Progesterone as an Anti-Inflammatory Drug and Immunomodulator: New Aspects in Hormonal Regulation of the Inflammation.

The specific regulation of inflammatory processes by steroid hormones has been actively studied in recent years, especially by progesterone (P4) and progestins. The mechanisms of the anti-inflammatory and immunomodulatory P4 action are not fully clear. The anti-inflammatory effects of P4 can be defined as nonspecific, associated with the inhibition of NF-κB and COX, as well as the inhibition of prostaglandin synthesis, or as specific, associated with the regulation of T-cell activation, the regulation of the production of pro- and anti-inflammatory cytokines, and the phenomenon of immune tolerance. The specific anti-inflammatory effects of P4 and its derivatives (progestins) can also include the inhibition of proliferative signaling pathways and the antagonistic action against estrogen receptor beta-mediated signaling as a proinflammatory and mitogenic factor. The anti-inflammatory action of P4 is accomplished through the participation of progesterone receptor (PR) chaperones HSP90, as well as immunophilins FKBP51 and FKBP52, which are the validated targets of clinically approved immunosuppressive drugs. The immunomodulatory and anti-inflammatory effects of HSP90 inhibitors, tacrolimus and cyclosporine, are manifested, among other factors, due to their participation in the formation of an active ligand-receptor complex of P4 and their interaction with its constituent immunophilins. Pharmacological agents such as HSP90 inhibitors can restore the lost anti-inflammatory effect of glucocorticoids and P4 in chronic inflammatory and autoimmune diseases. By regulating the activity of FKBP51 and FKBP52, it is possible to increase or decrease hormonal signaling, as well as restore it during the development of hormone resistance. The combined action of immunophilin suppressors with steroid hormones may be a promising strategy in the treatment of chronic inflammatory and autoimmune diseases, including endometriosis, stress-related disorders, rheumatoid arthritis, and miscarriages. Presumably, the hormone receptor- and immunophilin-targeted drugs may act synergistically, allowing for a lower dose of each.

New Properties and Mitochondrial Targets of Polyphenol Agrimoniin as a Natural Anticancer and Preventive Agent.

Agrimoniin is a polyphenol from the group of tannins with antioxidant and anticancer activities. It is assumed that the anticancer action of agrimoniin is associated with the activation of mitochondria-dependent apoptosis, but its mitochondrial targets have not been estimated. We examined the direct influence of agrimoniin on different mitochondrial functions, including the induction of the mitochondrial permeability transition pore (MPTP) as the primary mechanism of mitochondria-dependent apoptosis. Agrimoniin was isolated from Agrimonia pilosa Ledeb by multistep purification. The content of agrimoniin in the resulting substance reached 80%, as determined by NMR spectroscopy. The cytotoxic effect of purified agrimoniin was confirmed on the cultures of K562 and HeLa cancer cells by the MTT assay. When tested on isolated rat liver mitochondria, agrimoniin at a low concentration (10 µM) induced the low-amplitude swelling, which was inhibited by the MPTP inhibitors ADP and cyclosporine A, activated the opening of MPTP by calcium ions and stimulated the respiration supported by succinate oxidation. Also, agrimoniin reduced the electron acceptor DCPIP in a concentration-dependent manner and chelated iron ions. Owing to all these properties, agrimoniin can stimulate apoptosis or activate mitochondrial functions, which can be helpful in the prevention and elimination of stagnant pathological states.

Progestins as Anticancer Drugs and Chemosensitizers, New Targets and Applications.

Progesterone and its synthetic analogues, progestins, participate in the regulation of cell differentiation, proliferation and cell cycle progression. Progestins are usually applied for contraception, maintenance of pregnancy, and hormone replacement therapy. Recently, their effectiveness in the treatment of hormone-sensitive tumors was revealed. According to current data, the anticancer activity of progestins is mainly mediated by their cytotoxic and chemosensitizing influence on different cancer cells. In connection with the detection of previously unknown targets of the progestin action, which include the membrane-associated progesterone receptor (PR), non-specific transporters related to the multidrug resistance (MDR) and mitochondrial permeability transition pore (MPTP), and checkpoints of different signaling pathways, new aspects of their application have emerged. It is likely that the favorable influence of progestins is predominantly associated with the modulation of expression and activity of MDR-related proteins, the inhibition of survival signaling pathways, especially TGF-ß and Wnt/ß-catenin pathways, which activate the proliferation and promote MDR in cancer cells, and the facilitation of mitochondrial-dependent apoptosis. Biological effects of progestins are mediated by the inhibition of these signaling pathways, as well as the direct interaction with the nucleotide-binding domain of ABC-transporters and mitochondrial adenylate translocase as an MPTP component. In these ways, progestins can restore the proliferative balance, the ability for apoptosis, and chemosensitivity to drugs, which is especially important for hormone-dependent tumors associated with estrogen stress, epithelial-to-mesenchymal transition, and drug resistance.

Protectors of the Mitochondrial Permeability Transition Pore Activated by Iron and Doxorubicin.

AIM: The study is aimed at examining of action of iron, DOX, and their complex on the Mitochondrial Permeability Transition Pore (MPTP) opening and detecting of possible protectors of MPTP in the conditions close to mitochondria-dependent ferroptosis. BACKGROUND: The Toxicity of Doxorubicin (DOX) is mainly associated with free iron accumulation and mitochondrial dysfunction. DOX can provoke ferroptosis, iron-dependent cell death driven by membrane damage. The Mitochondrial Permeability Transition Pore (MPTP) is considered as a common pathway leading to the development of apoptosis, necrosis, and, possibly, ferroptosis. The influence of DOX on the Ca2+ -induced MPTP opening in the presence of iron has not yet been studied. OBJECTIVE: The study was conducted on isolated liver and heart mitochondria. MPTP and succinate- ubiquinone oxidoreductase were studied as targets of DOX in mitochondria-dependent ferroptosis. The iron chelator deferoxamine (DFO), the lipid radical scavenger butyl-hydroxytoluene (BHT), and rutenium red (Rr), as a possible inhibitor of ferrous ions uptake in mitochondria, were tested as MPTP protectors. The role of medium alkalization was also examined. METHODS: Changes of threshold calcium concentrations required for MPTP opening were measured by a Ca2+ selective electrode, mitochondrial membrane potential was registered by tetraphenylphosphonium (TPP+)-selective electrode, and mitochondrial swelling was recorded as a decrease in absorbance at 540 nm. The activity of Succinate Dehydrogenase (SDH) was determined by the reduction of the electron acceptor DCPIP. CONCLUSION: MPTP and the respiratory complex II are identified as the main targets of the iron-dependent action of DOX on the isolated mitochondria. All MPTP protectors tested abolished or weakened the effect of iron and a complex of iron with DOX on Ca2+ -induced MPTP opening, acting in different stages of MPTP activation. These data open new approaches to the modulation of the toxic influence of DOX on mitochondria with the aim to reduce their dysfunction.

The mitochondrial NO-synthase/guanylate cyclase/protein kinase G signaling system underpins the dual effects of nitric oxide on mitochondrial respiration and opening of the permeability transition pore.

The available data on the involvement of nitric oxide (NO) and mitochondrial calcium-dependent NO synthase (mtNOS) in the control of mitochondrial respiration and the permeability transition pore (mPTP) are contradictory. We have proposed that the mitochondrial mtNOS/guanylate cyclase/protein kinase G signaling system (mtNOS-SS) is also implicated in the control of respiration and mPTP, providing the interplay between NO and mtNOS-SS, which, in turn, may result in inconsistent effects of NO. Therefore, using rat liver mitochondria, we applied specific inhibitors of the enzymes of this signaling system to evaluate its role in the control of respiration and mPTP opening. Steady-state respiration was supported by pyruvate, glutamate, or succinate in the presence of hexokinase, glucose, and ADP. When applied at low concentrations, l-arginine (to 500 µm) and NO donors (to 50 µm) activated the respiration and increased the threshold concentrations of calcium and d,l-palmitoylcarnitine required for the dissipation of the mitochondrial membrane potential and pore opening. Both effects were eliminated by the inhibitors of NO synthase, guanylate cyclase, and kinase G, which denotes the involvement of mtNOS-SS in the activation of respiration and deceleration of mPTP opening. At high concentrations, l-arginine and NO donors inhibited the respiration and promoted pore opening, indicating that adverse effects induced by an NO excess dominate over the protection provided by mtNOS-SS. Thus, these results demonstrate the opposite impact of NO and mtNOS-SS on the respiration and mPTP control, which can explain the dual effects of NO.

Glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.

Chronic alcohol intoxication is associated with increased oxidative stress. However, the mechanisms by which ethanol triggers an increase in the production of reactive oxygen species (ROS) and the role of mitochondria in the development of oxidative stress has been insufficiently studied. The biochemical and proteomic data obtained in the present work suggest that one of the main causes of an increase in ROS generation is enhanced oxidation of glutamate in response to long-term alcohol exposure. In the course of glutamate oxidation, liver mitochondria from alcoholic rats generated more superoxide anion and H2O2 than in the presence of other substrates and more than control organelles. In mitochondria from alcoholic rats, rates of H2O2 production and NAD reduction in the presence of glutamate were almost twice higher than in the control. The proteomic study revealed a higher content of glutamate dehydrogenase in liver mitochondria of rats subjected to chronic alcohol exposure. Simultaneously, the content of mitochondrial catalase decreased compared to control. Each of these factors stimulates the production of ROS in addition to ROS generated by the respiratory chain complex I. The results are consistent with the conclusion that glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.

Mitochondrial metabolites in tissues as indicators of metabolic alterations during hibernation.

The decrease in metabolism is one of mechanisms for hibernating animals to resist hypoxia and oxidative stress. Assuming that the inhibition of mitochondria; respiration in torpor and its activation upon arousal are accompanied by changes in the content of mitochondrial substrates, we estimated the levels of endogenous metabolites of the tricarboxylic acid (TCA) cycle in the liver, brown adipose tissue, and the brain of the arctic ground squirrels as possible indicators of mitochondrial processes. The level of lactate in the same tissues and serum was determined as marker of hypoxia. It was found that the isocitrate (ISC) concentration in all tissues was one order of magnitude higher than that of alpha-ketoglutarate (KGL), while succinate was not detected in any of tissues, indicating the inhibition at the initial stages of the TCA cycle. During the torpor, the concentrations of ISC, KGL and lactate predominantly decreased in tissues. Serum lactate decreased five-fold in torpor and was restored in a temperature-dependent manner with a long period of persistence of stable concentration in the range of body temperature between 12 and 27°C upon arousal. The data obtained indicate the development of metabolic depression rather than hypoxia in these tissues.

Effect of progesterone and its synthetic analogues on the activity of mitochondrial permeability transition pore in isolated rat liver mitochondria.

The influence of progesterone and its synthetic analogues on the induction of the Ca(2+)-dependent mitochondrial permeability transition pore (MPTP) has been studied. The novel synthetic analogue of progesterone 17a-acetoxy-3b-butanoyloxy-6-methyl-pregna-4,6-diene-20-on (buterol) was compared with progesterone and medroxyprogesterone acetate (MPA). It was found that progesterone and buterol have opposite effects on the induction of MPTP opening by calcium ions. By contrast to progesterone, which decreased the calcium ion concentration necessary for pore opening, and MPA, which also, although at a lesser extent, activated the pore induction, buterol at a concentration of 20-100 microM blocked the pore opening and increased the calcium retention capacity of mitochondria more than twofold. The action of buterol is specific to the pore since it did not affect the respiration, whereas progesterone completely inhibited NAD-dependent respiration. MPA acted similar to progesterone but less effectively. The inhibitory effect of buterol was eliminated in the presence of carboxyatractyloside, which selectively binds the thiol groups of adenylate translocase and prevents the adenine nucleotide binding. These data indicate that buterol interacts with thiol groups, which explains its inhibitory effect not only on the mitochondrial pore but also on the transport system of xenobiotics in tumor cells in which buterol reduces the multidrug resistance.

Chemically assisted microbial production of succinic acid by the yeast Yarrowia lipolytica grown on ethanol.

A new two-step process of production of succinic acid (SA) has been developed, which includes the microbial synthesis of alpha-ketoglutaric acid by the yeast Yarrowia lipolytica (step 1) and subsequent oxidation of the acid by hydrogen peroxide to SA (step 2). The maximum concentration of SA and its yield were found to be 63.4 g l(-1) and 58% of the ethanol consumed, respectively. The purity of the SA isolated from the culture liquid filtrate reached 100%. The yield of SA was as high as 82% of its amount in the culture liquid filtrate. The quality of the SA produced by the invented method meets the biochemical grade definitions, as is evident from the respiratory and other relevant parameters of rat liver mitochondria upon the oxidation of this SA.

Nonezymatic formation of succinate in mitochondria under oxidative stress.

The products of the reactions of mitochondrial 2-oxo acids with hydrogen peroxide and tert-butyl hydroperoxide (tert-BuOOH) were studied in a chemical system and in rat liver mitochondria. It was found by HPLC that the decarboxylation of alpha-ketoglutarate (KGL), pyruvate (PYR), and oxaloacetate (OA) by both oxidants results in the formation of succinate, acetate, and malonate, respectively. The two latter products do not metabolize in rat liver mitochondria, whereas succinate is actively oxidized, and its nonenzymatic formation from KGL may shunt the tricarboxylic acid (TCA) cycle upon inactivation of alpha-ketoglutarate dehydrogenase (KGDH) under oxidative stress, which is inherent in many diseases and aging. The occurrence of nonenzymatic oxidation of KGL in mitochondria was established by an increase in the CO(2) and succinate levels in the presence of the oxidants and inhibitors of enzymatic oxidation. H(2)O(2) and menadione as an inductor of reactive oxygen species (ROS) caused the formation of CO(2) in the presence of sodium azide and the production of succinate, fumarate, and malate in the presence of rotenone. These substrates were also formed from KGL when mitochondria were incubated with tert-BuOOH at concentrations that completely inhibit KGDH. The nonenzymatic oxidation of KGL can support the TCA cycle under oxidative stress, provided that KGL is supplied via transamination. This is supported by the finding that the strong oxidant such as tert-BuOOH did not impair respiration and its sensitivity to the transaminase inhibitor aminooxyacetate when glutamate and malate were used as substrates. The appearance of two products, KGL and fumarate, also favors the involvement of transamination. Thus, upon oxidative stress, nonenzymatic decarboxylation of KGL and transamination switch the TCA cycle to the formation and oxidation of succinate.

Evidence of the glyoxylate cycle in the liver of newborn rats.

BACKGROUND: It is generally accepted that the glyoxylate cycle exists in microorganisms and higher plants but absent in higher animals. the hypodhesis of the glyoxylate cycle in the tissues of higher animals with a high level of physiological activity was first proposed by Kondrashova and Rodionova in 1971. The goal of this work was yo verifv this in newborn rats, which possess a 2.5-fold hygher physiological activity and oxygen consumption rate than adult rats. MATERIAL/METHODS: Newborn (7-day-old) anradult 1 ats were used for this experiment. The activities of the key enzymes of the glyoxylate cocle (isecitrate lyse and nmalate synthase) were measured by HPLC and spectroscopic methods. The activities of isocitrate lyase and malate synthase were found in the liver homogenates prepared from newborn rats, but not from adult rats. The activities of the enzymes common to both the Krebs cycle and the glyoxylate cycle (citrate synthase, aconitase, and malate dehydrogenase) were 20-40% higher in newborn than in adult rats. CONCLUSIONS: These data indicate the existence of the glyoxylate cycle in animal tissues.