Regulation of steroid hormone action in target cells by specific hormone-inactivating enzymes.

The target cell sensitivity of steroid hormones is determined by the concerted action of specific hormone receptors and steroid-inactivating enzymes. In recent years, a considerable amount of knowledge has been obtained on hormone receptor concentration-based target cell sensitivity. However, an equal understanding of the role of specific steroid-inactivating enzymes in hormone action is absent. This review highlights the importance of specific steroid-inactivating enzymes in the control of target cell sensitivity of mineralocorticoids, glucocorticoids, androgens, and estrogens. Two classes of enzymes that are actively involved in this process are hydroxysteroid dehydrogenases and hydroxysteroid sulfotransferases. Some of the target cells in which the critical roles of these enzymes have been extensively characterized are those of the kidney, endometrium, and liver. cDNA for many of these enzymes have already been cloned, and rapid progress in the elucidation of this component of steroid hormone action is anticipated.     

Sex steroid effects at target tissues: mechanisms of action

Our understanding of the mechanisms of sex hormone action has changed dramatically over the last 10 years. Estrogens, progestins, and androgens are the steroid hormones that modulate reproductive function. Recent data have shown that many other tissues are targets of sex hormones in addition to classical reproductive organs. This review outlines new advances in our understanding of the spectrum of steroid hormone ligands, newly recognized target tissues, structure-function relationships of steroid receptors, and, finally, their genomic and nongenomic actions. Sex-based specific effects are often related to the different steroid hormone mileu in men compared with women. Understanding the mechanisms of sex steroid action gives insight into the differences in normal physiology and disease states.     

Intracellular flow of particulate steroid-receptor complexes in steroid target cells

The structural expression of nuclear transportation of receptor-bound oestradiol was investigated by immunoelectron microscopy on human breast cancer cells. In oestradiol-treated cells during transportation, oestradiol antibodies attached to cytoplasmic oestradiol-bearing particles which seemed to interact with the nuclear membrane. These particles, subserving the intracellular flow of steroid-receptor complexes, could constitute a special cellular system of macromolecular transport, communication and compartmentalisation.     

p53 as a unique target of action of cisplatin in acute leukaemia cells

Acute promyelocytic leukaemia (APL) occurs in approximately 10% of acute myeloid leukaemia patients. Arsenic trioxide (ATO) has been for APL chemotherapy, but recently several ATO‐resistant cases have been reported worldwide. Cisplatin (CDDP) enhances the toxicity of ATO in ovarian, lung cancer, chronic myelogenous leukaemia, and HL‐60 cells. Hence, the goal of this study was to investigate a novel target of CDDP action in APL cells, as an alternate option for the treatment of ATO‐resistant APL patients. We applied biochemical, molecular, confocal microscopy and advanced gene editing (CRISPR‐Cas9) techniques to elucidate the novel target of CDDP action and its functional mechanism in APL cells. Our main findings revealed that CDDP activated p53 in APL cells through stress signals catalysed by ATM and ATR protein kinases, CHK1 and CHK2 phosphorylation at Ser 345 and Thr68 residues, and downregulation and dissociation of MDM2‐DAXX‐HAUSP complex. Our functional studies confirmed that CDDP‐induced repression of MDM2‐DAXX‐HAUSP complex was significantly reversed in both nutilin‐3‐treated KG1a and p53‐knockdown NB4 cells. Our findings also showed that CDDP stimulated an increased number of promyelocytes with dense granules, activated p53 expression, and downregulated MDM2 in liver and bone marrow of APL mice. Principal conclusion of our study highlights a novel mode of action of CDDP targeting p53 expression which may provide a basis for designing new anti‐leukaemic compounds for treatment of APL patients.     

Membranes as modulators of amyloid protein misfolding and target of toxicity

Abnormal protein aggregation is a hallmark of various human diseases. α-Synuclein, a protein implicated in Parkinson's disease, is found in aggregated form within Lewy bodies that are characteristically observed in the brains of PD patients. Similarly, deposits of aggregated human islet amyloid polypeptide (IAPP) are found in the pancreatic islets in individuals with type 2 diabetes mellitus. Significant number of studies have focused on how monomeric, disaggregated proteins transition into various amyloid structures leading to identification of a vast number of aggregation promoting molecules and processes over the years. Inasmuch as these factors likely enhance the formation of toxic, misfolded species, they might act as risk factors in disease. Cellular membranes, and particularly certain lipids, are considered to be among the major players for aggregation of α-synuclein and IAPP, and membranes might also be the target of toxicity. Past studies have utilized an array of biophysical tools, both in vitro and in vivo, to expound the membrane-mediated aggregation. Here, we focus on membrane interaction of α-synuclein and IAPP, and how various kinds of membranes catalyze or modulate the aggregation of these proteins and how, in turn, these proteins disrupt membrane integrity, both in vitro and in vivo. The membrane interaction and subsequent aggregation has been briefly contrasted to aggregation of α-synuclein and IAPP in solution. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells

Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 anti-PR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [³H]R5020, [³H]dexamethasone, [³H]OH-tamoxifen or [³H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4–6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presense of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found.

During primary culture, the effects of progesterone (P) and estradiol (E₂) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by E₂. Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.     

Alkyl-type glycerolipids as modulators of tumor cells destruction

The review summarizes current information on the biological activity and search of the antineoplastic mechanism of action among alkyl glycerolipids. Special attention is paid to following problems considered in experimental papers published during the last decade: selective ability phosphorus alkyl glycerolipids towards tumor cells using edelfosine and its analogues. The review contains modern information available in the literature, on a possible mechanism cytotoxic effect of such compounds.     

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells

Raloxifene and idoxifene areselective estrogen receptor modulators (SERMs) that exhibittissue-specific agonist or antagonist properties via interactions withthe estrogen receptor (ER). Both compounds are similarlyosteoprotective in the ovariectomized rat in vivo as assessed bymeasurement of bone mineral density, urinary pyridinium cross-links,and serum osteocalcin, suggesting a similar mechanism of action.However, we have identified a fundamental difference in this mechanismvia the estrogen response element (ERE) in osteoblast-like cells. Withthe use of ERE-luciferase reporter constructs, raloxifene, like thecomplete ER-antagonist ICI-182780, acts as an antagonist viathe ERE in osteoblastic cells. In contrast, idoxifene, like17-estrogen itself and 4-OH-tamoxifen, acts as an agonist inosteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780and raloxifene inhibited the ERE-dependent agonist activity of17-estradiol and idoxifene in osteoblastic cells. In contrast, inbreast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 hadno agonist activity and, indeed, raloxifene and idoxifene were potentantagonists of ERE-mediated 17-estradiol action, indicating anERE-dependent mode of action in these cells. Although these SERMsexhibit a similar antagonist activity profile in breast cells, they canbe distinguished mechanistically in osteoblastic cells.

     

Chalcogenopyrylium dyes as inhibitors/modulators of P-glycoprotein in multidrug-resistant cells

A series of chalcogenopyrylium dyes were evaluated as modulators/inhibitors of P-glycoprotein (Pgp). Their ability to inhibit verapamil (VER)-dependent ATPase activity (IC(50) values) in lipid-activated, mouse Cys-less mdr3 Pgp was determined. Their ability to promote calcein-AM (CAM) uptake in MDCKII-MDR1 cells and their capacity to be transported by Pgp in monolayers of MDCKII-MDR1 cells were also evaluated. The chalcogenopyrylium dyes promoted CAM uptake with values of EC(50) between 5 x 10(-6) and 3.5 x 10(-5)M and 7 of the 9 dyes examined in transport studies were substrates for Pgp with efflux ratios (P(BA/AB)) between 14 and 390. Binding of three compounds (1-S, 3-S, and 4-S) to Pgp was also assessed by fluorescence. These three thiopyrylium dyes showed increased fluorescence upon binding to Pgp, giving apparent binding constants, K(app), on the order of 10(-7) to 10(-6)M. Compound 8-Te was particularly intriguing since it appeared to influence Pgp at low micromolar concentrations as evidenced by its influence on VER-stimulated ATPase activity (IC(50) of 1.2 x 10(-6)M), CAM uptake (EC(50) of 5.4 x 10(-6)M), as well as [(3)H]-vinblastine transport by Pgp in cells (IC(50) of 4.3 x 10(-6)M) and within inside-out membrane vesicles (IC(50) of 9.6 x 10(-6)M). Yet, Pgp did not influence the distribution of 8-Te in MDCKII-MDR1 monolayers suggesting that 8-Te may bind to an allosteric site.     

Effect of steroid hormones on vaginal epithelial cells: Anin vitro model for steroid hormone action

Conditions have been standardized to maintain rat vaginal epithelial cellsin vitro with more than 95% viability. Cultured epithelial cells were used to study the effects of normal fetal calf scrum, estradiol and progesterone on the incorporation of [³H]-uridine in RNA and incorporation of [¹⁴C]-aminoacids in proteins. While fetal calf serum and estradiol stimulate the incorporation of both uridine and afno acids, progesterone did not show any effect. Estradiol treated vaginal cells show typical fcroridges (indicative of keratinization of cells) in contrast to estradiol deprived cells, which show microvilli on cell surface when examined in scanning electron microscope.     

Responsiveness and mechanisms of action of steroid hormones in human endometrial adenocarcinoma cells

The responsiveness and action mechanisms of steroid hormones and epidermal growth factor on human endometrial carcinoma cells are analyzed by using in vitro culture system. 1) The Ishikawa cells, derived from a well differentiated endometrial adenocarcinoma and possess ER and PR, are shown to respond to estrogens by increasing a variety of parameters, viz cell proliferation, PR levels, ALP and DNA polymerase activities. 2) ER and PR of those cells are localized in the nuclei by immunocytochemical staining using the monoclonal antibodies against to ER and PR, confirming the correctness of Gorski and Greene's one step theory involving the action mechanisms of steroid hormones. 3) Progestins reduced the ER level and stimulate E2DH activities and glycogen content, which are completely abolished by anti-progestin (RU486), suggesting that PR of those cells should be functional. 4) These responses to steroid hormones of Ishikawa cells are synergistically enhanced or appeared earlier by addition of EGF. 5) The main metabolite of E2 incubated with Ishikawa cells is E2-3-sulfate instead of E1, indicate that the higher estrogenic status may be persisted in endometrial cancer tissues.     

From the structure of steroid receptors to their assessment by immunocytochemistry in target cells

Immunohistochemical studies with antibodies to steroid hormone receptors provide new insight in the mechanism of action of steroid hormones. Immunologically reactive estrogen and progesterone receptors are found exclusively in cell nuclei of target cells even in the absence of the hormonal ligand. A hormonal treatment inducing receptor transformation and "translocation" to the nucleus does not modify the intracellular distribution of the receptor. This result is in contradiction with most biochemical studies which show a displacement of receptor from the cytosolic fraction to the nuclear fraction after hormone-receptor complex formation. We propose that different affinity levels of the non-transformed and hormone-complexed receptor molecules for nuclear structure produce unequal losses of nuclear receptor during homogenization. A lesser loss appears as an increase in nuclear binding sites or immunologically reactive receptor. The glucocorticosteroid receptor differs from the others in that it shows an increase of nuclear immunoreactive receptor after hormone administration. This result was accepted as evidence for a nuclear translocation in the sense initially proposed for all steroid hormones. Alternatively, one may propose another explanation based on the same experimental artefact as invoked for the estrogen and progesterone cytosol receptors. A higher affinity of the hormone-complexed receptor entails a lesser loss from the nucleus during tissue processing, and consequently an apparent increase in nuclear staining. Such a possibility is currently tested in parallel with the progesterone receptor.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone, all had IC(50) values between 1 and 5microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

G-quadruplex binders as cytostatic modulators of innate immune genes in cancer cells

G-quadruplexes (G4s) are non-canonical nucleic acid structures involved in fundamental biological processes. As G4s are promising anticancer targets, in past decades the search for effective anticancer G4 binders aimed at the discovery of more cytotoxic ligands interfering with specific G4 structures at oncogenes or telomeres. Here, we have instead observed a significant activation of innate immune genes by two unrelated ligands at non-cytotoxic concentrations. The studied G4 binders (pyridostatin and PhenDC3) can induce an increase of micronuclei triggering the activation of the cytoplasmic STING (stimulator of interferon response cGAMP interactor 1) signaling pathway in human and murine cancer cells. Ligand activity can then lead to type I interferon production and innate immune gene activation. Moreover, specific gene expression patterns mediated by a G4 binder in cancer cells correlate with immunological hot features and better survival in human TCGA (The Cancer Genome Atlas) breast tumors. The findings open to the development of cytostatic G4 binders as effective immunomodulators for combination immunotherapies in unresponsive tumors.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100 microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100 microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1 microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone all had IC(50) values between 1 and 5 microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

Role of macrophages as modulators but not as autonomous accessory cells in primary antibody response

The role of macrophages (M phi) in the in vitro primary antibody response of murine lymphocytes to sheep erythrocytes was investigated. Peritoneal M phi were activated to express Ia antigens either in vitro or in vivo. Nonactivated Ia- M phi were also examined. We observed that only Ia- M phi but also Ia+ M phi failed to trigger the antibody response, in contrast with splenic dendritic cells (DC) which served as potent and autonomous accessory cells, but that M phi modulated the level of response which was dependent primarily on the DC content of culture. The modulation appeared to incline to suppression rather than enhancement, when M phi were allowed to remain throughout the culture period for 4 days. A highly enhancing capacity of M phi, however, could be revealed by removing M phi 2 days after the initiation of culture, indicating that M phi exerted their suppressive effect more strongly in the late phase than in the early phase of in vitro antibody response. The modulatory activity seemed higher in Ia+ M phi than in Ia- M phi.     

Mechanism of action of levonorgestrel: in vitro metabolism and specific interactions with steroid receptors in target organs.

Levonorgestrel (LNG) is a synthetic steroid that displays potent progestional and androgenic effects but it lacks estrogen-like activity. To examine the mode of action of this progestin, we studied its metabolism in vitro in target organs and the specific interactions of LNG and its metabolites with putative steroid receptors. The results demonstrated that [³H]LNG was efficiently converted to A-ring reduced derivatives when incubated with rat hypothalamus and pituitary. Under optimal incubation conditions, [³H]5-dihydro LNG (5-LNG) and [³H]3-5-tetrahydro LNG (3,5-LNG) were identified as the major metabolic conversion products, while [³H]3ß, 5-LNG formation occured to a lesser extent. A-ring reduction of LNG was NADPH-dependent. Assessment of the relative binding affinities of LNG and its derivatives to progesterone (PR), androgen (AR) and estrogen (ER) receptors by displacement analysis revealed that unchanged LNG binds with high affinity to PR and AR but not to ER. 5-LNG exhibited a diminished though significant interactions with PR and an enhanced binding affinity for AR as compared with LNG, indicating that 5-reduction of LNG increases its affinity for AR. The most striking finding was that further reduction of the 5-LNG molecule at C-3 abolished its binding activity to PR, AR, and even to ER. The overall data provides a plausible explanation for the lack of estrogen agonistic action of LNG and for its potent progestational and androgenic effects.