In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

New insights into cancer cell-specific biological pathways are urgently needed to promote development of rationally targeted therapeutics. Reactive oxygen species (ROS) and their role in cancer cell response to growth factor signaling and hypoxia are emerging as verdant areas of exploration on the road to discovering cancer's Achilles heel. One of the distinguishing and near-universal hallmarks of cancer growth is hypoxia. Unregulated cellular proliferation leads to formation of cellular masses that extend beyond the resting vasculature, resulting in oxygen and nutrient deprivation. The resulting hypoxia triggers a number of critical adaptations that enable cancer cell survival, including apoptosis suppression, altered glucose metabolism, and an angiogenic phenotype. Ironically, recent investigations suggest that oxygen depletion stimulates mitochondria to elaborate increased ROS, with subsequent activation of signaling pathways, such as hypoxia inducible factor 1alpha, that promote cancer cell survival and tumor growth. Because mitochondria are key organelles involved in chemotherapy-induced apoptosis induction, the relationship between mitochondria, ROS signaling, and activation of survival pathways under hypoxic conditions has been the subject of increased study. Insights into mechanisms involved in ROS signaling may offer novel avenues to facilitate discovery of cancer-specific therapies. Preclinical and clinical evaluation of agents that modify ROS signaling in cancer offers a novel avenue for intervention. This review will cover recent work in ROS-mediated signaling in cancer cells and its potential as a target for developmental therapeutics.

/pdf/reactive-oxygen-species-a-breath-of-life-or-death-41pgxu2yqb.pdf

Reactive Oxygen Species: A Breath of Life or Death?

The journal of pharmacology and experimental therapeutics

The generation of reactive oxygen species (ROS) and an altered redox status are common biochemical aspects in cancer cells. ROS can react with the polyunsaturated fatty acids of lipid membranes and induce lipid peroxidation. The end products of lipid peroxidation, 4-hydroxynonenal (HNE), have been considered to be a second messenger of oxidative stress. Beyond ROS involvement in carcinogenesis, increased ROS level can inhibit tumor cell growth. Indeed, in tumors in advanced stages, a further increase of oxidative stress, such as that occurs when using several anticancer drugs and radiation therapy, can overcome the antioxidant defenses of cancer cells and drive them to apoptosis. High concentrations of HNE can also induce apoptosis in cancer cells. However, some cells escape the apoptosis induced by chemical or radiation therapy through the adaptation to intrinsic oxidative stress which confers drug resistance. This paper is focused on recent advances in the studies of the relation between oxidative stress, lipid peroxidation products, and cancer progression with particular attention to the pro-oxidant anticancer agents and the drug-resistant mechanisms, which could be modulated to obtain a better response to cancer therapy.

/pdf/oxidative-stress-and-lipid-peroxidation-products-in-cancer-4lkjscnwon.pdf

Oxidative stress and lipid peroxidation products in cancer progression and therapy.

Background—Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic β2-adrenergic receptor (β2AR) from canonical stimulatory G-protein–activated cardiostimulant to inhibitory G-protein–activated cardiodepressant pathways. Methods and Results—We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via Gi inactivation by pertussis toxin pretreatment. β2AR number and functional responses were greater...

High Levels of Circulating Epinephrine Trigger Apical Cardiodepression in a β2-Adrenergic Receptor/Gi–Dependent Manner A New Model of Takotsubo Cardiomyopathy

Objectives
Diabetic cardiomyopathy (DCM), characterized by myocardial structural and functional changes, is an independent cardiomyopathy that develops in diabetic individuals. The present study was sought to investigate the effect of curcumin on modulating DCM and the mechanisms involved.

/pdf/curcumin-alleviates-diabetic-cardiomyopathy-in-experimental-13ppar5bi2.pdf

Curcumin Alleviates Diabetic Cardiomyopathy in Experimental Diabetic Rats

Aim: The present study was designed to determine the possible pathway underlying the enhancement of apoptosis induced by the combined use of arsenic trioxide (As 2 O 3 ) and ascorbic acid (AA).
 Methods: The level of intracellular reactive oxygen species (ROS) was detected by means of flow cytometry analysis with an oxidation-sensitive fluorescent probe (6-carboxy-2',7'dichlorodihydrofluorescein diacetate) uploading. The activity of glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were detected by biochemical methods. The mitochondrial membrane potential was measured by flow cytometry analysis with rhodamine 123 staining. Bcl-2, Bax, and p 17 subunit of caspase-3 were analyzed using the Western blot method. The apoptosis rate was determined by flow cytometry with annexin-V/propidium iodide staining.
 Results: Compared with As 2 O 3 (2.0 μmol/L) treated alone, As 2 O 3 (2.0 μmol/L) in combination with AA (100 μmol/L) decreased intracellular GSH content from 101.30±5.76 to 81.91±3.12 mg/g protein, and increased ROS level from 127.61±5.12 to 152.60±5.88, which was represented by the 2, 7-dichlorofluorescein intensity. The loss of mitochondria membrane potential was increased from 1269.97±36.11 to 1540.52±52.63, which was presented by fluorescence intensity. The p17 subunit of caspase-3 expression was increased approximately 2-fold. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax were equal to that of As 2 O 3 treated alone ( P >0.05). When the ROS scavenger, N/em>-acetyl- L/em>-cysteine, was added to As 2 O 3 and AA combined treatment group, the apoptosis rate decreased from 15.60 %±1.14% to 9.48%±0.67%, and the ROS level decreased from 152.60±5.88 to 102.77±10.25.
 Conclusion: AA potentiated As 2 O 3 -induced apoptosis through the oxidative pathway by increasing the ROS level. This may be the result of depleting intracellular GSH. It may influence the downstream cascade following ROS, including mitochondria depolarization and caspase-3 activation. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax influenced by As 2 O 3 was not found to be potentiated by AA.

/pdf/role-of-oxidative-stress-in-the-apoptosis-of-hepatocellular-ylwjmhr35d.pdf

Jizhou Xiang

Papers

Curcumin Alleviates Diabetic Cardiomyopathy in Experimental Diabetic Rats

Role of oxidative stress in the apoptosis of hepatocellular carcinoma induced by combination of arsenic trioxide and ascorbic acid.

Protective effect of resveratrol derived from Polygonum cuspidatum and its liposomal form on nigral cells in parkinsonian rats.

Erythropoietin protects PC12 cells from β-amyloid25–35-induced apoptosis via PI3K/Akt signaling pathway

Protective effect of erythropoietin on β-amyloid-induced PC12 cell death through antioxidant mechanisms