The neuro-steroid, 3beta androstene 17alpha diol exhibits potent cytotoxic effects on human malignant glioma and lymphoma cells through different programmed cell death pathways.

The neuro-steroids 3beta-androstene-17alpha-diol (17alpha-AED), 3beta-androstene-17beta-diol (17beta-AED), 3beta-androstene-7alpha,-17beta-triol (7alpha-AET) and 3beta-androstene-7beta,-17beta-triol (7beta-AET) are metabolites of dehydroepiandrosterone and are produced in neuro-ectodermal tissue. Both epimers of androstenediols (17alpha-AED and 17beta-AED) and androstenetriols (7alpha-AET and 7beta-AET) have markedly different biological functions of their chemical analogue. We investigated the cytotoxic activity of these neuro-steroids on human T98G and U251MG glioblastoma and U937 lymphoma cells. Proliferation studies showed that 17alpha-AED is the most potent inhibitor, with an IC(50) approximately 15 microM. For T98G glioma, 90% inhibition was achieved with 25 muM of 17alpha-AED. Other neuro-steroids tested only marginally suppressed cell proliferation. Reduced cell adherence and viability could be detected after 18 h of 17alpha-AED exposure. Treatment with 17alpha-AED induced a significant level of apoptosis in U937 lymphoma cells, but not in the glioma cells. Cytopathology of 17alpha-AED-treated T98G cells revealed the presence of multiple cytoplasmic vacuoles. Acridine orange staining demonstrated the formation of acidic vesicular organelles in 17alpha-AED-treated T98G and U251MG, which was inhibited by bafilomycin A1. These findings indicate that 17alpha-AED bears the most potent cytotoxic activity of the neuro-steroids tested, and the effectiveness may depend on the number of hydroxyls and their position on the androstene molecule. These cytotoxic effects may utilize a non-apoptotic pathway in malignant glioma cells.

17 alpha androstenediol inhibition of breast tumor cell proliferation in estrogen receptor-positive and -negative cell lines.

Androstene-3beta, 17alpha-diol (17alpha-AED) inhibits DNA synthesis and induces apoptosis in several myeloid cancer cell lines. The purpose of this study was to determine if 17alpha-AED inhibition of human breast carcinoma cell proliferation is dependent on the estrogen or androgen receptor. At concentrations of 12.5 to 50 x 10(-9) M 17alpha-AED inhibited the proliferation of ZR75-1, estrogen receptor-positive (ER+) cells, by 54% to 68%. Further, 17alpha-AED inhibited MDA-MB231, estrogen receptor-negative (ER-) cells, by 33.6% to 56.0%. The inhibitory effect was dose dependent with a minimal effective inhibitory dose at 12.5x10(-9) M for both cell lines. Both 17beta-AED and estradiol potentiate the inhibitory effect of 17alpha-AED on ER+ cells at lower doses (3.13 to 6.25 x 10(-9) M) where 17alpha-AED alone was not inhibitory. The inhibitory action of 17alpha-AED on human mammary carcinomas appears to be independent of either the alpha estrogen or the androgen receptors.

Androstenediol-induced restoration of responsiveness to influenza vaccination in mice.

Androstenediol (AED), a metabolite of dehydroepiandrosterone (DHEA) regulates innate and adaptive immune responses. To examine whether AED could effectively reverse the age-associated decline of antiviral immunity, 3-, 10-, and 22-month-old mice were treated with AED-sulfate (AED-S) for 45 days beginning 10 days prior to vaccination. Subsequently, mice were primed and boosted with suboptimal doses of a commercially-available trivalent influenza vaccine. Treatment of 10-month-old animals with AED-S during vaccination increased the titer of circulating antiviral immunoglobulin G to levels comparable with those in 3-month-old mice. Furthermore, AED-S treatment protected 10-month-old animals from intranasal challenge with a lethal dose of influenza virus 21 days after secondary vaccination. Although AED-S treatment of 22-month-old mice did not enhance vaccine responses and failed to protect against lethal challenge, the data from the 10-month-old animals suggest that treatment with AED-S will prevent the early manifestations of immunosenescence.

Androstenediol stimulates myelopoiesis and enhances resistance to infection in gamma-irradiated mice.

The ionizing radiation-induced hemopoietic syndrome is characterized by defects in immune function and increased mortality due to infections and hemorrhage. Since the steroid 5-androstene-3beta, 17beta-diol (5-androstenediol, AED) modulates cytokine expression and increases resistance to bacterial and viral infections in rodents, we tested its ability to promote survival after whole-body ionizing radiation in mice. In unirradiated female B6D2F1 mice, sc AED elevated numbers of circulating neutrophils and platelets and induced proliferation of neutrophil progenitors in bone marrow. In mice exposed to whole-body (60)Co gamma-radiation (3 Gy), AED injected 1 h later ameliorated radiation-induced decreases in circulating neutrophils and platelets and marrow granulocyte-macrophage colony-forming cells, but had no effect on total numbers of circulating lymphocytes or erythrocytes. In mice irradiated (0, 1 or 3 Gy) and inoculated four days later with Klebsiella pneumoniae, AED injected 2 h after irradiation enhanced 30-d survival. Injecting AED 24 h before irradiation or 2 h after irradiation increased survival to approximately the same extent. In K. pneumoniae-inoculated mice (irradiated at 3-7 Gy) and uninoculated mice (irradiated at 8-12 Gy), AED (160 mg/kg) injected 24 h before irradiation significantly promoted survival with dose reduction factors (DRFs) of 1.18 and 1.26, respectively. 5-Androstene-3beta-ol-17-one (dehydroepiandrosterone, DHEA) was markedly less efficacious than AED in augmenting survival, indicating specificity. These results demonstrate for the first time that a DHEA-related steroid stimulates myelopoiesis, and ameliorates neutropenia and thrombocytopenia and enhances resistance to infection after exposure of animals to ionizing radiation.

Androstenetriol and androstenediol. Protection against lethal radiation and restoration of immunity after radiation injury.

Androstenetriol (AET) and Androstenediol (AED) upregulate host immunity, leading to increased resistance against infections. AET augments IL-2, IL-3, IFN gamma levels, and counteracts hydrocortisone immune suppression. AET and AED at a dose of 0.75 mg/- and 8.0 mg/25-g mouse, protected 60 and 70%, respectively, of C57/BL/6J mice irradiated with a lethal dose. These hormones also protected mice irradiated with 6 Gy and infected with a coxsackievirus B4 LD50. AET significantly increased spleen lymphocyte numbers at 7, 14, and 21 days after a 6-Gy exposure. Fluorescent activated cell-sorter analysis of irradiated mice, spleen, and bone marrow showed that AET significantly augmented the myeloid precursor markers, CD11b/Mac-1, and B220 (pan B), as well as the absolute numbers of CD4+/CD8+ cells over the 21 days of testing. Overall, the data are consistent with AET/AED inducing a more rapid recovery of all hematopoietic precursors from the small number of surviving stem cells.

Steroid hormone regulation of antiviral immunity.

Recent observations in both humans and animals have demonstrated that stress is immunomodulatory and can alter the pathogenesis of microbial infections to the extent that it may be adverse to health. Stress disrupts homeostasis, and the body responds through endocrine and nervous system interactions in an effort to re-establish the health of the host. However, the resulting physiologic changes associated with stress, such as the rise in serum glucocorticoids (GCs), are implicated in suppression of antiviral immunity. Therefore, it would be of significance to counterregulate stress-mediated immunosuppression during viral infection to improve immune responses and limit virus-mediated damage. The data in this study focus upon the antiglucocorticoid influence of a native steroid hormone that has been shown to augment immune function and protect animals against lethal viral infections. Androstenediol (5-androstene-3 beta,17 beta-diol, AED), a metabolite of dehydroepiandrosterone (DHEA), confers protection against lethal infection with influenza A virus. The protective activity appears to counterbalance the function of the regulatory GCs because AED prevents GC-mediated suppression of IL-1, TNF-alpha, and IL-2 secretion. Furthermore, AED inhibits GC-induced transcription of a GC-sensitive reporter gene.

Control of the immune response by DHEA and its metabolites.

The 17 keto steroid, Dehydroepiandrosterone (5-androsten-3 beta-17-one, DHEA) has been shown to protect mice from a variety of lethal infections. This includes, but is not limited to, infection with viruses (herpesvirus type 2, coxsackievirus B4-CVB4),bacteria (Enterococcus faecalis, Pseudomonas aeruginosa), and a parasite (Cryptosporidium parvum). We have reported that androstenediol (5-androsten-3 beta-17 beta-diol, beta AED), which is derived from DHEA, is at least 100x more effective in up-regulating systemic resistance against CVB4-infection than its precursor. Furthermore, androstenetriol (5-androstene-3 beta-7 beta-17 beta-triol beta AET) which is formed by 7 beta hydroxylation of beta AED, was more effective against CVB4-infection than its precursor beta AED. Neither steroid however has shown any significant direct antiviral effects. The in-vitro influences of DHEA, beta AED, and beta AET on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of concanavalin A (Con A) or lipopolysaccharide (LPS) activated cultures in a dose dependent manner. beta AED had little influence on the activation response. However, beta AET potentiated the response to both mitogens significantly above control. The regulation of interleukin-2 and interleukin-3 secretion from Con A-activated lymphocytes was analogous to these observations. These functions were suppressed by DHEA, unaffected by beta AED, and potently increased by beta AET. Moreover, the classic immuno-suppressive effects of hydro-cortisone on Con A-induced lymphocyte proliferation, as well as IL-2 and IL-3 production were unaffected by co-cultured with DHEA and only minimally counteracted by beta AED. In contrast, beta AET significantly counteracted the effect of hydrocortisone when co-cultured together. These results show that while in-vivo, DHEA, beta AED, and beta AET each function in a similar manner. In-vitro, their effects are dramatically different from one another with only beta AET potentiating the cellular response by increasing lymphocyte activation and counteracting the immuno-suppressive activity of hydrocortisone.

Endocrine regulation of murine macrophage function: effects of dehydroepiandrosterone, androstenediol, and androstenetriol.

In these studies, the in vitro influences of dehydroepiandrosterone (DHEA), androstenediol (AED), and androstenetriol (AET) on proinflammatory cytokine production from macrophages was examined. From physiologic to pharmacologic doses, DHEA suppressed secretion of each pro-inflammatory cytokine while AED had little influence on the responses. In sharp contrast, AET augmented TNF-alpha and IL-1 secretion while not influencing IL-6 production. Furthermore, the antiglucocorticoid activity of DHEA, AED, and AET was also investigated. Co-culture with AET counteracted the down-regulatory effect of hydrocortisone on LPS-induced TNF-alpha and IL-1 secretion. These data imply that AET is capable of regulating cytokine secretion from macrophages and may function to counterbalance glucocorticoid function.

Endocrine regulation of the immune response to influenza virus infection with a metabolite of DHEA-androstenediol.

In these studies the influence of androstenediol on the course of an experimental virus infection was examined. Pretreatment with 320 mg/kg AED protected male mice from lethal influenza virus infection. In addition, AED enhanced antigen-induced trafficking of mononuclear cells into the draining lymph node and augmented antigen-specific activation of helper-T cells, which are important for control of viral pathogenesis. Furthermore, AED prevented the characteristic increase in serum corticosterone noted during influenza A virus infection. Although steroid hormones, at least corticosteroids, typically suppress host immune and inflammatory responses in vivo, these data suggest that AED may function to augment host immune and inflammatory responses in contrast to corticosteroids.

Contrasting effects of alpha- and beta-androstenediol on oncogenic myeloid cell lines in vitro.

The in vitro effects of 17 alpha AED, the isomer of 5-androstene-3 beta,17 beta diol (17 beta AED) on the basal growth of murine RAW 264.7, P388D1, and human HL-60 cells were investigated. 17 alpha AED treatment of RAW cells for 48 h reduced total cell number without increasing cell death as detected by trypan blue exclusion. At these doses, DNA synthesis as measured by [3H]thymidine incorporation was suppressed by as much as 65%, P < 0.05. This effect was time- and dose-dependent and reversible on removal of the steroid. Similar results were obtained with P388D1 and human HL-60 cell lines. At 50 nM or above, 17 alpha AED induced apoptosis in RAW cells and HL-60 as detected by transmission electron microscopy and TUNEL assays. By contrast, treating cells with the isomer 17 beta AED had no such effect. These data suggest that the balance between the anti-proliferative effect of 17 alpha AED and the proliferative effects of 17 beta AED may determine the overall level of myelopoiesis.

Antiglucocorticoid function of androstenetriol.

The anti-inflammatory and immunosuppressive functions of corticosteroids have been well established and characterized. In contrast, a different group of native steroids, which include dehydroepiandrosterone (DHEA) and two of its metabolites, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-17 beta-triol, beta AET), function in vivo to up-regulate host immune response against infections and counteract stress-induced immunosuppression. Indeed, DHEA and particularly, AED and beta AET, have been shown to protect mice from viral, bacterial, and parasitic infections. In vivo, these three hormones are in opposition to the widely demonstrated immunosuppressive action of glucocorticoids, suggesting a possible new immune regulation mechanism. The individual activity in vitro of each of these steroids, i.e. DHEA, AED, and beta AET, on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of cultures activated with concanavalin A (ConA) or lipopolysaccharide (LPS) in a dose-dependent manner. AED had little influence on the activation response. However, beta AET potentiated the response to both mitogens significantly above control. The regulation of the cytokine secretion, of both interleukin-2 (IL-2) and interleukin-3 (IL-3), from ConA-activated lymphocytes was affected in the same manner. These functions were depressed by DHEA, unaffected by AED, and potently increased by beta AET. Moreover, the classic immunosuppressive effects of hydrocortisone on ConA-induced lymphocyte proliferation, as well as on IL-2 and IL-3 production, were unaffected by being co-cultured with DHEA and only minimally counteracted by AED at high doses. In contrast, co-culturing with beta AET significantly counteracted the immunosuppressive effects of hydrocortisone on lymphocyte proliferation and cytokine production. These data show that in-vivo, DHEA, AED, and beta AET may have some similar functions, while in vitro, their effects are dramatically different from one another. Only beta AET could markedly potentiate the cellular response by increasing lymphocyte activation and counteracting the immnosuppressive activity of hydrocortisone on lymphocyte proliferation and cytokine production.

Regulation of the immune response by dehydroepiandrosterone and its metabolites.

Dehydroepiandrosterone (5-androsten-3 beta-ol-17-one, DHEA) has been shown to protect mice from a variety of lethal infections. This includes, but is not limited to, infection with viruses (herpes virus type 2, coxsackie virus B4 (CB4)), bacteria (Enterococcus faecalis, Pseudomonas aeruginosa), and a parasite (Cryptosporidium parvum). We have previously reported that androstenediol (5-androstene-3 beta, 17 beta-diol, AED), derived from DHEA, is at least 100 x more effective in up-regulating systemic resistance against CB4 infection than its precursor. Furthermore, androstenetriol (5-androstene-3 beta,7 beta, 17 beta-triol, AET) which is formed by 7 beta hydroxylation of AED, was more effective against CB4 infection than its precursor, AED. Neither steroid, however, has shown any significant direct antiviral effects. The in vitro influences of DHEA, AED and AET on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of concanavalin A (ConA)- or lipopolysaccharide-activated cultures in a dose-dependent manner. AED had little influence on the activation response. However, AET potentiated the response to both mitogens significantly above the control level. The regulation of interleukin (IL)-2 and IL-3 secretion from ConA-activated lymphocytes was analogous to these observations. These functions were depressed by DHEA, unaffected by AED, and potently increased by AET. Moreover, the classic immunosuppressive effects of hydrocortisone on ConA-induced lymphocyte proliferation, as well as IL-2 and IL-3 production, were unaffected by co-culture with DHEA and only minimally counteracted by AED. In contrast. AET significantly counteracted the effect of hydrocortisone when co-cultured together. These data show that while DHEA, AED and AET each function in a similar manner in vivo, in vitro their effects are dramatically different from one another with only AET potentiating the cellular response by increasing lymphocyte activation and counteracting the immunosuppressive activity of hydrocortisone.

In vitro potentiation of lymphocyte activation by dehydroepiandrosterone, androstenediol, and androstenetriol.

Dehydroepiandrosterone (5-androstene-3 beta-ol-17-one, DHEA) has been shown to protect mice from lethal viral and bacterial infections. However, recently, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-triol, AET), metabolites of DHEA, have each shown to be more potent endocrine regulators of the immune response. In contrast to glucocorticosteroids, in vivo, these steroid hormones up-regulated the cellular immune response of the host to limit virus-mediated pathology. These experiments first examined the in vitro influences of DHEA, and AED, or AET on mitogen-stimulated activation of murine lymphocytes. From physiologic to pharmacologic doses, DHEA suppressed proliferation of mixed splenocyte cultures activated with Con A or LPS by 20 to 70% whereas AED had little influence on the response. In sharp contrast, AET potentiated the response with both mitogens to 50 to 70% above control. Analogous to these observations was the regulation of IL-2 and IL-3 secretion from Con A-activated lymphocytes by each of these hormones which again was depressed analyzed. The suppressive effects of hydrocortisone on Con A-induced lymphocyte proliferation and cytokine production were strongly counteracted by coculture with AET. DHEA did not counteract hydrocortisone activity whereas AED showed moderate antiglucocorticoid function.

Mobilization of cutaneous immunity for systemic protection against infections.

This laboratory reported that a single subcutaneous (SC) injection of the natural steroid hormone dehydroepiandrosterone (DHEA) resulted in significant protection against a lethal herpes virus type 2 encephalitis or a systemic coxsackievirus B4 infection. Our previous results have shown that SC injection of DHEA resulted in upregulation of the specific host immune response resulting in protection against a lethal infection. This hormone did not have any direct antiviral effects in vitro. Furthermore, results indicate that, in vivo, DHEA is not the agent directly mediating the upregulation of the immune response. In the skin, DHEA is converted to androstenediol (AED) and it, in turn, is converted to androstenetriol; this is a metabolic process which appears unique to the skin. This report demonstrates that SC injection of AED results in markedly greater resistance against both viral and bacterial infection. Both DHEA and AED appear to function by facilitating and upregulating host immune responses via mobilization of cutaneous immunity to obtain systemic protection against infections. Because these steroids are native to the host and are regulated by the central nervous system, it is suggested that they may be an integral element of neuroimmunomodulation.

Androstenediol regulates systemic resistance against lethal infections in mice.

We previously reported that subcutaneous injection of DHEA (5-androsten-3 beta-ol-17-one, dehydroepiandrosterone) protected mice from lethal infection. This included both a lethal herpes virus type 2 encephalitis and a lethal systemic coxsackievirus B4 (CB4) infection. Androstenediol (5-androsten-3 beta-17 beta-diol, AED), a metabolic product of DHEA is up to 100 x more effective in regulating systemic resistance against lethal infection with CB 4 than its precursor DHEA. Compared to DHEA, treatment with AED was markedly superior in protecting mice against virus induced myocardiopathy, pancreopathy, and mortality. In addition to its protective effect, AED but not DHEA, induced a 3-4 fold proliferation of the spleen and thymus in virus infected animals; this effect of AED was only seen above a certain threshold dose. Neither steroid, however, has shown any significant direct antiviral effect in vitro; similarly, virus tissues titers in vivo are not affected by the hormones. Additionally, both DHEA and AED protected against a lethal infection with Enterococcus faecalis. These observations demonstrate that the steroid hormones DHEA and AED provide a novel approach for prevention and protection of the host from a variety of infectious diseases.

Clinical variation in X-linked adrenoleukodystrophy: fatty acid and lipid metabolism in cultured fibroblasts.

To determine whether the clinical phenotype of ALD correlates with the extent of metabolic abnormality, we investigated VLFA metabolism in cultured fibroblasts from patients with the clinically severe childhood from of ALD and the milder AMN variant. No differences were seen in the content of neutral lipids or phospholipids, in incorporation of [1-14C]lignocerate into cellular lipids, or in the fatty acid composition of fibroblasts from patients with childhood ALD or AMN. [1-14C]Lignocerate oxidation was deficient to a similar extent (35-40% of normal) in both intact fibroblasts and cell homogenates from patients with childhood ALD and AMN. With the use of fibroblast homogenates, oxidation of lignocerate was partially inhibited by various long-chain fatty acids, and residual activity in ALD homogenates was more susceptible to inhibition by palmitate than normal. In the presence of competing palmitate, residual lignocerate oxidative activity in fibroblast homogenates was reduced to 20 +/- 4% of normal in childhood ALD and 24 +/- 2% of normal in AMN. These results indicate that residual VLFA oxidative activity, fatty acid composition, VLFA metabolism, and lipid content of cultured fibroblasts do not correlate with the clinical expression of the ALD gene.

Immunodeficiency as primary phenotype of diabetes mutation db. Studies with coxsackievirus B4.

Restriction of food intake (R) in the C57BL/KsJ db/db diabetic mutant mouse prevents phenotypic expression of diabetes, whereas ad libitum feeding (AL) results in spontaneous diabetes. Previous results showed that coxsackievirus B4 (CB4)-infected genetically identical db/db mice with and without diabetes could be distinguished by the levels of CB4-neutralizing antibody and virus-specific antibodies as determined by enzyme-linked immunosorbent assay and the numbers of splenic antibody-forming cells. Our results show that the diabetic genotype db/db R was deficient in total spleen lymphocytes and lymphocyte subsets and was unable to produce agglutinating antibody to sheep erythrocytes (SRBCs) or specific antibody to noninfectious CB4. The db/db AL mutant expressing the diabetic phenotype was not as deficient in spleen cell parameters. The response to noninfectious CB4 was delayed but substantial. The db/db AL mouse was also unique with its higher agglutinating antibody levels after virus infection than its uninfected control or the infected or uninfected db/db R mouse. In vitro SRBC immunization of spleen lymphocytes determined that this enhanced response was largely dependent on the diabetic milieu and was not a property of the cells. Genetic predisposition to diabetes is characterized by immunodeficiency as evident from inadequate levels of antibodies to infectious or noninfectious antigens and absolute and relative deficiency in spleen lymphocyte subsets and total numbers of spleen cells. Phenotypic expression of diabetes results in partial amelioration of the immunodeficiency evident in diabetic genotype db/db R without disease.

Evaluation of virus neutralization antibody levels in diabetic mutant mice.

Experiments were designed to determine whether genetic predisposition to diabetes or overt diabetes in the identical genotype had an effect on the level of neutralization antibodies to coxsackievirus B4 (CB4). Quantitation of neutralization antibody (NT) levels against CB4 was performed using both the 50% endpoint procedure and the multivariate Wilcoxon rank sum test. The results of these experiments demonstrate that the use of the multivariate procedure for the analysis of neutralization antibody levels provides both quantitative and qualitative information not evident when only the classical 50% end point procedure is used. Moreover, when comparison on antibody levels between different groups is being considered, the power of the comparison is markedly greater using the multivariate Wilcoxon rank sum test results. The present report provide an illustration of the difference in the qualitative and quantitative information obtained by the end-point procedure and the more comprehensive multivariate procedure for the analysis of neutralization antibody levels in diabetic mutant mice infected with coxsackievirus B4.