BNIP3 as a Regulator of Cisplatin-Induced Apoptosis.

BNIP3 is a member of Bcl-2 protein family involved in regulation of various forms of cell death. However, its role in these processes remains unclear and varies depending on the type of cancer cells and environmental factors (pH, O2 level, etc.). Here, the role of BNIP3 in apoptosis regulation in lung adenocarcinoma cells was investigated. The suppressed expression of BNIP3 caused inhibition of oxygen consumption and stimulated production of the mitochondrial reactive oxygen species, suggesting the role of BNIP3 in induction of mitochondrial dysfunction and its potential involvement in regulation of cell death. Indeed, cytochrome c release in the cells with BNIP3 knockout and knockdown was higher than in the wild-type (WT) upon apoptosis stimulation by cisplatin. Moreover, suppression of BNIP3 expression led to the increase in the caspase-3 activity and, as a consequence, accumulation of the apoptotic marker - p89 fragment of poly(ADP-ribose)-polymerase (PARP) - as compared to WT cells. Analysis of the SubG1 population by flow cytometry confirmed the elevated level of apoptosis in the BNIP3 knockout cells. Pretreatment with the antioxidant Trolox did not affect cell death, indicating that it was independent on reactive oxygen species. These data show that BNIP3 is involved in maintaining normal functioning of mitochondria and, as a result, can regulate the mitochondrial pathway of cell death.

Reactive Oxygen Species and Antioxidants in Carcinogenesis and Tumor Therapy.

Strictly regulated balance between the formation and utilization of reactive oxygen species (ROS) is the basis of normal functioning of organisms. ROS play an important role in the regulation of many metabolic processes; however, excessive content of ROS leads to the development of various disorders, including oncological diseases, as a result of ROS-induced mutations in DNA. In tumors, high levels of oxygen radicals promote cell proliferation and metastasis. On the other hand, high content of ROS can trigger cell death, a phenomenon used in the antitumor therapy. Water- and lipid-soluble antioxidants, as well as antioxidant enzyme systems, can inhibit ROS generation; however, they should be used with caution. Antioxidants can suppress ROS-dependent cell proliferation and metastasis, but at the same time, they may inhibit the death of tumor cells if the antitumor therapeutic agents stimulate oxidative stress. The data on the role of antioxidants in the death of tumor cells and on the effects of antioxidants taken as dietary supplements during antitumor therapy, are contradictory. This review focuses on the mechanisms by which antioxidants can affect tumor and healthy cells.

Inhibition of P-glycoprotein stimulates cell death under Hypoxia-mimicking conditions.

The most common drug resistance mechanism in tumor cells is expression on their surface of the energy-dependent pump like P-glycoprotein (P-gp) that expels chemotherapeutic agents from the interior. An imitation of the hypoxic condition by the iron chelator deferoxamine caused Hypoxia-inducible factor 1-alpha (HIF-1α) stabilization and inhibition of doxorubicin-induced apoptosis in colon cancer НСТ116 cells. P-gp blocker verapamil suppressed doxorubicin accumulation leading to cell death induction. Considering these results, P-gp may be used as a potential target to stimulate chemotherapeutic drugs activity that will contribute to more efficient tumor cells elimination.

Cellular Energetics as a Target for Tumor Cell Elimination.

Investigation of cancer cell metabolism has revealed variability of the metabolic profiles among different types of tumors. According to the most classical model of cancer bioenergetics, malignant cells primarily use glycolysis as the major metabolic pathway and produce large quantities of lactate with suppressed oxidative phosphorylation even in the presence of ample oxygen. This is referred to as aerobic glycolysis, or the Warburg effect. However, a growing number of recent studies provide evidence that not all cancer cells depend on glycolysis, and, moreover, oxidative phosphorylation is essential for tumorigenesis. Thus, it is necessary to consider distinctive patterns of cancer metabolism in each specific case. Chemoresistance of cancer cells is associated with decreased sensitivity to different types of antitumor agents. Stimulation of apoptosis is a major strategy for elimination of cancer cells, and therefore activation of mitochondrial functions with direct impact on mitochondria to destabilize them appears to be an important approach to the induction of cell death. Consequently, the design of combination therapies using acclaimed cytotoxic agents directed to induction of apoptosis and metabolic agents affecting cancer cell bioenergetics are prospective strategies for antineoplastic therapy.

[Induction of Unspecific Permeabilization of Mitochondrial Membrane and Its Role in Cell Death].

Mitochondria participate in various vital cellular processes. Violation of their functions can lead to the development of cardiovascular and neurodegenerative diseases and malignancies. One of the key events responsible for mitochondrial damage-induction of Ca^(2+)-dependent mitochondrial permeability transition, due to the opening of a nonspecific pore in the inner mitochondrial membrane. Despite active studies of pore components, its detailed structure has not yet been established. This review analyzes possible constituents and regulators of the pore, the role of the pore in various pathologies, and hypotheses that explain the organization of the pores. Elucidation of these questions can help developing strategies for the treatment of a wide range of pathologies-from Alzheimer and Parkinson's disease to cancer.

Etoposide and hypoxia do not activate apoptosis of multipotent mesenchymal stromal cells in vitro.

Incubation of multipotent mesenchymal stromal cells from human adipose tissue with etoposide for 24, 48, and 72 h under standard conditions (20% O(2)), at "physiological" oxygen content (5% O(2)), and under hypoxic conditions (1% O(2)) did not induce cell apoptosis and only slightly increased the number of necrotic cells. The absence of growth factors in the culture medium did not potentiate the damaging effect of etoposide on multipotent mesenchymal stromal cells under standard and hypoxic conditions. We concluded that etoposide produced no pro-apoptotic effect on multipotent mesenchymal stromal cells from the human adipose tissue.

[Effect of mitochondrial energetic state on radiation-induced DNA internucleosomal fragmentation in irradiated thymocytes]. / Vliianie énergeticheskogo sostoianiia mitokhondrii na mezhnukleosomnuiu fragmentatsiiu DNK v timotsitakh posle oblucheniia.

One of the earliest features of apoptosis is a decrease of mitochondrial membrane potential. Here we show that when apoptosis is induced in thymocytes by ionizing radiation, inhibitors of mitochondrial energy production suppress DNA internucleosomal fragmentation. The suppression of fragmentation by inhibitors does not correlate with their effect on mitochondrial membrane potential, as it was observed when membrane potential was decreased (in the presence of inhibitors of respiratory chain, uncouplers of oxidative phosphorylation) or non affected (in the presence of oligomycin, inhibitor of mitochondrial ATPase).

Investigation of calcium accumulation in mitochondria in cells undergoing apoptosis.

One of the earliest features of apoptosis is the induction of the mitochondrial permeability transition (MPT) due to opening of a pore in the mitochondrial membrane. We estimated the Ca2+ capacity of mitochondria (a threshold level of Ca2+ that induces the release of this cation from mitochondria) during apoptosis. Incubation of thymocytes at 37 degrees C for 4 h equally decreased the mitochondrial Ca2+ capacity both in the presence and the absence of dexamethasone, an inducer of apoptosis. At the same time, dexamethasone significantly stimulated internucleosomal DNA fragmentation, which is one of the manifestations of apoptosis. Cyclosporin A prevented the time-dependent decrease in the Ca2+ capacity of mitochondria but did not affect internucleosomal DNA fragmentation. Therefore, induction of apoptosis assessed by internucleosomal DNA fragmentation is not mediated by the mitochondrial permeability transition.

[Physiologic activation of peroxidation by negative air ions]. / Fiziologicheskaia aktivatsiia perekisnogo okisleniia otritsatel'nymi aéroionami.

The effect of negative air ions (NAI) was investigated on physiological processes of peroxidative oxidation in mitochondria preserving their native structure organization in associations in rat liver homogenate. Tchijevsky lustre generating superoxide was served as a source of NAI. The investigated objects were put into solutions pretreated with NAI flow. An increase of peroxide induced Ca2+ release from mitochondria loaded with the cation was found. Under prolonged action of NAI flow on homogenates kept in ice followed by incubation in media pretreated with NAI an elevation of products of peroxide lipid oxidation (POL) was found in case of their low initial level and decrease of POL products in case of their high initial level. The range of changes is considerably less than under pathogeneous rise of POL. The discovered mild activation of peroxidative oxidation within physiological range of POL concentrations is considered as a primary physico-chemical mechanism of beneficial biological action of NAI.

Inhibitors of mitochondrial energy production prevent DNA internucleosomal fragmentation in thymocytes.

Apoptosis or programmed cell death is a gene-controlled process of cell self-destruction. One of the earliest manifestations of apoptosis, which precedes morphological changes, is a decrease of mitochondrial membrane potential. Here we show that neither inhibitors of the mitochondrial respiratory chain (rotenone and antimycin) nor an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), agents which decrease the mitochondrial membrane potential, induce DNA internucleosomal fragmentation, but all of them markedly prevent fragmentation induced either by glucocorticoids or the Ca2+ ionophore A23187. A similar effect was also observed in the presence of a mitochondrial ATPase inhibitor (oligomycin). The inhibition of DNA internucleosomal fragmentation can be explained by the ability of inhibitors to prevent the mitochondrial permeability transition--a key event in apoptosis induction.

[The effect of superhigh doses of gamma radiation on the energetics of rat liver mitochondria]. / Vliianie sverkhvysokikh doz gamma-radiatsii na énergetiku mitokhonrii pecheni krys.

The effect of high dose of gamma radiation (200 Gy) on energy of rat liver mitochondria was studied. 1 hour after irradiation state 3 (V3) increased by 32 +/- 7%, state 4 (V4)- by 25 +/- 8%, the rate of ATP synthesis by 52 +/- 11%. 48 hours after irradiation these parameters decreased to control levels inspite of accumulation of lipid peroxidation products.

Thermoregulatory, carboxyatractylate-sensitive uncoupling in heart and skeletal muscle mitochondria of the ground squirrel correlates with the level of free fatty acids.

Thermoregulatory uncoupling of oxidative phosphorylation has been studied in heart and skeletal muscle mitochondria of ground squirrels. The respiratory rate of mitochondria in the presence of oligomycin was found to be much higher in winter (in hibernating, arousing, or aroused animals) than in summer. This additional respiration is strongly (arousing animals) or completely (hibernating and aroused animals) inhibited by carboxyatractylate (CAtr) and bovine serum albumin (BSA). The CAtr- and BSA-induced decreases in the rate of respiration are accompanied by membrane potential increases. The rate of the CAtr- and BSA-sensitive respiration is proportional to the content of free fatty acids which, in the heart, decreases in the order: arousing greater than aroused = hibernating greater than summer animals. Maximal respiratory rates observed in the presence of dinitrophenol (arousing greater than aroused greater than summer greater than hibernating animals) do not parallel the fatty acid level. It is assumed that some heat production in the winter animals is due to fatty acid-induced, ATP/ADP-antiporter-mediated uncoupling in heart and skeletal muscle mitochondria. The peak of heat production during arousal after hibernation also includes some other stimulatory effect on mitochondrial respiration.

The role of lipid peroxidation products in cumene hydroperoxide-induced Ca2+ efflux from mitochondria.

Cumene hydroperoxide-induced calcium release from mitochondria has been studied. Activation of lipid peroxidation by increasing concentrations of cumene hydroperoxide does not enhance calcium efflux induced by low (up to 50 microM) concentration of cumene hydroperoxide. It is concluded that cumene hydroperoxide-induced calcium release depends mainly on processes coupled to hydroperoxide reduction by an endogenous enzyme system.

[Participation of phospholipase A2 in uncoupling in rat liver mitochondria induced by products of lipid peroxidation]. / Uchastie fosfolipazy A2 v indutsiruemom produktami perekisnogo okisleniia lipidov razobshchenii mitokhondrii pecheni krys.

Accumulation of lipid peroxidation products induced by cumol hydroperoxide (230 microM) results in the loss of the membrane potential only in calcium-loaded mitochondria. The phospholipase A2 inhibitor, p-bromophenylacylbromide, prevents mitochondrial uncoupling but has no effect on the accumulation of lipid peroxidation products.

The role of phospholipase A2 in lipid peroxidation-induced fall of membrane potential of rat liver mitochondria.

Cumene hydroperoxide (230 microM)-induced fall of the membrane potential takes place only in Ca2(+)-loaded mitochondria. Inhibitor of phospholipase A2 p-bromphenacyl bromide prevents uncoupling of mitochondria, having no effect on the accumulation of lipid peroxidation products.

The effect of butylated hydroxytoluene, an inhibitor of lipid peroxidation, on the calcium-induced uncoupling of rat liver mitochondria.

The accumulation of Ca2+ by rat liver mitochondria in the presence of Pi results in spontaneous activation of respiration, accompanied by progressive loss of the accumulated cation. The lipid peroxidation inhibitor, butylated hydroxytoluene, completely prevents and reverses the loss of accumulated Ca2+ and restores respiration to the state 4 level, but exerts no effect on the rate of Ca2+ accumulation and respiration in the presence of the uncoupler. The strong inhibition by butylated hydroxytoluene of ruthenium red-insensitive Ca2+ efflux has also been observed. No correlation between the BHT-sensitive Ca2+ loss and the formation of malonic dialdehyde in mitochondria has been found. The data obtained suggest that the Ca2+-induced uncoupling of mitochondria is mainly due to the appearance of electrogenic ion fluxes that are controlled by the initial steps of lipid peroxidation.

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus.

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.

[Biochemical bases of the inhibition and activation of liver mitochondrial respiration in hibernating susliks]. / Biokhimicheskie osnovy tormozheniia i aktivatsii dykhaniia mitokhondrii pecheni giberniruiushchikh suslikov.

The possible mechanisms in regulation of the respiration rate of mitochondria from liver of hibernating ground squirrels have been investigated. The inhibition of respiration has been shown to be mainly due to the inhibition of electron transfer to the respiratory chain from flavoproteins to cytochrome c. Calcium ions evoke the gradual increase in the respiration rate of mitochondria from liver of hibernating ground squirrels which is abolished by adding albumin, ruthenium red and sovcaine. The lower content of free fatty acids and decreased rate of the oxidation of exogenic NADH in the external pathway indicate the decrease in the activity of phospholipase A2 in mitochondria from liver of hibernating ground squirrels. The decreased calcium capacity of mitochondria indicates the higher sensitivity to calcium ions. A conclusion concerning the leading role of phospholipase A2 in Ca-induced activation of respiration of mitochondria from liver of hibernating ground squirrels is made.

[An antioxidant prevents and reverses calcium-induced uncoupling of rat liver mitochondria]. / Antioksidant predotvrashchaet i obrashchaet indutsiruemoe ionami kal'tsiia razobshchenie mitokhondrii pecheni krys.

Accumulation of Ca2+ by rat liver mitochondria in the presence of inorganic phosphate results in spontaneous activation of respiration accompanied by a progressive loss of the accumulated cation. The lipid peroxidation inhibitor, ionol, completely prevents and reverses the Ca2+/phosphate-induced loss of accumulated Ca2+ and restores the respiration to state 4 level without having any effect on the rate of Ca2+ accumulation and respiration in the presence of an uncoupler. No correlation between the ionol-dependent loss of Ca2+ and the formation of malonic dialdehyde in mitochondria was found. The measurements of delta psi across the inner mitochondrial membrane during a progressive loss of Ca2+ suggest that the Ca2+/phosphate-induced "uncoupling" is mainly due to the appearance of electrogenic fluxes (but not Ca2+ cycling) which is under control of some products of initial steps of lipid peroxidation.

Participation of endogenous fatty acids in Ca2+ release activation from mitochondria.

A correlation between the rate of H+/Ca2+ exchange and the content of free fatty acids in mitochondria has been found. Fatty acids were isolated from mitochondria with different activities of H+/Ca2+ exchange. It has been shown that these free fatty acids are able to induce Ca2+ release in exchange to protons after being added to freshly isolated mitochondria.