The Effects of the Steroids 5-Androstenediol and Dehydroepiandrosterone and Their Synthetic Derivatives on the Viability of K562, HeLa, and Wi-38 Cells and the Luminol-Stimulated Chemiluminescence of Peripheral Blood Mononuclear Cells from Healthy Volunteers.

In order to evaluate the role of substituents at 3-C and 17-C in the cytotoxic and cytoprotective actions of DHEA and 5-AED molecules, their derivatives were synthesized by esterification using the corresponding acid anhydrides or acid chlorides. As a result, seven compounds were obtained: four DHEA derivatives (DHEA 3-propionate, DHEA 3-butanoate, DHEA 3-acetate, DHEA 3-methylsulfonate) and three 5-AED derivatives (5-AED 3-butanoate, 5-AED 3,17-dipropionate, 5-AED 3,17-dibutanoate). All of these compounds showed micromolar cytotoxic activity toward HeLa and K562 human cancer cells. The maximum cytostatic effect during long-term incubation for five days with HeLa and K562 cells was demonstrated by the propionic esters of the steroids: DHEA 3-propionate and 5-AED 3,17-dipropionate. These compounds stimulated the growth of normal Wi-38 cells by 30-50%, which indicates their cytoprotective properties toward noncancerous cells. The synthesized steroid derivatives exhibited antioxidant activity by reducing the production of reactive oxygen species (ROS) by peripheral blood mononuclear cells from healthy volunteers, as demonstrated in a luminol-stimulated chemiluminescence assay. The highest antioxidant effects were shown for the propionate ester of the steroid DHEA. DHEA 3-propionate inhibited luminol-stimulated chemiluminescence by 73% compared to the control, DHEA, which inhibited it only by 15%. These data show the promise of propionic substituents at 3-C and 17-C in steroid molecules for the creation of immunostimulatory and cytoprotective substances with antioxidant properties.

Possible Participation of Adenine Nucleotide Translocase ANT1 in the Cytotoxic Action of Progestins, Glucocorticoids, and Diclofenac on Tumor Cells.

A comparative analysis of the cytostatic effects of progestins (gestobutanoyl, megestrol acetate, amol, dienogest, and medroxyprogesterone acetate), glucocorticoids (hydrocortisone, dexamethasone), and diclofenac on tumor cells was carried out in order to confirm their in silico predicted probabilities experimentally. The results showed the different sensitivity of HeLa, MCF-7, Hep-2, K-562, and Wi-38 cell lines to progestins, glucocorticoids, and diclofenac. The minimum IC50 was found for progestin gestobutanoyl (GB) as 18 µM for HeLa cells, and varied from 31 to 38 µM for MCF-7, Hep-2, and K-562. Glucocorticoids and diclofenac were much less cytotoxic in the HeLa, MCF-7, and Hep-2 cell lines than progestins, with IC50 values in the range of 150-3000 µM. Myelogenous leukemia K-562 cells were the least sensitive to the action of progestins and glucocorticoids but the most sensitive to diclofenac, which showed a pronounced cytotoxic effect with an IC50 of 31 µM. As we have shown earlier, progestins can uniquely modulate MPTP opening via the binding of adenine nucleotide translocase. On this basis, we evaluated the expression of adenylate nucleotide translocase ANT1 (SLC25 A4) as a possible participant in cytotoxic action in these cell lines after 48 h incubation with drugs. The results showed that progestins differently regulated ANT1 expression in different cell lines. Gestobutanoyl had the opposite effect on ANT1 expression in the HeLa, K562, and Wi-38 cells compared with the other progestins. It increased the ANT1 expression more than twofold in the HeLa and K562 cells but had no influence on the Wi-38 cells. Glucocorticoids and diclofenac increased ANT1 expression in the Wi-38 cells and decreased it in the K562, MCF-7, and Hep-2 cells. The modulation of ANT1 expression discovered in our study can be a new explanation of the cytotoxic and cytoprotective effects of hormones, which can vary depending on the cell type. ANT isoforms in normal and cancerous cells could be a new target for steroid hormone and anti-inflammatory drug action.

The Effect of Progestins on Cytokine Production in the Peripheral Blood Mononuclear Cells of Menopausal Women and Their Luminol-Dependent Chemiluminescence.

Steroid hormones are the key regulators of inflammatory and autoimmune processes. The role of steroid hormones is mostly inhibitory in these processes. The expression of IL-6, TNFα, and IL-1ß, as markers of inflammation, and TGFß, as a marker of fibrosis, could be useful tools to predict the response of an individual's immune system to the different progestins suitable for the treatment of menopausal inflammatory disorders, including endometriosis. In this study, the progestins P4 and MPA, as well as the novel progestin gestobutanoyl (GB), which possess potent anti-inflammatory properties towards endometriosis, were studied at a fixed concentration of 10 µM. Their influence on the production of the above cytokines in PHA-stimulated peripheral blood mononuclear cells (PBMCs) during 24 h incubation was evaluated by ELISA. It was found that synthetic progestins stimulated the production of IL-1ß, IL-6, and TNFα and inhibited TGFß production, while P4 inhibited IL-6 (33% inhibition) and did not influence TGFß production. In the MTT-viability test, P4 also decreased PHA-stimulated PBMC viability by 28% during 24 h incubation, but MPA and GB did not have any inhibitory or stimulatory effects. The luminol-dependent chemiluminescence (LDC) assay revealed the anti-inflammatory and antioxidant properties of all the tested progestins, as well as some other steroid hormones and their antagonists: cortisol, dexamethasone, testosterone, estradiol, cyproterone, and tamoxifen. Of these, tamoxifen showed the most pronounced effect on the oxidation capacity of PBMC but not on that of dexamethasone, as was expected. Collectively, these data demonstrate that PBMCs from menopausal women respond differently to P4 and synthetic progestins, most likely due to distinct actions via various steroid receptors. It is not only the progestin affinity to nuclear progesterone receptors (PR), androgen receptors, glucocorticoid receptors, or estrogen receptors that is important for the immune response, but also the membrane PR or other nongenomic structures in immune cells.

Specific Features of Mitochondrial Dysfunction under Conditions of Ferroptosis Induced by t-Butylhydroperoxide and Iron: Protective Role of the Inhibitors of Lipid Peroxidation and Mitochondrial Permeability Transition Pore Opening.

Recent studies have indicated the critical importance of mitochondria in the induction and progression of ferroptosis. There is evidence indicating that tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide, is capable of inducing ferroptosis-type cell death. We investigated the effect of TBH on the induction of nonspecific membrane permeability measured by mitochondrial swelling and on oxidative phosphorylation and NADH oxidation assessed by NADH fluo rescence. TBH and iron, as well as their combinations, induced, with a respective decrease in the lag phase, the swelling of mitochondria, inhibited oxidative phosphorylation and stimulated NADH oxidation. The lipid radical scavenger butylhydroxytoluene (BHT), the inhibitor of mitochondrial phospholipase iPLA2γ bromoenol lactone (BEL), and the inhibitor of the mitochondrial permeability transition pore (MPTP) opening cyclosporine A (CsA) were equally effective in protecting these mitochondrial functions. The radical-trapping antioxidant ferrostatin-1, a known indicator of ferroptotic alteration, restricted the swelling but was less effective than BHT. ADP and oligomycin significantly decelerated iron- and TBH-induced swelling, confirming the involvement of MPTP opening in mitochondrial dysfunction. Thus, our data showed the participation of phospholipase activation, lipid peroxidation, and the MPTP opening in the mitochondria-dependent ferroptosis. Presumably, their involvement took place at different stages of membrane damage initiated by ferroptotic stimuli.

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.

Involvement of Multidrug Resistance Modulators in the Regulation of the Mitochondrial Permeability Transition Pore.

The permeability transition pore in mitochondria (MPTP) and the ATP-binding cassette transporters (АВС transporters) in cell membranes provide the efflux of low-molecular compounds across mitochondrial and cell membranes, respectively. The inhibition of ABC transporters, especially of those related to multi drug resistance (MDR) proteins, is an actively explored approach to enhance intracellular drug accumulation and increase thereby the efficiency of anticancer therapy. Although there is evidence showing the simultaneous effect of some inhibitors on both MDR-related proteins and mitochondrial functions, their influence on MPTP has not been previously studied. We examined the participation of verapamil and quinidine, classified now as the first generation of MDR modulators, and avermectin, which has recently been actively studied as an MDR inhibitor, in the regulation of the MPTP opening. In experiments on rat liver mitochondria, we found that quinidine lowered and verapamil increased the threshold concentrations of calcium ions required for MPTP opening, and that they both decreased the rate of calcium-induced swelling of mitochondria. These effects may be associated with the positive charge of the drugs and their aliphatic properties. Avermectin not only decreased the threshold concentration of calcium ions, but also by itself induced the opening of MPTP and the mitochondrial swelling inhibited by ADP and activated by carboxyatractyloside, the substrate and inhibitor of adenine nucleotide translocase (ANT), which suggests the involvement of ANT in the process. Thus, these data indicate an additional opportunity to evaluate the effectiveness of MDR modulators in the context of their influence on the mitochondrial-dependent apoptosis.

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.

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.