Structural Stereochemistry of Androstene Hormones Determines Interactions with Human Androgen, Estrogen, and Glucocorticoid Receptors.

DHEA, 17α-AED, 17ß-AED, and 17ß-AET exhibit strong biological activity that has been attributed to androgenic, estrogenic, or antiglucocorticoid activity in vivo and in vitro. This study compared DHEA, 17α-AED, 17ß-AED, and 17ß-AET for their ability to activate the human AR, ER, and GR and determine the relative androgenicity, estrogenicity, and glucocorticoid activity. The results show that, at the receptor level, these androstene hormones are weak AR and even weaker ER activators. Direct androstene hormone activation of the human AR, ERα, and ERß may not be essential for their biological function. Similarly, these hormones indirectly activated the human GR, only in the presence of high dexamethasone concentrations. These results underscore the major difference between androstene hormone interactions with these nuclear receptors and their biological effects.

17α-androstenediol-mediated oncophagy of tumor cells by different mechanisms is determined by the target tumor.

Δ5-androstene-3ß,17α-diol (17α-AED) mediates oncophagy of human myeloid, glioma, and breast tumor cells by apoptotic- and autophagic-programmed cell death pathways, whereas the 17ß-epimer does not. In hematologically derived myeloid tumor cells, 17α-AED induced apoptosis, as determined by TUNEL staining, caspase, PARP activation, and electron microscopy. In contrast, 17α-AED treatment of glioma cells of neuroectodermal lineaged induced autophagy, evident by the presence of acidic vesicular organelles, LC3 processing, and upregulation of beclin-1. Proliferation inhibition studies on primary and established glioma cells demonstrated that the IC-50 of the steroid is ∼15 µM. In the case of breast cancer cells, the bioactivity of 17α-AED is independent of the expression of estrogen or androgen receptors. Collectively, oncophagy is induced by 17α-AED treatment in human tumor cells and proceeds by the induction of either autophagy or apoptosis. The neoplastic cell determines which oncophagic pathway is utilized.

Modulation of traumatic brain injury using progesterone and the role of glial cells on its neuroprotective actions.

TBI is a complex disease process caused by a cascade of systemic events. Attention is now turning to drugs that act on multiple pathways to enhance survival and functional outcomes. Progesterone has been found to be beneficial in several animal species, different models of brain injury, and in two preliminary human clinical trials. It holds promise as a treatment for TBI. Progesterone's multiple mechanisms of action may work synergistically to prevent the death of neurons and glia, leading to reduced morbidity and mortality. This review highlights the importance of glial cells as mediators of progesterone's actions on the CNS and describes progesterone's pleiotrophic effects on immune enhancement and neuroprotection in TBI.

Beta androstenediol mitigates the damage of 1 GeV/n Fe ion particle radiation to the hematopoietic system.

Space exploration is associated with exposure to 1-3 Gy solar particle radiation and galactic cosmic radiation that could increase cancer rates. Effective nontoxic countermeasures to high linear energy transfer (LET) radiation exposure are highly desirable but currently not available. The aim was to determine whether a single subcutaneous injection of androstenediol (Δ(5) androsten-3ß, 17ß-diol [AED]) could mitigate and restore the mouse hematopoetic system from the radiation-mediated injury of 3 Gy whole-body high LET (56)Fe(26+) exposure. The findings show that postradiation AED treatment has an overall positive and significant beneficial effect to restore the levels of hematopoeitic elements (p<0.001). Androstenediol treatment significantly increased monocyte levels at days 4, 7, and 14 and, similarly, increased red blood cell, hemoglobin, and platelet counts. Flow cytometry analysis 14 days after radiation and AED treatment demonstrated an increase (p<0.05) in bone marrow cells counts. Ex vivo osteoclastogenesis studies show that AED treatment is necessary and advantageous for the development and restoration of osteoclastogenesis after radiation exposure. These findings clearly show that androstenediol functions as a countermeasure to remedy hematopoeitic injury mediated by high LET iron ion radiation. Presently, no other agent has been shown to have such properties.

The neuro-steroid, 5-androstene 3ß,17α diol; induces endoplasmic reticulum stress and autophagy through PERK/eIF2α signaling in malignant glioma cells and transformed fibroblasts.

In this study, we identified a mechanism by which the neuro-steroid, 5-androstene 3ß,17α diol (17α-AED) induces autophagy in human malignant glioma cells and transformed fibroblasts. 17α-AED treatment induced endoplasmic reticulum (ER) stress, identified by the partial activation of an unfolded protein response in T98G, U87MG, U251MG, LN-18, LN-229 and LN-Z308 glioma cell lines. In this regard, there were increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein of 78kDa transcripts but no splicing of X-box-binding protein 1 mRNA or processing of activating transcription factor-6 in glioma cells treated with the neuro-steroid. 17α-AED induced eukaryotic translational initiation factor 2α (eIF2α) phosphorylation in glioma cells which correlated with microtubule-associated protein-light chain 3 (LC3) conversion from LC3-I to -II. In transformed murine embryonic fibroblasts (MEFs) that are deficient of eIF2α function or T98G glioma cells transfected with a dominant-negative eIF2α construct, 17α-AED induced LC3 conversion was significantly reduced as compared to control cells. Neuro-steroid treatment caused the activation of the eIF2α kinase, protein kinase-like ER kinase (PERK) but not other eIF2α kinases in glioma cells. Moreover, eIF2α phosphorylation and LC3 conversion, in response to 17α-AED treatment, was blocked in MEFs that lacked PERK activity. T98G cells transfected with a dominant-negative PERK construct exhibited an attenuated response to neuro-steroid treatment in terms of decreases in: eIF2α activation; CHOP expression; the incidence of autophagy; and cytotoxicity. These results demonstrate that ER stress is linked to 17α-AED induced autophagy by PERK/eIF2α signaling in human malignant glioma cells and transformed fibroblasts.

Effects of dehydroepiandrosterone-sulfate (DHEA-S) and benznidazole treatments during acute infection of two different Trypanosoma cruzi strains.

A significant role for hormones in regulating the balance of Th1- and Th2-associated cytokines with a role in modulating diseases has been accumulating. Previously, we reported that dehydroepiandrosterone (DHEA), the most abundant steroid hormone synthesized by the adrenal cortex, markedly reduced the blood and tissue parasites in experimentally Trypanosoma cruzi-infected rats. Based on these findings, the main purpose of this study was to investigate the effect of dehydroepiandrosterone-sulfate ester (DHEA-S) therapy alone or in combination with benznidazole (BNZ) (recommended in Brazil for the treatment of T. cruzi infection) will be effective during the acute phase of two different lineages of T. cruzi strains: type I (Y strain) and type II (Bolivia strain) of T. cruzi. Administration of either DHEA-S or BNZ alone or in combination significantly reduced the Y strain parasite load as compared with untreated. Furthermore treatment with DHEA-S resulted in Bolivia strain clearance. This protective effect of DHEA-S was associated with the host's immune response, as evidence by enhanced levels of interferon-gamma and interleukin-2. DHEA-S treatment also increased peritoneal macrophages levels and nitrite production. DHEA-S treatment was effective in reducing the mortality rate as compared to BNZ alone or to combiner DHEA-S+BNZ treatment of T. cruzi Bolivia strain infected animals. These findings suggest that hormonal therapy may have a protective effect in the treatment of T. cruzi infection.

The anti-tumor effects of androstene steroids exhibit a strict structure-activity relationship dependent upon the orientation of the hydroxyl group on carbon-17.

Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene-diols or -triols in alpha- and beta-epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Delta(5)cycloperhydrophenanthrene ring. 5-Androstene-3beta,17beta-diol (3beta,17beta-AED) functions to upregulate immunity and the addition of a third hydroxyl group at C-7 in the alpha- or beta-orientation (3beta,7alpha,17beta-AET and 3beta,7beta,17beta-AET, respectively) enhances the immunological activity of the molecule. In contrast, 5-androstene-3beta,17alpha-diol (3beta,17alpha-AED) possesses potent anti-tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C-7 to make 5-androstene-3beta,7alpha,17alpha-triol (3beta,7alpha,17alpha-AET) and compared the anti-tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3beta,17alpha-AED. The ranking of the anti-tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3beta,17alpha-AED ( approximately 10 microm) > 3beta,7alpha,17alpha-AET ( approximately 30 microm) " 3beta,7alpha,17beta-AET ( approximately 150 microm)> 3beta,7beta,17beta-AET (not achievable) >or= 3beta,17beta-AED (not achievable). 3beta,17alpha-AED and 3beta,7alpha,17alpha-AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C-17 dictates the anti-tumor activity of the androstenes and must be in the alpha-configuration, demonstrating a strict structure-activity relationship.

Androstenediol reverses steroid-inhibited wound healing.

It is well recognized that stress of any nature will cause a delay in the wound healing response. This delayed healing response appears closely associated with immune regulators. In this study, CD-1 mice were injected with a long acting form of methyl prednisolone to cause a steroid-induced immune suppression. After 24 hours, two 6-mm full thickness wounds were placed on the animals' backs and one group of animals received the immune-regulating hormone, androstenediol. Wound contraction was quantified by planimetry for the subsequent 14 days. Animals that were stressed with methyl prednisolone but receiving androstenediol contracted their open wounds at faster rates compared with methyl prednisolone-stressed animals treated with the vehicle alone. These findings suggest that restoration of immune regulation by androstenediol can reverse the delayed open wound contraction secondary to steroid stress.

Autophagy and the functional roles of Atg5 and beclin-1 in the anti-tumor effects of 3beta androstene 17alpha diol neuro-steroid on malignant glioma cells.

In this study, we demonstrate that the anti-tumor activity of the neuro-steroid, 3beta androstene 17alpha diol (17alpha-AED) on malignant glioma cells is mediated by the induction of autophagy. 17alpha-AED can inhibit the proliferation an induce cell death of multiple, unrelated gliomas with an IC(50) between 8 and 25muM. 17alpha-AED treatment induced the formation of autophagosomes and acidic vesicular organelles in human malignant gliomas which was blocked by bafilomycin A1 or 3-methyladenine. Cleavage of microtubule-associated protein-light chain 3 (LC3), an essential step in autophagosome formation, was detected in human malignant glioma cells exposed to 17alpha-AED. In 17alpha-AED treated T98G glioma cells there was an increase in the autophagy related proteins Atg5 and beclin-1. Silencing of ATG5 or beclin-1 with small interfering RNA significantly reduced the incidence of autophagy in 17alpha-AED treated malignant gliomas and attenuated the cytotoxic effects of the neuro-steroid indicating that the induction of autophagy mediates the anti-glioma activity of 17alpha-AED rather than serving as a cyto-protective response. These results demonstrate that 17alpha-AED possesses significant anti-glioma activity when used at pharmacologically relevant concentrations in vitro and the cytotoxic effects are resultant from the induction of autophagy.

Beta-androstenes and resistance to viral and bacterial infections.

This report illustrates that the beta-androstenes are indeed able to upregulate the host immune response to a level that enables the host to resist lethal infection by viruses or bacteria. These agents consist of a subgroup of steroids, which also mediates a rapid recovery of hematopoietic precursor cells after whole-body lethal radiation injury. In vivo, the androstenes increase the levels of the Th1 cytokines such as IL-2, IL-3, and IFN. Similar to hydrocortisone, they suppress inflammation, but without immune suppression, and have a role in the maintenance of the Th1/Th2 balance and immune homeostasis.

Effects of alpha/beta-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts.

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17beta-androstenediol (beta-AED), 7beta,17beta-androstenetriol (beta-AET) or the 17alpha-androstenediol (alpha-AED), and 7alpha,17beta-androstenetriol (alpha-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The alpha-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again alpha-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-alpha and -gamma, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-gamma. Analysis of PPAR-gamma mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased PPAR-gamma activation by 19%, while beta-AED conferred a 37% decrease in PPAR-gamma activation. In conclusion, alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-gamma that in turn caused mild apoptosis of FOB-9 cells.

Androstenetriol immunomodulation improves survival in a severe trauma hemorrhage shock model.

BACKGROUND: Traumatic shock activates the hypothalamic-pituitary-adrenal axis (HPA) to mediate a cascade of defensive mechanisms that often include overwhelming inflammatory response and immunosuppression, which may lead to multiple organ failure. Androstenetriol (5 androstene, 3beta, 7beta, 17beta triol-AET) is a metabolite of dehydroepiandrosterone that markedly up regulates host immune response, prevents immune suppression, modulates inflammation and improves survival after lethal infections by pathogens and lethal radiation. HYPOTHESIS: AET-induced immune modulation will improve survival in a conscious rodent model of traumatic shock. METHODS: A relevant traumatic shock rodent model that applies to both combat and civilian sectors was used. After creation of a midline laparotomy (soft tissue trauma), animals were hemorrhaged to a mean arterial pressure of 35-40 mm Hg. Resuscitation was initiated sixty minutes later with crystalloid fluid and packed red blood cells and animals were observed for two days. In a randomized and blinded fashion, AET or vehicle was administered subcutaneously at the beginning of resuscitation. RESULTS: In the vehicle group 5 out of 16 animals survived, (31%). In contrast, 9 out of 13 animals who received AET survived (69%), (Fisher Exact Test p < 0.05). Survival in the AET treatment group was associated with reduced levels of IL-6, IL-10, and IL-18, and enhanced IFN-gamma and IL-2 levels. CONCLUSION: : The results indicate that AET provides a significant protective effect and improves survival in a clinically relevant model of traumatic hemorrhagic shock. AET protective effects are associated with an elevation of Th1 and reduction of Th2 cytokines.

Androstenetriol improves survival in a rodent model of traumatic shock.

UNLABELLED: Trauma results in activation of the hypothalamic-pituitary-adrenal axis to mediate a cascade of neurohormonal changes as a defensive mechanism. Its prolongation, however, leads to a hypermetabolic, hypoperfused, and immunosuppressed state, setting the stage for subsequent sepsis and organ failure. Androstenetriol (5-androstene-3beta, 7beta, 17betatriol - AET), a metabolite of dehydroepiandrosterone, up-regulates the host immune response markedly, prevents immune suppression and controls inflammation, leading to improved survival after lethal infections by several diverse pathogens and lethal radiation. Such actions may be useful in improving survival from traumatic shock. HYPOTHESIS: The neurosteroid AET will increase survival following traumatic shock. METHODS: A combat relevant model of traumatic shock was used. Male Sprague-Dawley rats were anesthetized, catheterized and subjected to soft tissue injury (laparotomy). Animals were allowed to regain consciousness over the next 0.5 h and then bled 40% of their blood volume over 15 min. Forty-five minutes after the onset of hemorrhage animals were randomized to receive either a single subcutaneous dose of AET (40 mg/kg, sc) or vehicle (methylcellulose). Volume resuscitation consisted of l-lactated Ringer's (three times the shed blood volume), followed by packed red blood cells (one-third shed red cell volume). Animals were observed for three days. RESULTS: A total of 24 animals were studied. Of the 12 animals randomized to receive AET, all (100%) survived compared to 9 of 12 animals (75%) randomized to receive the vehicle (p < 0.05). CONCLUSION: AET significantly improved survival when administered subcutaneously in a single dose in this rodent model of traumatic shock. Further survival and mechanism studies are warranted.

Immune up-regulation and tumor apoptosis by androstene steroids.

beta Androstenes steroid up-regulates immunity to increase resistance against lethal infection and lethal radiation, and mediates a rapid recovery of hematopoietic precursor cells after radiation injury. beta Androstenetriol increases the levels of the TH(1) cytokines, IL-2, IL-3, IFN gamma and counteracts hydrocortisone mediated immune suppression. In contrast, 17 alpha androstenediol inhibits proliferation and mediates apoptosis in tumor cells of murine and human origin. Its epimer 17beta androstenediol does not. The antiproliferative functions of 17 alpha androstenediol are not dependent on either the estrogen or androgen receptors. Our findings show that beta androstenes and analogs protect the host from lethal infection by DNA or RNA viruses such as, herpesvirus type 2, coxsackievirus B4, influenza, and arthropod borne viruses. These androstenes also protected the host from lethal bacterial infections by Enterococcus faecalis, Pseudomonas aeruginosa, and Klebsiella pneumonia and from parasites infections, i.e. Cryptosporidium parvum, and malaria. In vivo, the level of potency follows the order: dehydroepiandrosterone<<

Androstenediol and dehydroepiandrosterone protect mice against lethal bacterial infections and lipopolysaccharide toxicity.

The protective effects of the hormones androstenediol (androstene-3beta, 17beta,-diol; AED) and dehydroepiandrosterone (5-androsten-3beta-ol-17-one; DHEA) on the pathophysiology of two lethal bacterial infections and endotoxin shock were examined. The infections included a gram-positive organism (Enterococcus faecalis) and a gram-negative organism (Pseudomonas aeruginosa). Both hormones protected mice from the lethal bacterial infections and from lipopolysaccharide (LPS) challenge. Treatment of animals lethally infected with P. aeruginosa with DHEA resulted in a 43% protection whereas treatment with AED gave a 67% protection. Both hormones also protected completely animals infected with an LD50 dose of E. faecalis. Similarly, the 88% mortality rate seen in LPS challenge was reduced to 17% and 8.5%, by treatment with DHEA and AED, respectively. The protective influences of both steroids were shown not to be directly antibacterial, but primarily an indirect antitoxin reaction. DHEA appears to mediate its protective effect by a mechanism that blocks the toxin-induced production of pathophysiological levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1. AED usually had greater protective effects than DHEA; however, the AED effect was independent of TNF-alpha suppression, both in vivo and in vitro. The data suggest that both DHEA and AED may have a role in the neuro-endocrine regulation of antibacterial immune resistance.