Different mitochondrial response to cisplatin and hyperthermia treatment in human AGS,Caco-2 and T3M4 cancer cell lines

Gastrointestinal cancers (gastric, pancreatic and colorectal) are life-threatening diseases, which easily spread to peritoneal cavity (Juhl et al. in Int J Cancer 57:330–335, 1994; Schneider et al. in Gastroenterology 128:1606–1625, 2005; Geer and Brennan in Am J Surg 165:68–72 1993). Application of hyperthermal intraperitoneal chemotherapy (HIPEC) is one of the choices treating these malignancies and prolonging patient survival time. Despite numbers of clinical trials showing positive effects of HIPEC against various types of cancer, the question whether hyperthermia significantly potentiate the cytotoxicity of cisplatin remains unanswered. Little information is available on the HIPEC effect at the level of mitochondria. To define the effect of hyperthermia (40 °C and 43 °C) to cisplatin treated human gastric AGS, pancreatic T3M4 and colorectal Caco-2 cancer cells, we established an in vitro experiment, which mimics clinical HIPEC conditions. Giving the importance of mitochondrial energy metabolism in cancer, we investigated the effect of cisplatin and hyperthermia on mitochondrial Complex-I (glutamate/malate) and complex-II (succinate) dependent respiratory rates, the coupling of oxidative phosphorylation, the proton permeability of mitochondrial inner membrane and on the integrity of mitochondrial outer membrane in Caco-2, AGS and T3M4 cancer cell lines. Our main findings are: 1) treatment of cells with cisplatin causes the impairment of mitochondrial functions – the increase in the proton permeability of mitochondrial inner membrane and decrease in the oxidative phosphorylation efficiency in Caco-2, AGS and T3M4 cancer cells; 2) hyperthermia (40 °C and 43 °C) increased state 2 respiration rate only in AGS cells without any effects on Caco-2 and T3M4 cells; 3) hyperthermia in combination with cisplatin doesn’t enhance cisplatin effect neither in Caco-2 and T3M4 nor in AGS cells. Thus, our results show the different mitochondrial response of gastric AGS, pancreatic T3M4 and colorectal Caco-2 cancer cells to cisplatin or/and hyperthermia – treatment. Further studies are needed to find the mechanisms of cell line - specific mitochondrial response to cisplatin and hyperthermia.     

Inhibition of tumor propellant glutathione peroxidase 4 induces ferroptosis in cancer cells and enhances anticancer effect of cisplatin

Glutathione peroxidase 4 (GPX4) has been confirmed to inhibit ferroptosis in cancer cells, however, whether GPX4 serves as an oncogene is not clear. In this study, the expression of GPX4 and its influence to survival of patients with cancer were analyzed via public databases. Furthermore, the epigenetic regulation of GPX4 and the relation between GPX4 and chemoresistance of different anticancer drugs was also detected. Most importantly, cytological assays were performed to investigate the function of GPX4 in cancer cells. The results showed that GPX4 was higher expressed in cancer tissues than normal and was negatively associated with prognosis of patients. Furthermore, at upstream of GPX4 there was low DNA methylation sites and enhanced level of H3K4me3 and H3K27ac, indicating that high level of GPX4 in cancer may resulted from epigenetic regulation. Moreover, GPX4 was positively related to chemoresistance of anticancer drugs L-685458, lapatinib, palbociclib, and topotecan. In addition, GPX4 may potentially be involved in translation of protein, mitochondrial respiratory chain complex I assembly, electron transport oxidative phosphorylation, nonalcoholic fatty liver disease, and metabolic pathways. Finally, we detected that GPX4 inhibited ferroptosis in cancer cells, the inhibition of GPX4 via RSL3 could enhance the anticancer effect of cisplatin in vitro and in vivo. In conclusion, GPX4 acts as an oncogene and inhibits ferroptosis in cancer cells, the anticancer effect of cisplatin can be enhanced by GPX4 inhibition.     

AIF deficiency compromises oxidative phosphorylation

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

The molecular archaeology of a mitochondrial death effector: AIF in Drosophila

Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (DeltaN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. DeltaN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that DeltaN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.     

Novel role of NOX in supporting aerobic glycolysis in cancer cells with mitochondrial dysfunction and as a potential target for cancer therapy

Elevated aerobic glycolysis in cancer cells (the Warburg effect) may be attributed to respiration injury or mitochondrial dysfunction, but the underlying mechanisms and therapeutic significance remain elusive. Here we report that induction of mitochondrial respiratory defect by tetracycline-controlled expression of a dominant negative form of DNA polymerase γ causes a metabolic shift from oxidative phosphorylation to glycolysis and increases ROS generation. We show that upregulation of NOX is critical to support the elevated glycolysis by providing additional NAD+. The upregulation of NOX is also consistently observed in cancer cells with compromised mitochondria due to the activation of oncogenic Ras or loss of p53, and in primary pancreatic cancer tissues. Suppression of NOX by chemical inhibition or genetic knockdown of gene expression selectively impacts cancer cells with mitochondrial dysfunction, leading to a decrease in cellular glycolysis, a loss of cell viability, and inhibition of cancer growth in vivo. Our study reveals a previously unrecognized function of NOX in cancer metabolism and suggests that NOX is a potential novel target for cancer treatment.     

Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts

To study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min of ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20-min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30-min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30-min ischemic hearts elevated LVEDP, attenuated LVDP, and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP-to-O ratio, as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants [N-acetyl-L-cysteine or N-(2-mercaptopropionyl)-glycine]. Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical-generating system (xanthine plus xanthine oxidase) and an oxidant (H(2)O(2)) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.     

Cowchock Syndrome Is Associated with a Mutation in Apoptosis-Inducing Factor

Cowchock syndrome (CMTX4) is a slowly progressive X-linked recessive disorder with axonal neuropathy, deafness, and cognitive impairment. The disease locus was previously mapped to an 11 cM region at chromosome X: q24-q26. Exome sequencing of an affected individual from the originally described family identified a missense change c.1478A>T (p.Glu493Val) in AIFM1, the gene encoding apoptosis-inducing factor (AIF) mitochondrion-associated 1. The change is at a highly conserved residue and cosegregated with the phenotype in the family. AIF is an FAD-dependent NADH oxidase that is imported into mitochondria. With apoptotic insults, a N-terminal transmembrane linker is cleaved off, producing a soluble fragment that is released into the cytosol and then transported into the nucleus, where it triggers caspase-independent apoptosis. Another AIFM1 mutation that predicts p.Arg201del has recently been associated with severe mitochondrial encephalomyopathy in two infants by impairing oxidative phosphorylation. The c.1478A>T (p.Glu493Val) mutation found in the family reported here alters the redox properties of the AIF protein and results in increased cell death via apoptosis, without affecting the activity of the respiratory chain complexes. Our findings expand the spectrum of AIF-related disease and provide insight into the effects of AIFM1 mutations.     

Disruption of oxidative phosphorylation and synaptic Na,K-ATPase activity by pristanic acid in cerebellum of young rats

Aims

Peroxisomal biogenesis disorders (PBD) are inherited disorders clinically manifested by neurological symptoms and brain abnormalities, in which the cerebellum is usually involved. Biochemically, patients affected by these neurodegenerative diseases accumulate branched-chain fatty acids, including pristanic acid (Prist) in the brain and other tissues.

Main methods

In the present investigation we studied the in vitro influence of Prist, at doses found in PBD, on oxidative phosphorylation, by measuring the activities of the respiratory chain complexes I–IV and ATP production, as well as on creatine kinase and synaptic Na⁺, K⁺-ATPase activities in rat cerebellum.

Key findings

Prist significantly decreased complexes I–III (65%), II (40%) and especially II–III (90%) activities, without altering the activities of complex IV of the respiratory chain and creatine kinase. Furthermore, ATP formation and synaptic Na⁺, K⁺-ATPase activity were markedly inhibited (80–90%) by Prist. We also observed that this fatty acid altered mitochondrial and synaptic membrane fluidity that may have contributed to its inhibitory effects on the activities of the respiratory chain complexes and Na⁺, K⁺-ATPase.

Significance

Considering the importance of oxidative phosphorylation for mitochondrial homeostasis and of Na⁺, K⁺-ATPase for the maintenance of cell membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission in cerebellum. We postulate that these pathomechanisms may contribute to the cerebellar alterations observed in patients affected by PBD in which Prist is accumulated.     

Supramolecular structure of the mitochondrial oxidative phosphorylation system

The protein complexes of the mitochondrial oxidative phosphorylation system were recently reported to form supramolecular assemblies termed respiratory supercomplexes or respirasomes. These supercomplexes are considered to be of great functional importance. Here we review new insights into supercomplex structure and physiology.     

Impaired OXPHOS complex III in breast cancer

We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis.     

Assembly of the oxidative phosphorylation system in humans: what we have learned by studying its defects

Assembly of the oxidative phosphorylation (OXPHOS) system in the mitochondrial inner membrane is an intricate process in which many factors must interact. The OXPHOS system is composed of four respiratory chain complexes, which are responsible for electron transport and generation of the proton gradient in the mitochondrial intermembrane space, and of the ATP synthase that uses this proton gradient to produce ATP. Mitochondrial human disorders are caused by dysfunction of the OXPHOS system, and many of them are associated with altered assembly of one or more components of the OXPHOS system. The study of assembly defects in patients has been useful in unraveling and/or gaining a complete understanding of the processes by which these large multimeric complexes are formed. We review here current knowledge of the biogenesis of OXPHOS complexes based on investigation of the corresponding disorders.     

Pyruvate modifies glycolytic and oxidative metabolism of rat embryonic spinal cord astrocyte cell lines and prevents their spontaneous transformation

This study aimed to provide detailed data on mitochondrial respiration of normal astrocyte cell lines derived from rat embryonic spinal cord. Astrocytes in early passages (EP), cultured without pyruvate for more than 35 passages, defined here as late passages (LP), undergo spontaneous transformation. To study initial steps in cell transformation, EP data were compared with those of LP cells. LP cells had reduced glycolysis, fewer mitochondria and extremely low oxidative rates, resulting from a dysfunction of complexes I and II + III of the respiratory chain. Treatment of EP cells with pyruvate until they were, by definition, LP cultures prevented transformation of these cells. Pyruvate-treated EP cells had more mitochondria than normal cells but slightly lower respiratory rates. The increase of mitochondrial content thus appears to act as a compensatory effect to maintain oxidative phosphorylation in these LP 'non-transformed' cells, in which mitochondrial function is reduced. However, pyruvate treatment of transformed LP cells during additional passages did not significantly restore their oxidative metabolism. These data highlight changes accompanying spontaneous astrocyte transformation and suggest potential targets for the control of astrocyte proliferation and reaction to various insults to the central nervous system.     

Endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, specific respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase 2 null mice

Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alpha-ketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment.     

Cathepsin L deficiency results in reactive oxygen species (ROS) accumulation and vascular cells activation

Recent evidence suggests a link between cathepsin L (CTSL) and vascular diseases. However, its contribution to reactive oxygen species (ROS) homeostasis in the vasculature remains unknown. p66shc is a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals. In this study, we explored the relationship between CTSL and oxidative damage in vasculature and whether the oxidative damage is mediated by p66shc.Carotid arteries from aged mice (24 months old) showed a reduction in CTSL expression compared with young wild-type mice (4 months old). Local knockdown of CTSL in carotid arteries of young mice by adenoviral vector encoding the short hairpin RNA targeting CTSL leading to premature vascular aging, as shown by mitochondrial disruption, increased β-galactosidase–positive cells, reduced telomerase activity, and up-regulation of p66shc. Knockdown of CTSL decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, III, and IV, leading to increased mitochondrial ROS and hyperpolarization of the mitochondrial membrane in vitro. Furthermore, knockdown of CTSL also stimulated ROS production and senescence in vascular cells, accompanied by the up-regulation of p66shc.However, p66shc knockdown blunted the alteration in ROS production, and senescence in CTSL knockdown vascular cells. This study suggests that CTSL knockdown partially induces vascular cells damage via increased ROS production and up-regulation of p66shc.