The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia.

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF1, that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O2) unexpectedly showed that Δψm is preserved independently of the presence of IF1 and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing ΔµΗ+ with the FCCP uncoupler, the IF1-silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF1 overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF1 overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction.

Is supercomplex organization of the respiratory chain required for optimal electron transfer activity?

The supra-molecular assembly of the main respiratory chain enzymatic complexes in the form of "super-complexes" has been proved by structural and functional experimental evidence. This evidence strongly contrasts the previously accepted Random Diffusion Model stating that the complexes are functionally connected by lateral diffusion of small redox molecules (i.e. Coenzyme Q and cytochrome c). This review critically examines the available evidence and provides an analysis of the functional consequences of the intermolecular association of the respiratory complexes pointing out the role of Coenzyme Q and of cytochrome c as channeled or as freely diffusing intermediates in the electron transfer activity of their partner enzymes.

Hypoxia decreases ROS level in human fibroblasts.

We have previously demonstrated that cells adapt to hypoxia using different metabolic reprogramming mechanisms depending on metabolism. We now investigate how the different adapting mechanisms affect reactive oxygen species (ROS) levels, and how ROS levels and cellular metabolism are linked. We show that when skin fibroblasts grew under short-term hypoxia (1% oxygen tension) ROS level markedly decreased (-50%) whatever substrate was available to the cells. Indeed, cellular ROS level linearly and directly decreased with oxygen tension. However, these relationships cannot explain the progressive ROS level decrease observed after prolonged cells hypoxia exposure. In glucose-enriched medium reduced mitochondrial mass and greater fragmentation are observed, both clear-cut indications of mitophagy suggesting that this is responsible for cellular ROS level decrease. Otherwise, in glucose-free medium exposure to prolonged hypoxia resulted in only minor mass reduction, but significantly enhanced expression of antioxidant enzymes. Interestingly, cellular ROS levels were lower in glucose-free compared to glucose-enriched medium under either normoxic or hypoxic conditions. Taken together, these findings reveal that in primary human fibroblasts hypoxia induces a decline in ROS and that different metabolism-dependent mechanisms contribute it, besides the major oxygen concentration decrease. In addition, the present data support the notion that metabolisms generating fewer ROS are associated with lower HIF-1α stabilization.

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.

Resolution of the circular dichroism spectra of the mitochondrial cytochrome bc1 complex.

The circular dichroic spectrum of the mitochondrial cytochrome bc1 complex isolated from bovine heart has been resolved into the contributions from the prosthetic groups: cytochrome c1, the 'Rieske' iron-sulphur centre and the two b cytochromes. It is apparent that firstly, the circular dichroism (CD) properties of cytochrome c1 within the bc1 complex differ from those found in the isolated cytochrome c1 and secondly, both the oxidized and reduced b cytochromes exhibit an intense spectrum of bilobic shape, with the wavelengths of the cross-over points closely corresponding to those of the maxima in the optical absorbance spectra. These latter CD features are discussed in relation to the proposed structure of cytochrome b.

Temperature-induced states of isolated F1-ATPase affect catalysis, enzyme conformation and high-affinity nucleotide binding sites.

Isolated, nucleotide-depleted bovine-heart F1-ATPase exhibits a break in Arrhenius plot with a 2.7-fold increase in activation energy of ATP hydrolysis below 18-19 degrees C. Analysis of intrinsic tyrosine fluorescence and of the circular dichroism of F1-ATPase showed an abrupt and reversible conformational change occurring at the break temperature, characteristic of a structural tightening at low temperature. Analysis of catalytic nucleotide binding sites using fluorescent ADP analog, 3'-O-(1-naphthoyl)adenosine diphosphate did not show any significant change in affinity of nucleotide binding around the transition temperature but the bound fluorophore exerted a more restricted motion and slower rotation at temperature below the break, indicating a change in the mobility of groups in the close neighbourhood. It is concluded that, as a result of temperature, two kinetically distinct states of F1-ATPase are induced, due to a change in enzyme conformation, which influences directly the properties of catalytic nucleotide binding sites.

Increased state 4 mitochondrial respiration and swelling in early post-ischemic reperfusion of rat heart.

Isolated rat hearts were exposed to 30 min ischemia or to 30 min ischemia followed by 2, 5 or 40 min reperfusion and mitochondria were isolated at these different time points. ADP-stimulated, succinate-dependent respiration rate (state 3) was not significantly changed at the different time points examined. In contrast, state 4 (non-ADP-stimulated) respiration rate was significantly increased after 30 min ischemia, and it increased further during the first post-ischemic reperfusion period. Mitochondrial swelling, as evaluated under conditions of the major controlled ion channels (i.e. permeability transition pore and ATP-dependent mitochondrial K(+) channel) closed, significantly increased in parallel. It is suggested that the inner mitochondrial membrane permeability is increased under exposure of the heart to ischemia and early reperfusion, and that the phenomenon is reversible upon subsequent long periods of reperfusion.

Effects of cholesterol on the kinetics of mitochondrial ATPase.

Enrichment of the inner mitochondrial membrane with cholesterol induces an increase in ATPase activity with a decrease in the Km for ATP. Cholesterol also abolishes the discontinuity normally found in the Arrhenius plot of ATPase activity. Since no change is detected in the rate of proton translocation through the ATPase membrane sector, it is concluded that cholesterol incorporation induces changes in the hydrolytic step of ATPase via a conformational change transmitted from the membrane sector to the catalytic sector F1.

Temperature-dependent conformational changes in isolated oligomycin-sensitive ATPase.

Isolated oligomycin-sensitive ATPase undergoes a kinetic change at 20-25 degrees C with a higher activation energy and a lower Km for ATP below this temperature range. This observation has been correlated with temperature-dependent structural changes detected by circular dichroism in the UV region in the isolated enzyme. The negative ellipticities in the 208-225 nm region, which are proportional to the alpha-helix content, increase with rise in temperature to a maximum above 25 degrees C.

Lack of major changes in ATPase activity in mitochondria from liver, heart, and skeletal muscle of rats upon ageing.

ATP hydrolase activity has been investigated in mitochondria from liver, heart, and skeletal muscle from adult (6 months) and aged (24 months) rats. No significant changes in total ATPase activity were observed in the three tissues, but the oligomycin sensitivity was slightly decreased in heart mitochondria of aged rats. The bicarbonate-induced stimulation of hydrolytic activity was somewhat decreased in mitochondria from aged rats, particularly in liver. No significant change was observed in ATPase activity after release of the endogenous inhibitor protein, IF1. It is concluded that no activity changes to be directly ascribed to the catalytic sector F1 of the enzyme occur upon ageing, but it cannot be excluded that changes in the membrane sector F0 occur as a consequence of mtDNA mutations.

Effects of niridazole and 5-nitroimidazoles on heart mitochondrial respiration.

2.3 micromoles/mg protein of MFNI induced a 60% decrease in the heart mitochondrial ADP-stimulated oxygen uptake using glutamate-malate as substrate. The same amount of niridazole, ipronidazole, DA 3851 and ornidazole led to falls of less than 20% in the oxygen uptake, whilst metronidazole was ineffective. State 3 and state 3 mu (uncoupled) respiration were affected to the same extent. Oxygen-uptake using succinate as substrate was not inhibited indicating that the action was exerted at the NADH oxidation level. The relationship between electroreduction potentials of the test compounds and inhibition of respiration has been studied.

Mitochondrial cytochrome c oxidase subunit III is selectively down-regulated by aluminum exposure in PC12S cells.

Aluminum (Al) has been implicated in several neurological diseases including dialysis dementia and Alzheimer's disease (AD). One possible mechanism of Al neurotoxicity could involve alteration of mitochondrial gene expression. We exposed PC12 cells to 0.1-100 microM AlCl3 for 6h at pH 7.4. Internalized Al, measured by atomic absorption spectrometry, was linearly proportional to the extracellular Al concentration. Northern blot analyses showed that cytochrome c oxidase subunit III (COX III) mRNA was significantly reduced by 70% after addition of 1 microM AlCl3. Higher concentrations of AlCl3 did not show a significant further effect. These results suggest that Al neurotoxicity involves a specific impairment of cytochrome c oxidase.

Mechanisms for reduction of endothelial activation by oleate: inhibition of nuclear factor-kappaB through antioxidant effects.

In a model of early atherogenesis based on cultured endothelial cells, we observed that the incorporation of oleic acid in cellular lipids decreases the stimulated expression of several endothelial adhesion molecules and soluble products typically expressed during endothelial activation and involved in monocyte recruitment. We investigated possible mechanisms for this effect assessing the stimulated induction of nuclear factor-kappaB. In parallel, we also measured glutathione (GSH) content and the activity of antioxidant enzymes after oleate treatment and cytokine stimulation. Oleate prevented the stimulated depletion of GSH without any change in the activity of antioxidant enzymes. These results suggest an antioxidant mechanism by which oleate may exert direct vascular atheroprotective effects.

Dietary lipid effects on microsome fatty acid composition of liver and brain, on liver glucose-6-phosphatase, and on brain 5'-nucleotidase activity in the rat.

The effects of incorporation of dietary oils with different n6/n3 ratio and polyunsaturated fatty acids content into rat liver and brain microsomes has been studied. The investigation of membrane fatty acid composition of liver microsomes and that of brain microsomes gave different results. In particular, liver microsomes of rats fed fish oil showed a relatively higher content of 20:5n3 and 22:6n3, and a lower content of 20:4n6. Under these conditions, a reduced glucose-6-phosphatase activity was measured. Brain microsomal fatty acid composition was only slightly affected by dietary lipid intake. The 5'-nucleotidase activity of those particles was similar, although statistically different values were found in fish-oil-fed rats and in olive-oil-fed rats. The effects of membrane fatty acid composition on membrane-bound enzyme activity are discussed.

Effects of 4'-O-tetrahydropyranyl-doxorubicin on isolated perfused rat heart and cardiac mitochondrial cytochrome C oxidase activity.

The acute cardiac effects of the antitumour anthracycline 4'-O-tetrahydropyranyl-doxorubicin (THP) were studied on isolated, perfused, spontaneously beating rat hearts and on cardiac mitochondrial cytochrome c oxidase activity. Perfusion for 1 hour with 2.5 or 5.0 micrograms/ml of THP was associated with a marked widening of the QRS complex and the S alpha T segment, as well as with a reduction of the R- and T-wave amplitude, the heart rate, the isometric systolic tension and the coronary flow. Heart perfusion with equimolar doses of doxorubicin (2.2 or 4.4 micrograms/ml) induced a significant enlargement of the SaT segment and a reduction in the heart rate and the coronary flow. Both anthracyclines inhibited the cytochrome c oxidase activity in a dose-dependent manner; however, THP exerted a significant inhibition at concentrations higher than doxorubicin (20 microM). Results demonstrate that THP induces a higher degree of acute cardiotoxicity on isolated rat hearts compared with doxorubicin. The reduced inhibitory effect of THP on the cytochrome c oxidase activity of isolated heart mitochondria is consistent with the lower degree of chronic cardiotoxicity displayed by THP in animals and humans.

The inhibition of endothelial activation by unsaturated fatty acids.

Dietary long-chain fatty acids (FA) may influence pathological processes involving endothelial activation and leukocyte-endothelial interactions, such as inflammation and atherosclerosis. We previously showed that the n-3 FA docosahexaenoate (22:6n-3, DHA) inhibits cytokine-stimulated expression of endothelial-leukocyte adhesion molecules and soluble cytokines in the range of nutritionally achievable plasma concentrations. More recently we assessed structural determinants of VCAM-1 inhibition by FA. Cultured endothelial cells were incubated first with various saturated, monounsaturated, n-6 or n-3 polyunsaturated FA alone and then together with interleukin-1 or tumor necrosis factor. Saturated FA did not inhibit cytokine-induced endothelial activation, while a progressive increase in inhibitory activity was observed, for the same chain length, with the increase in double bonds accompanying the transition from monounsaturates to n-6 and, further, to n-3 FA. Comparison of various FA indicated no role of the double-bond position or configuration; the greater number of double bonds could explain the greater inhibitory activity of n-3 vs. n-6 FA. In order to ascertain mechanisms for these effects, we demonstrated inhibition of nuclear factor-kappaB (NF-kappaB) activation by DHA in parallel with a reduction in hydrogen peroxide (a critical mediator of NF-kappaB activation) released by endothelial cells either extracellularly or intracellularly. This suggests that a property related to fatty acid peroxidability (the presence of multiple double bonds) is related to inhibitory properties of hydrogen peroxide release and, consequently, of endothelial activation.

Studies on the effects of anthracyclines on mitochondrial respiration in vitro.

Aclacinomycin, 4'-epi-doxorubicin and 4'-epi-tetrahydropyranyl-adriamycin, three novel anthraquinone derivatives under investigation for their antitumour activity, showed an inhibitory effect on the in vitro respiration of mitochondria from rat hearts. The inhibition proved to be concentration-dependent in the range 0.05 to 1.40 mM and both the NADH-oxidase and the succinate oxidase systems were affected to different extents. Among the compounds tested, 4'-epi-tetrahydropyranyl-adriamycin appeared to be the least powerful effector, requiring a significantly higher concentration for 50% inhibition of oxidation than doxorubicin and the other analogues examined.

Inhibitory effects of several anthracyclines on mitochondrial respiration and coenzyme Q10 protection.

A set of three novel anthracyclines, active as antitumour agents, has been examined for their possible effects on rat heart mitochondrial respiration. The in vitro inhibiting effects of the compounds have been compared with that of the older doxorubicin. Aclacinomycin was generally more inhibitory than doxorubicin, 4'-epi-doxorubicin and 4'-epi-tetrahydropyranyl-adriamycin, with both succinate and NAD+-linked substrates. Attempts to prevent anthracycline from inhibiting the succinate oxidase activity by means of adding exogenous coenzyme Q10 gave encouraging results, the inhibiting effect being in fact reduced.

Polyamines reduce heart injury caused by doxorubicin.

Isolated rat heart was perfused with 18 microM doxorubicin in the recirculating buffer. This treatment resulted in progressive contractile impairment. Aortic flow and minute work (i.e the product of cardiac output and peak systolic pressure) were reduced up to 20 and 29% of basal values, respectively. These parameters were partially restored in hearts perfused with 400 microM spermine, a naturally occurring polyamine, and 18 microM doxorubicin in the recirculating buffer. The results suggest the need for an investigation of the possible antagonistic role of polyamines against anthracycline cardiotoxicity.

Cardiotoxic effects of adriamycin and mitochondrial oxidation in rat cardiac tissue.

Rats were subjected to chronic treatment with adriamycin (ADR). Significant alterations of ECG tracings were induced, starting from the third week of treatment. These alterations were related to mitochondrial damage of the heart tissue. A decrease of respiration rate in phosphorylating conditions was observed in isolated organelles over a period of three weeks. Meanwhile, adriamycinol (ADRol) concentration in heart extracts increased during chronic treatment.