Human Steroid Research Articles

(−)‐Epicatechin attenuates prednisolone induced‐hyperglycemia

The chronic use of steroids in humans is known to increase blood glucose levels. The consumption of (−)‐epicatechin (Epi) rich cocoa has been associated with improvements in insulin sensitivity and increases in mitochondrial volume and cristae density. During 15 days, rats were treated with prednisolone (Pred) (10 mg/Kg/day, SC) and received either Epi (1 mg/kg/twice a day) or water by gavage, to evaluate the effects of Epi on Pred‐induced hyperglycemia and glucose control systems. Rats not treated with Pred and not receiving Epi served as controls. Epi treated rats demonstrated lower fasting glucose levels than Pred only rats (p<0.05). Epi treated rats also displayed glucose tolerance comparable to that of controls. Western blot analysis of skeletal muscle demonstrated minor changes in proteins levels involved in the insulin‐signaling pathway by Epi. Pred reduced protein levels of the oxidative phosphorylation (OXPHOS) respiratory complexes. However, Epi treatment was able to maintain OXPHOS levels similar to controls. The protein levels of the catalytic subunit E1α of the pyruvate dehydrogenase were increased by Epi. These findings suggest that Epi protection against Pred‐induced hyperglycemia may be due to an improvement in mitochondrial function and glucose metabolism.

3D-QSAR CoMFA and CoMSIA studies for design of potent human steroid 5α-reductase inhibitors

3D-QSAR studies were performed on a set of sixty-one 6-azasteroidal human steroid 5α-reductase inhibitors. The models developed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methodologies employing atom and centroid based alignment were reliable and significant having good predictive r 2 value. CoMSIA model developed by a combination of steric, electrostatic, hydrophobic and hydrogen bond donor showed good relative and predictive properties. The predictive power of the models was assessed using an external test set of 10 compounds. The best model had shown cross validation r 2 (0.702) with 7 optimum components, non-cross-validation r 2 (0.956), F value (86.158), predicted r 2 (0.704), standard error of estimate (0.165). Further the CoMFA/CoMSIA contour plots show a preference toward the C-17 substituents and indicate that bulky group at C-17 is an important requirement for exhibiting 5α-reductase inhibition. These developed models could be used to design and optimise the more potent 6-aza steroidal inhibitors of 5α-reductase enzyme. The 3D-QSAR studies involving comparative molecular field analysis and comparative molecular similarity indices analysis on a series of 6-azasteroids as 5α-reductase inhibitors is reported.

Structural architecture of the human long non-coding RNA, steroid receptor RNA activator

While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.

Occurrence of androgens in sewage treatment plants influents is associated with antagonist activities on other steroid receptors

The occurrence of endocrine disrupting chemicals such as estrogens in raw urban sewage is well documented. By contrast, the presence of other steroidal activities in wastewater has been poorly studied, although they can cause undesirable biological responses in the environment. In this work, extracts of raw wastewater were tested for agonist and antagonist activities on estrogen, androgen, progesterone, mineralocorticoid and glucocorticoid receptors. We detected strong estrogenic activities that correlated well with the concentration of natural estrogens (estrone, estriol and 17β-estradiol) measured by chemical analysis. We also measured strong androgenic activities which were not due to estrogen receptor ligands based on the use of recombinant estrogen receptor α affinity columns. Several molecules with androgenic activities were identified in wastewater samples, testosterone, dihydrotestosterone and epiandrosterone being the most abundant. However, they explain only a small part of the detected androgenic activity, as indicated by the comparison of the detected biological responses with the results of the targeted chemical analysis. Finally, we found that our samples also contained strong antagonist activities on progesterone, glucocorticoid and mineralocorticoid receptors. Very interestingly, we identified pregnenolone (the precursor to all steroid hormones in humans) as a major endocrine disrupting chemical which accounts for most of the anti-mineralocorticoid activities present in raw wastewater. In conclusion, this study demonstrates the occurrence of androgen agonists as well as other steroid receptor antagonists such as pregnenolone in raw wastewater. Further research is needed to assess the fate of such compounds during sewage treatment and their potential effect on living organisms.

Orteronel (TAK-700), a novel non-steroidal 17,20-lyase inhibitor: Effects on steroid synthesis in human and monkey adrenal cells and serum steroid levels in cynomolgus monkeys

Surgical or pharmacologic methods to control gonadal androgen biosynthesis are effective approaches in the treatment of a variety of non-neoplastic and neoplastic diseases. For example, androgen ablation and its consequent reduction in circulating levels of testosterone is an effective therapy for advanced prostate cancers. Unfortunately, the therapeutic effectiveness of this approach is often temporary because of disease progression to the ‘castration resistant’ (CRPC) state, a situation for which there are limited treatment options. One mechanism thought to be responsible for the development of CRPC is extra-gonadal androgen synthesis and the resulting impact of these residual extra-gonadal androgens on prostate tumor cell proliferation. An important enzyme responsible for the synthesis of extra-gonadal androgens is CYP17A1 which possesses both 17,20-lyase and 17-hydroxylase catalytic activities with the 17,20-lyase activity being key in the androgen biosynthetic process. Orteronel (TAK-700), a novel, selective, and potent inhibitor of 17,20-lyase is under development as a drug to inhibit androgen synthesis. In this study, we quantified the inhibitory activity and specificity of orteronel for testicular and adrenal androgen production by evaluating its effects on CYP17A1 enzymatic activity, steroid production in monkey adrenal cells and human adrenal tumor cells, and serum levels of dehydroepiandrosterone (DHEA), cortisol, and testosterone after oral dosing in castrated and intact male cynomolgus monkeys. We report that orteronel potently suppresses androgen production in monkey adrenal cells but only weakly suppresses corticosterone and aldosterone production; the IC50 value of orteronel for cortisol was ∼3-fold higher than that for DHEA. After single oral dosing, serum levels of DHEA, cortisol, and testosterone were rapidly suppressed in intact cynomolgus monkeys. In castrated monkeys treated twice daily with orteronel, suppression of DHEA and testosterone persisted throughout the treatment period. In both in vivo models and in agreement with our in vitro data, suppression of serum cortisol levels following oral dosing was less than that seen for DHEA. In terms of human CYP17A1 and human adrenal tumor cells, orteronel inhibited 17,20-lyase activity 5.4 times more potently than 17-hydroxylase activity in cell-free enzyme assays and DHEA production 27 times more potently than cortisol production in human adrenal tumor cells, suggesting greater specificity of inhibition between 17,20-lyase and 17-hydroxylase activities in humans vs monkeys. In summary, orteronel potently inhibited the 17,20-lyase activity of monkey and human CYP17A1 and reduced serum androgen levels in vivo in monkeys. These findings suggest that orteronel may be an effective therapeutic option for diseases where androgen suppression is critical, such as androgen sensitive and CRPC.

Development of humanized steroid and xenobiotic receptor mouse by homologous knock-in of the human steroid and xenobiotic receptor ligand binding domain sequence

The human steroid and xenobiotic receptor (SXR), (also known as pregnane X receptor PXR, and NR1I2) is a low affinity sensor that responds to a variety of endobiotic, nutritional and xenobiotic ligands. SXR activates transcription of Cytochrome P450, family 3, subfamily A (CYP3A) and other important metabolic enzymes to up-regulate catabolic pathways mediating xenobiotic elimination. One key feature that demarcates SXR from other nuclear receptors is that the human and rodent orthologues exhibit different ligand preference for a subset of toxicologically important chemicals. This difference leads to a profound problem for rodent studies to predict toxicity in humans. The objective of this study is to generate a new humanized mouse line, which responds systemically to human-specific ligands in order to better predict systemic toxicity in humans. For this purpose, the ligand binding domain (LBD) of the human SXR was homologously knocked-in to the murine gene replacing the endogenous LBD. The LBD-humanized chimeric gene was expressed in all ten organs examined, including liver, small intestine, stomach, kidney and lung in a pattern similar to the endogenous gene expressed in the wild-type (WT) mouse. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the human-selective ligand, rifampicin induced Cyp3a11 and Carboxylesterase 6 (Ces6) mRNA expression in liver and intestine, whereas the murine-selective ligand, pregnenolone-16-carbonitrile did not. This new humanized mouse line should provide a useful tool for assessing whole body toxicity, whether acute, chronic or developmental, induced by human selective ligands themselves and subsequently generated metabolites that can trigger further toxic responses mediated secondarily by other receptors distributed body-wide.

Interactions of insulin and estrogen in the regulation of cell proliferation and carcinogenesis

Equilibrium of sexual steroids and metabolic processes has close correlations. Insulin is a potent regulator of human sexual steroid hormone production and modulates their signals at receptor level. Insulin resistance and excessive insulin production provoke hyperandrogenism and estrogen deficiency in women resulting not only in anovulatory dysfunction but also a high risk for cardiovascular diseases and cancer. Physiologic functions of all female organs have higher estrogen demand as compared with men. In healthy women estrogen predominance against androgens is a favor in their reproductive period, which means a strong defense against insulin resistance and its complications. However, in postmenopausal cases the increasing prevalence of insulin resistance and type-2 diabetes associated with estrogen deficiency and androgen excess, result in a gender specific higher risk for precancerous lesions and cancer as compared with men. Estrogen has beneficial effect on the energy metabolism, glucose homeostasis and on the lipid metabolism of liver and of peripheral tissues as well. A moderate or severe decrease in serum estrogen level enhances the prevalence of insulin resistant states. In premenopausal women long or irregular menstrual cycles are predictors for the risk of insulin resistance and type-2 diabetes. Moreover, in postmenopausal estrogen deficient cases elevated fasting glucose, increased body weight and abdominal fat deposition are often observed progressively with age in correlation with an impaired glucose tolerance. In the rare cases of estrogen deficient men severe type-2 diabetes seems to be a characteristic complication. Upon becoming familiar with the cancer risk of insulin resistance and estrogen deficiency, there would be plenty of possibilities for primary cancer prevention. In patients with cancer the treatment of hormonal and metabolic disturbances may become effective adjuvant therapy.

Preclinical pharmacological profile of nomegestrol acetate, a synthetic 19-nor-progesterone derivative

BackgroundNomegestrol acetate (NOMAC), a synthetic progestogen derived from 19-nor-progesterone, recently completed clinical trials for use with 17beta-estradiol in a new monophasic combined oral contraceptive. In this review, published as well as previously unpublished preclinical studies that detail the effects of NOMAC on estrogenic, progestogenic, and androgenic systems, as well as mineralocorticoid, glucocorticoid, bone, and metabolic indices are described.MethodsIn vitro assays to determine NOMAC structure-activity relationships used tissue derived from rat uteri. Transactivation profiles were performed using Chinese hamster ovary (CHO) cells transfected with cDNAs encoding human steroid receptors. Estrogenic and anti-estrogenic activities were monitored in vivo in rats as well as in vitro in human breast cancer cells. Standard in vivo techniques were used in rats to determine progestational activity; antigonadotropic, androgenic, mineralocorticoid, and glucocorticoid activities; as well as effects on bone and other metabolic indices. Ovulation inhibition was monitored in rats and primates. NOMAC’s effects on cardiovascular systems were determined in dogs and primates.ResultsNOMAC was without significant agonistic or antagonistic activity for estrogen receptor alpha or beta in vitro, and inhibited ovulation in rats and monkeys (2.5 mg/kg and 1 mg/kg, respectively). NOMAC lacked androgenic, antimineralocorticoid, glucocorticoid, and metabolic activity and exhibited moderate anti-androgenic activity in rats. NOMAC did not affect bone mineral density (BMD) in rats or hemodynamic and electrophysiologic parameters in dogs and primates.ConclusionsNOMAC is a selective progestogen structurally similar to progesterone that has modest anti-androgenic activity and does not affect lipid or carbohydrate metabolism, BMD, or many cardiovascular parameters in selected animal models.

Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2

The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.

Hydroxysteroid sulfotransferase 2B1b expression and localization in normal human brain

Steroid sulfonation in the human brain has not been well characterized. The major sulfotransferase (SULT) isoforms that conjugate steroids in humans are SULT1E1, SULT2A1, and SULT2B1b. SULT2B1b catalyzes the sulfonation of 3β-hydroxysteroids, including neurosteroids dehydroepiandrosterone and pregnenolone, as well as cholesterol and several hydroxycholesterols. SULT2B1b mRNA and protein expression were detected in adult and fetal human brain sections, whereas neither mRNA, nor protein expression were identified for SULT1E1 or SULT2A1. Using immunohistochemical analysis, SULT2B1b expression was detected in neurons and oligodendrocytes in adult brain and in epithelial tissues in 28-week-old fetal brain. Sulfonation of cholesterol, oxysterols, and neurosteroids in the brain is apparently catalyzed by SULT2B1b since expression of neither SULT2A1 nor SULT1E1 was detected in human brain sections. SULT2B1b mRNA and protein were also detected in human U373-MG glioblastoma cells. Both mRNA and protein expression of liver X receptor (LXR)-β, but not LXR-α, were detected in U373-MG cells, and LXR-β activation resulted in a decrease in SULT2B1b protein expression. Since hydroxycholesterols are important physiological LXR activators, this suggests a role for regulation of sterol metabolism by LXR and SULT2B1b. Therefore, elucidating key enzymes in the metabolism of cholesterol and neurosteroids could help define the properties of steroid conjugation in the human brain.

Metabolic studies with promagnon, methylclostebol and methasterone in the uPA +/+-SCID chimeric mice

The chimeric uPA +/+-SCID mouse model, transplanted with human hepatocytes, was previously validated as an alternative tool to study in vivo the human steroid metabolism. This humanized mouse model was now applied, in the framework of anti-doping research, to test different nutritional supplements containing steroids. These steroids, intentionally or accidentally added to a nutritional supplement, usually are derivatives of testosterone. Information about the metabolism of these derivatives, which is important to assure their detection, is quite limited. However, due to ethical constraints, human volunteers cannot be used to perform experimental excretion studies. Therefore the chimeric mice were selected to perform three separated excretion studies with superdrol (methasterone), promagnon and also methylclostebol. The urine of the humanized mice was collected 24 h after a single dose administration and analyzed by gas chromatography–mass spectrometry (GC–MS). The results indicated the presence of several metabolites including a 3-keto reduced metabolite and numerous hydroxylated metabolites. Also phase 2 metabolism was investigated to update the complete picture of their metabolism.

Neuroactive steroids: focus on human brain

Studies in experimental animals have revealed important roles of neuroactive steroids in the control of central nervous system functions during physiological and pathological conditions, suggesting that they may represent good candidates for the development of neuroprotective strategies for neurodegenerative and psychiatric disorders. Even if the characterization of the roles played by neuroactive steroids in humans is still at the beginning, several data are already available showing that they may be synthesized within the human CNS. Among the different enzymes, a prominent role is dedicated to aromatase that synthesizes estradiol whose neuroprotective effects have been described in experimental animals. Neuroactive steroid levels are modified by neurodegenerative conditions (i.e. Alzheimer's and Parkinson's diseases, multiple sclerosis) or in other mental diseases (i.e. schizophrenia), and may have an important role in physiological conditions, as the reorganization of grey and white matter during human puberty and adolescence or as a consequence of emotional responses. The interaction of some neuroactive steroids (i.e., allopregnanolone and isopregnanolone) with GABA-A receptor is particularly important in mood disorders. The presumptive role of estradiol and progesterone in neuroprotection is here discussed by comparing contradictory data that have been collected in humans. In conclusion, the state of the art of our knowledge of the role of neuroactive steroids in the normal and pathological human brain suggests several lines of future therapeutic developments in the treatments of neurological, neurodegenerative and affective disorders. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.

From steroid receptors to cytokines: The thermodynamics of self-associating systems

Since 1987, the Gibbs Conference on Biothermodynamics has maintained a focus on understanding the quantitative aspects of gene regulatory systems. These studies coupled rigorous techniques with exact theory to dissect the linked reactions associated with bacterial and lower eukaryotic gene regulation. However, only in the last ten years has it become possible to apply this approach to clinically relevant, human gene regulatory systems. Here we summarize our work on the thermodynamics of human steroid receptors and their interactions with multi-site promoter sequences, highlighting results not available from more traditional biochemical and structural approaches. Noting that the Gibbs Conference has also served as a vehicle to promote the broader use of thermodynamics in understanding biology, we then discuss collaborative work on the hydrodynamics of a cytokine implicated in tumor suppression, prostate derived factor (PDF).

Evaluation of sulfatase-directed quinone methide traps for proteomics

Sulfatases hydrolytically cleave sulfate esters through a unique catalytic aldehyde, which is introduced by a posttranslational oxidation. To profile active sulfatases in health and disease, activity-based proteomic tools are needed. Herein, quinone methide (QM) traps directed against sulfatases are evaluated as activity-based proteomic probes (ABPPs). Starting from a p-fluoromethylphenyl sulfate scaffold, enzymatically generated QM-traps can inactivate bacterial aryl sulfatases from Pseudomonas aeruginosa and Klebsiella pneumoniae, and human steroid sulfatase. However, multiple enzyme-generated QMs form, diffuse, and non-specifically label purified enzyme. In complex proteomes, QM labeling is sulfatase-dependent but also non-specific. Thus, fluoromethylphenyl sulfates are poor ABPPs for sulfatases.

Biotechnological Production of 20-alpha-Dihydrodydrogesterone at Pilot Scale

The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α-dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190μMday(-1) was determined for a total biotransformation time of 136h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3 ± 1.4g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale.

Abstract 1014: Induction of human steroid sulfatase by TNF-α through PI3-K/Akt signaling pathway in PC-3 human prostate cancer cells

Abstract Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates and has a pivotal role in regulating the formation of biologically active estrogens. STS may help support the growth of hormone-dependent cancers, including prostate cancers and is considered as a new promising drug target for treating estrogen-mediated cancer. However, the molecular mechanism of STS expression is still not well-known. Previously, cytokines such as TNF-α and IL-6 were known to increase the activity of STS but the changes in expression have not been completely elucidated. To investigate whether cytokines are able to regulate transcription of STS gene, we studied the effect of TNF-α on STS expression in human prostate cancer PC-3 cells. Western blot and RT-PCR analyses showed that treatment with TNF-α significantly induced expression of mRNA level and protein level of STS in concentration- and time-dependent manners. Treatment with TNF-α resulted in a strong increase in the phosphorylation of Akt on Ser 473 and when the cells were treated with PI3-K inhibitors such as LY294002 or wortmannin or Akt inhibitor (Akt inhibitor IV) together, STS expression induced by TNF-α was significantly blocked. Moreover, inhibition of Akt activation by transfecting dominant-negative Akt plasmid also prevented TNF-α -mediated STS gene expression. Taken together, these data strongly suggest that TNF-α induces STS expression through PI3-K/Akt signaling pathway in PC-3 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2011-1014

Dioxin levels in the breast milk and estradiol and androgen levels in the saliva of Vietnamese primiparae

Human exposure to polychlorinated dibenzodioxins (PCDD) and dibenzofurans (PCDF), especially 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), was investigated in Vietnam since initial severe adverse health effects were reported in the late 1970s. The purpose of this study was to determine the effects of dioxin exposure on steroid hormones of primiparae in an Agent Orange/dioxin hot-spot and a non-exposed area in Vietnam. Sixteen primiparae (8 at each site), all of whom were aged between 20 and 30 years with infants aged between 4 and 16 weeks, agreed to participate in this study. The mean dioxin levels in breast milk of primiparae from the hot-spot area, in terms of PCDD, PCDF, and PCDD + PCDF toxic equivalents (TEQ), were significantly higher than those for the non-exposed area. PCDD TEQ, PCDF TEQ, and PCDD + PCDF TEQ levels showed a significant correlation with dehydroepiandrosterone (DHEA), androstenedione (A-dione), and estradiol (E2) in the saliva of primiparae in a combination of hot-spot and non-exposed areas in Vietnam. The dose–response curve between salivary E2 or A-dione levels and dioxin levels was U-shaped in humans. This study provides an overview of studies regarding dioxin hot-spots and effects on human health and steroid hormone levels in particular, with a focus on the toxicity attributed to dioxins and furans. Furthermore, causal evidence regarding the effects of dioxins on endocrine disruption in humans is provided.

Light at Night Activates IGF-1R/PDK1 Signaling and Accelerates Tumor Growth in Human Breast Cancer Xenografts

Regulation of diurnal and circadian rhythms and cell proliferation are coupled in all mammals, including humans. However, the molecular mechanisms by which diurnal and circadian rhythms regulate cell proliferation are relatively poorly understood. In this study, we report that tumor growth in nude rats bearing human steroid receptor-negative MCF-7 breast tumors can be significantly accelerated by exposing the rats to light at night (LAN). Under normal conditions of an alternating light/dark cycle, proliferating cell nuclear antigen (PCNA) levels in tumors were maximal in the early light phase but remained at very low levels throughout the daily 24-hour cycle period monitored. Surprisingly, PCNA was expressed in tumors continually at a high level throughout the entire 24-hour period in LAN-exposed nude rats. Daily fluctuations of Akt and mitogen activated protein kinase activation in tumors were also disrupted by LAN. These fluctuations did not track with PCNA changes, but we found that activation of the Akt stimulatory kinase phosphoinositide-dependent protein kinase 1 (PDK1) directly correlated with PCNA levels. Expression of insulin-like growth factor 1 receptor (IGF-1R), an upstream signaling molecule for PDK1, also correlated with fluctuations of PDK1/PCNA in the LAN group. In addition, circulating IGF-1 concentrations were elevated in LAN-exposed tumor-bearing nude rats. Finally, RNAi-mediated knockdown of PDK1 led to a reduction in PCNA expression and cell proliferation in vitro and tumor growth in vivo, indicating that PDK1 regulates breast cancer growth in a manner correlated with PCNA expression. Taken together, our findings demonstrate that LAN exposure can accelerate tumor growth in vivo, in part through continuous activation of IGF-1R/PDK1 signaling.

The role of estrogen deficiency in skin ageing and wound healing

The links between hormonal signalling and lifespan have been well documented in a range of model organisms. For example, in C.elegans or D.melanogaster, lifespan can be modulated by ablating germline cells, or manipulating reproductive history or pregnenolone signalling. In mammalian systems, however, hormonal contribution to longevity is less well understood. With increasing age human steroid hormone profiles change substantially, particularly following menopause in women. This article reviews recent links between steroid sex hormones and ageing, with special emphasis on the skin and wound repair. Estrogen, which substantially decreases with advancing age in both males and females, protects against multiple aspects of cellular ageing in rodent models, including oxidative damage, telomere shortening and cellular senescence. Estrogen's effects are particularly pronounced in the skin where cutaneous changes post-menopause are well documented, and can be partially reversed by classical Hormone Replacement Therapy (HRT). Our research shows that while chronological ageing has clear effects on skin wound healing, falling estrogen levels are the principle mediator of these effects. Thus, both HRT and topical estrogen replacement substantially accelerate healing in elderly humans, but are associated with unwanted deleterious effects, particularly cancer promotion. In fact, much current research effort is being invested in exploring the therapeutic potential of estrogen signalling manipulation to reverse age-associated pathology in peripheral tissues. In the case of the skin the differential targeting of estrogen receptors to promote healing in aged subjects is a real therapeutic possibility.

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10(8) clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR-L3 and CDR-H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three-dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire.