Free radicals, metals and antioxidants in oxidative stress-induced cancer

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Fish bioaccumulation and biomarkers in environmental risk assessment: a review

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

The accidental discovery of the anticancer properties of cisplatin and its clinical introduction in the 1970s represent a major landmark in the history of successful anticancer drugs. Although carboplatin--a second-generation analogue that is safer but shows a similar spectrum of activity to cisplatin--was introduced in the 1980s, the pace of further improvements slowed for many years. However, in the past several years interest in platinum drugs has increased. Key developments include the elucidation of mechanisms of tumour resistance to these drugs, the introduction of new platinum-based agents (oxaliplatin, satraplatin and picoplatin), and clinical combination studies using platinum drugs with resistance modulators or new molecularly targeted drugs.

The resurgence of platinum-based cancer chemotherapy

The glutathione S-transferases (GST) represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C, synthetase, are encoded by single genes and both have arisen separately from the soluble GST. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress.A description of the mechanisms of transcriptional and posttranscriptional regulat...

The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.

The Nrf2 regulatory network provides an interface between redox and intermediary metabolism

Increases in the intracellular levels of reactive oxygen species (ROS), frequently referred to as oxidative stress, represents a potentially toxic insult which if not counteracted will lead to membrane dysfunction, DNA damage and inactivation of proteins. Chronic oxidative stress has numerous pathological consequences including cancer, arthritis and neurodegenerative disease. Glutathione-associated metabolism is a major mechanism for cellular protection against agents which generate oxidative stress. It is becoming increasingly apparent that the glutathione tripeptide is central to a complex multifaceted detoxification system, where there is substantial inter-dependence between separate component members. Glutathione participates in detoxification at several different levels, and may scavenge free radicals, reduce peroxides or be conjugated with electrophilic compounds. Thus, glutathione provides the cell with multiple defences not only against ROS but also against their toxic products. This article discusses how glutathione biosynthesis, glutathione peroxidases, glutathione S-transferases and glutathione S-conjugate efflux pumps function in an integrated fashion to allow cellular adaption to oxidative stress. Co-ordination of this response is achieved, at least in part, through the antioxidant responsive element (ARE) which is found in the promoters of many of the genes that are inducible by oxidative and chemical stress. Transcriptional activation through this enhancer appears to be mediated by basic leucine zipper transcription factors such as Nrf and small Maf proteins. The nature of the intracellular sensor(s) for ROS and thiol-active chemicals which induce genes through the ARE is described. Gene activation through the ARE appears to account for the enhanced antioxidant and detoxification capacity of normal cells effected by many cancer chemopreventive agents. In certain instances it may also account for acquired resistance of tumours to cancer chemotherapeutic drugs. It is therefore clear that determining the mechanisms involved in regulation of ARE-driven gene expression has enormous medical implications.

Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress

Glutathione S‐transferases

https://www.jbc.org/content/285/29/22576.full.pdf

p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription.

John D. Hayes

Papers

The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance.

The Nrf2 regulatory network provides an interface between redox and intermediary metabolism

Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress

Glutathione S‐transferases

p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription.