Reactive oxygen species‐provoked mitochondria‐dependent cell death during ageing of elm (Ulmus pumila L.) seeds

Previous studies have shown that controlled deterioration treatment (CDT) induces programmed cell death in elm (Ulmus pumila L.) seeds, which undergo certain fundamental processes that are comparable to apoptosis in animals. In this study, the essential characteristics of mitochondrial physiology in elm seeds during CDT were identified by cellular ultrastructural analysis, whole‐body optical imaging, Western blotting and semi‐quantitative RT–PCR. The alteration in mitochondrial morphology was an early event during CDT, as indicated by progressive dynamic mitochondrial changes and rupture of the mitochondrial outer membrane; loss of mitochondrial transmembrane potential (Δψ_m) ensued, and mitochondrial ATP levels decreased. The mitochondrial permeability transition pore inhibitor cyclosporine A effectively suppressed these changes during ageing. The in situ localization of production of reactive oxygen species (ROS), and evaluation of the expression of voltage‐dependent anion‐selective channel and cyclophilin D indicated that the levels of mitochondrial permeability transition pore components were positively correlated with ROS production, leading to an imbalance of the cellular redox potential and ultimately to programmed cell death. Pre‐incubation with ascorbic acid slowed loss of mitochondrial Δψ_m, and decreased the effect of CDT on seed viability. However, there were no significant changes in multiple antioxidant elements or chaperones in the mitochondria during early stages of ageing. Our results indicate that CDT induces dynamic changes in mitochondrial physiology via increased ROS production, ultimately resulting in an irreversible loss of seed viability.     

Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell^?1 s^?1, and 5 and 35 amol cell^?1 s^?1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies.     

ON THE PARADIGM OF ALTRUISTIC SUICIDE IN THE UNICELLULAR WORLD

Altruistic suicide is best known in the context of programmed cell death (PCD) in multicellular individuals, which is understood as an adaptive process that contributes to the development and functionality of the organism. After the realization that PCD‐like processes can also be induced in single‐celled lineages, the paradigm of altruistic cell death has been extended to include these active cell death processes in unicellular organisms. Here, we critically evaluate the current conceptual framework and the experimental data used to support the notion of altruistic suicide in unicellular lineages, and propose new perspectives. We argue that importing the paradigm of altruistic cell death from multicellular organisms to explain active death in unicellular lineages has the potential to limit the types of questions we ask, thus biasing our understanding of the nature, origin, and maintenance of this trait. We also emphasize the need to distinguish between the benefits and the adaptive role of a trait. Lastly, we provide an alternative framework that allows for the possibility that active death in single‐celled organisms is a maladaptive trait maintained as a byproduct of selection on pro‐survival functions, but that could—under conditions in which kin/group selection can act—be co‐opted into an altruistic trait.     

Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin‐induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL‐60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL‐60 cells which were also more sensitive and contained autophagic vesicles. Studies in Rho⁰ cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti‐proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy, and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance. J. Cell. Biochem. 111: 574–584, 2010. © 2010 Wiley‐Liss, Inc.     

Cyclosporin A does not protect the disruption of the inner mitochondrial membrane potential induced by potassium ionophores in intact K562 cells

Mitochondrial dysfunction has been widely associated with programmed cell death. Studies of intact cells are important for the understanding of the process of cell death and its relation to mitochondrial physiology. Using cytofluorometric approaches we studied the mitochondrial behavior in an erythroleukemic cell line. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), potassium exchanger (nigericin), potassium ionophore (valinomycin), Na+K+-ATPase inhibitor (ouabain) and mitochondrial permeability transition pore inhibitor (cyclosporin A) were evaluated. Cyclosporin A (CSA) was very effective in attenuating the disruption of inner mitochondrial membrane potential induced by CCCP. However, CSA failed to protect the loss of inner mitochondrial membrane potential induced by potassium intracellular flux manipulation. Our findings suggest that mitochondrial cyclophilin is not involved in the cell events mediated by deregulation of potassium flux, underlining the need for further studies in intact tumor cells for a better understanding of the involvement of mitochondria physiology in cell death events.     

In EXOG‐depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain

Depletion of mitochondrial endo/exonuclease G‐like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease G‐like‐depleted cells, and this is marked by a decrease in mitochondrial reserve capacity. Neurohormonal stimulation with phenylephrine (PE) did not have an additive effect on the hypertrophic response induced by endo/exonuclease G‐like depletion. Interestingly, PE‐induced atrial natriuretic peptide (ANP) gene expression was completely abolished in endo/exonuclease G‐like‐depleted cells, suggesting a reverse signaling function of endo/exonuclease G‐like. Endo/exonuclease G‐like depletion initially resulted in increased mitochondrial OCR, but this declined upon PE stimulation. In particular, the reserve capacity of the mitochondrial respiratory chain and maximal respiration were the first indicators of perturbations in mitochondrial respiration, and these marked the subsequent decline in mitochondrial function. Although pathological stimulation accelerated these processes, prolonged EXOG depletion also resulted in a decline in mitochondrial function. At early stages of endo/exonuclease G‐like depletion, mitochondrial ROS production was increased, but this did not affect mitochondrial DNA (mtDNA) integrity. After prolonged depletion, ROS levels returned to control values, despite hyperpolarization of the mitochondrial membrane. The mitochondrial dysfunction finally resulted in cell death, which appears to be mainly a form of necrosis. In conclusion, endo/exonuclease G‐like plays an essential role in cardiomyocyte physiology. Loss of endo/exonuclease G‐like results in diminished adaptation to pathological stress. The decline in maximal respiration and reserve capacity is the first sign of mitochondrial dysfunction that determines subsequent cell death.     

Nitric oxide affects plant mitochondrial functionality in vivo

In this report, we show that nitric oxide affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway. The residual respiration depends on the alternative pathway and novel synthesis of alternative oxidase occurs. These modifications are associated with depolarisation of the mitochondrial membrane potential and release of cytochrome c from mitochondria, suggesting a conserved signalling pathway in plants and animals. This signal cascade is triggered at the mitochondrial level and induces about 20% of cell death. In order to achieve a higher level of cell death, the addition of H(2)O(2) is necessary.     

Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus

Dengue virus infection affects millions of people all over the world. Although the clinical manifestations of dengue virus-induced diseases are known, the physiopathological mechanisms involved in deteriorating cellular function are not yet understood. In this study we evaluated for the first time the associations between dengue virus-induced cell death and mitochondrial function in HepG2, a human hepatoma cell line. Dengue virus infection promoted changes in mitochondrial bioenergetics, such as an increase in cellular respiration and a decrease in DeltaPsim. These alterations culminated in a 20% decrease in ATP content and a 15% decrease in the energy charge of virus-infected cells. Additionally, virus-infected cells showed several ultrastructural alterations, including mitochondria swelling and other morphological changes typical of the apoptotic process. The alterations in mitochondrial physiology and energy homeostasis preceded cell death. These results indicate that HepG2 cells infected with dengue virus are under metabolic stress and that mitochondrial dysfunction and alterations in cellular ATP balance may be related to the pathogenesis of dengue virus infection.     

Antiproliferative and cytotoxic effects of purple pitanga (Eugenia uniflora L.) extract on activated hepatic stellate cells

The presence of phenolic compounds in fruit‐ and vegetable‐rich diets has attracted researchers' attention due to their health‐promoting effects. The objective of this study was to evaluate the effects of purple pitanga (Eugenia uniflora L.) extract on cell proliferation, viability, mitochondrial membrane potential, cell death and cell cycle in murine activated hepatic stellate cells (GRX). Cell viability by 3‐(4,5‐dimethylthiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay was significantly decreased on cells treated with 50 and 100 µg ml^?1 of purple pitanga extract for 48 and 72 h, and the percentage of dead cell stained with 7‐amino‐actinomycin D was significantly higher in treated cells. The reduction of cell proliferation was dose dependent, and we also observed alterations on cell cycle progression. At all times studied, GRX cells treated with 50 and 100 µg ml^?1 of purple pitanga showed a significant reduction in cellular mitochondrial content as well as a decrease in mitochondrial membrane potential. Furthermore, our results indicated that purple pitanga extract induces early and late apoptosis/necrosis and necrotic death in GRX cells. This is the first report describing the antiproliferative, cytotoxic and apoptotic activity for E. uniflora fruits in hepatic stellate cells. The present study provides a foundation for the prevention and treatment of liver fibrosis, and more studies will be carried to elucidate this effect. Copyright © 2013 John Wiley & Sons, Ltd.     

Localization of the anti‐cancer peptide EGFR‐lytic hybrid peptide in human pancreatic cancer BxPC‐3 cells by immunocytochemistry

Cationic lytic‐type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti‐cancer epithelial growth factor receptor (EGFR)‐lytic hybrid peptide, a 32‐amino‐acid peptide composed of an EGFR‐binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR‐lytic hybrid peptide in BxPC‐3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR‐lytic peptide compared with non‐treated cells. In confocal laser scanning microscopy, immunostaining of EGFR‐lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR‐lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR‐lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR‐lytic peptide; however, no effect on cell death after EGFR‐lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR‐lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Shifts in growth strategies reflect tradeoffs in cellular economics

The growth rate‐dependent regulation of cell size, ribosomal content, and metabolic efficiency follows a common pattern in unicellular organisms: with increasing growth rates, cell size and ribosomal content increase and a shift to energetically inefficient metabolism takes place. The latter two phenomena are also observed in fast growing tumour cells and cell lines. These patterns suggest a fundamental principle of design. In biology such designs can often be understood as the result of the optimization of fitness. Here we show that in basic models of self‐replicating systems these patterns are the consequence of maximizing the growth rate. Whereas most models of cellular growth consider a part of physiology, for instance only metabolism, the approach presented here integrates several subsystems to a complete self‐replicating system. Such models can yield fundamentally different optimal strategies. In particular, it is shown how the shift in metabolic efficiency originates from a tradeoff between investments in enzyme synthesis and metabolic yields for alternative catabolic pathways. The models elucidate how the optimization of growth by natural selection shapes growth strategies.     

Apoptosis and necrosis mediate skeletal muscle fiber loss in age‐induced mitochondrial enzymatic abnormalities

Sarcopenia, the age‐induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age‐dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age‐dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC‐abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.     

Mitochondria: releasing power for life and unleashing the machineries of death

The mitochondrion has long been known both as a chemical powerplant and as a cellular compartment housing various biosynthetic pathways. However, studies on the function of mitochondria in apoptotic cell death have revealed a versatility and complexity of these organelles previously unsuspected. The mechanisms proposed for mitochondrial involvement in cell death are diverse and highly controversial. In one model, mitochondria are seen as passive containers that can be made to leak out cytotoxic proteins. In other scenarios, however, certain more or less familiar aspects of mitochondrial physiology, such as oxidative phosphorylation, generation of oxygen radicals, dynamic morphological rearrangements, calcium overload, and permeability transition, are proposed to play crucial roles. In this review, we examine a few promising mechanisms that have been gaining attention recently.     

Evidence that a mitochondrial death spiral underlies antagonistic pleiotropy

The antagonistic pleiotropy (AP) theory posits that aging occurs because alleles that are detrimental in older organisms are beneficial to growth early in life and thus are maintained in populations. Although genes of the insulin signaling pathway likely participate in AP, the insulin‐regulated cellular correlates of AP have not been identified. The mitochondrial quality control process called mitochondrial autophagy (mitophagy), which is inhibited by insulin signaling, might represent a cellular correlate of AP. In this view, rapidly growing cells are limited by ATP production; these cells thus actively inhibit mitophagy to maximize mitochondrial ATP production and compete successfully for scarce nutrients. This process maximizes early growth and reproduction, but by permitting the persistence of damaged mitochondria with mitochondrial DNA mutations, becomes detrimental in the longer term. I suggest that as mitochondrial ATP output drops, cells respond by further inhibiting mitophagy, leading to a further decrease in ATP output in a classic death spiral. I suggest that this increasing ATP deficit is communicated by progressive increases in mitochondrial ROS generation, which signals inhibition of mitophagy via ROS‐dependent activation of insulin signaling. This hypothesis clarifies a role for ROS in aging, explains why insulin signaling inhibits autophagy, and why cells become progressively more oxidized during aging with increased levels of insulin signaling and decreased levels of autophagy. I suggest that the mitochondrial death spiral is not an error in cell physiology but rather a rational approach to the problem of enabling successful growth and reproduction in a competitive world of scarce nutrients.     

Mitochondrial c-Jun N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.     

Mitochondrial shape changes: orchestrating cell pathophysiology

Mitochondria are highly dynamic organelles, the location, size and distribution of which are controlled by a family of proteins that modulate mitochondrial fusion and fission. Recent evidence indicates that mitochondrial morphology is crucial for cell physiology, as changes in mitochondrial shape have been linked to neurodegeneration, calcium signalling, lifespan and cell death. Because immune cells contain few mitochondria, these organelles have been considered to have only a marginal role in this physiological context—which is conversely well characterized from the point of view of signalling. Nevertheless, accumulating evidence shows that mitochondrial dynamics have an impact on the migration and activation of immune cells and on the innate immune response. Here, we discuss the roles of mitochondrial dynamics in cell pathophysiology and consider how studying dynamics in the context of the immune system could increase our knowledge about the role of dynamics in key signalling cascades.     

Structure,function, and regulation of mitofusin‐2 in health and disease

Mitochondria are highly dynamic organelles that constantly migrate, fuse, and divide to regulate their shape, size, number, and bioenergetic function. Mitofusins (Mfn1/2), optic atrophy 1 (OPA1), and dynamin‐related protein 1 (Drp1), are key regulators of mitochondrial fusion and fission. Mutations in these molecules are associated with severe neurodegenerative and non‐neurological diseases pointing to the importance of functional mitochondrial dynamics in normal cell physiology. In recent years, significant progress has been made in our understanding of mitochondrial dynamics, which has raised interest in defining the physiological roles of key regulators of fusion and fission and led to the identification of additional functions of Mfn2 in mitochondrial metabolism, cell signalling, and apoptosis. In this review, we summarize the current knowledge of the structural and functional properties of Mfn2 as well as its regulation in different tissues, and also discuss the consequences of aberrant Mfn2 expression.     

Ferroptosis: Final destination for cancer?

Ferroptosis is a recently defined, non‐apoptotic, regulated cell death (RCD) process that comprises abnormal metabolism of cellular lipid oxides catalysed by iron ions or iron‐containing enzymes. In this process, a variety of inducers destroy the cell redox balance and produce a large number of lipid peroxidation products, eventually triggering cell death. However, in terms of morphology, biochemistry and genetics, ferroptosis is quite different from apoptosis, necrosis, autophagy‐dependent cell death and other RCD processes. A growing number of studies suggest that the relationship between ferroptosis and cancer is extremely complicated and that ferroptosis promises to be a novel approach for the cancer treatment. This article primarily focuses on the mechanism of ferroptosis and discusses the potential application of ferroptosis in cancer therapy.