interpretation of high-throughput gene expression, proteomic and metabolomic experiments.

Take home message: Integrated literature-derived protein and chemical knowledge bases can rationalize many aspects of drug development process including drug repositioning and biomarker design.     

In vitro assays and techniques utilized in anticancer drug discovery

Development of a cancer is a multistep process and six major hallmarks of cancer that are known to control malignant transformation have been described. Anticancer drug development is a tedious process, requiring a number of in vitro, in vivo and clinical studies. In vitro assays provide an initial platform for cancer drug discovery approaches. A wide range of in vitro assays/techniques have been developed to evaluate each hallmark feature of cancer and selection of a particular in vitro assay or technique mainly depends on the specific research question (s) to be examined. In the present review, we have described some commonly utilized in vitro assays and techniques used to examine cell viability/proliferation, apoptosis, cellular senescence, invasion and migration, oxidative stress and antioxidant effects, gene and protein expression, angiogenesis and genomic alterations in cancer drug discovery. Additionally, uses of modern techniques such as high throughput screening, high content screening and reporter gene assays in cancer drug discovery have also been described.     

GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress

The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC–MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.