A gain-of-function mutation in DHT synthesis in castration-resistant prostate cancer.

Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation that leads to a gain-of-function in 3ß-hydroxysteroid dehydrogenase type 1 (3ßHSD1), which catalyzes the initial rate-limiting step in conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3ßHSD1 is a valid target for the treatment of CRPC.

Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians.

Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases1-3. Here we show that centenarians have a distinct gut microbiome that is enriched in microorganisms that are capable of generating unique secondary bile acids, including various isoforms of lithocholic acid (LCA): iso-, 3-oxo-, allo-, 3-oxoallo- and isoallolithocholic acid. Among these bile acids, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from the faecal microbiota of a centenarian, we identified Odoribacteraceae strains as effective producers of isoalloLCA both in vitro and in vivo. Furthermore, we found that the enzymes 5α-reductase (5AR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSDH) were responsible for the production of isoalloLCA. IsoalloLCA exerted potent antimicrobial effects against Gram-positive (but not Gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and Enterococcus faecium. These findings suggest that the metabolism of specific bile acids may be involved in reducing the risk of infection with pathobionts, thereby potentially contributing to the maintenance of intestinal homeostasis.

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

Structure-activity relationship and mechanistic study of organotins as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases.

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3ß-HSD2 with IC50 values of 114.79, 106.98, and 5.40 µM, respectively. For pig 3ß-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 µM, respectively. Similarly, for rat 3ß-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 µM, respectively. They were mixed inhibitors of pig and rat 3ß-HSD, while triphenyltin was identified as a competitive inhibitor of human 3ß-HSD2. The mechanism underlying the inhibition of organotins on 3ß-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3ß-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

Cholesterol synthesis enzyme SC4MOL is fine-tuned by sterols and targeted for degradation by the E3 ligase MARCHF6.

Cholesterol biosynthesis is a highly regulated pathway, with over 20 enzymes controlled at the transcriptional and posttranslational levels. While some enzymes remain stable, increased sterol levels can trigger degradation of several synthesis enzymes via the ubiquitin-proteasome system. Of note, we previously identified four cholesterol synthesis enzymes as substrates for one E3 ubiquitin ligase, membrane-associated RING-CH-type finger 6 (MARCHF6). Whether MARCHF6 targets the cholesterol synthesis pathway at other points is unknown. In addition, the posttranslational regulation of many cholesterol synthesis enzymes, including the C4-demethylation complex (sterol-C4-methyl oxidase-like, SC4MOL; NAD(P)-dependent steroid dehydrogenase-like, NSDHL; hydroxysteroid 17-beta dehydrogenase, HSD17B7), is largely uncharacterized. Using cultured mammalian cell lines (human-derived and Chinese hamster ovary cells), we show SC4MOL, the first acting enzyme of C4-demethylation, is a MARCHF6 substrate and is rapidly turned over and sensitive to sterols. Sterol depletion stabilizes SC4MOL protein levels, while sterol excess downregulates both transcript and protein levels. Furthermore, we found SC4MOL depletion by siRNA results in a significant decrease in total cell cholesterol. Thus, our work indicates SC4MOL is the most regulated enzyme in the C4-demethylation complex. Our results further implicate MARCHF6 as a crucial posttranslational regulator of cholesterol synthesis, with this E3 ubiquitin ligase controlling levels of at least five enzymes of the pathway.

Arabidopsis 3ß-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development.

The biosynthesis of C27-29 sterols from their C30 precursor squalene involves C24-alkylation and the removal of three methyl groups, including two at the C4 position. The two C4 demethylation reactions require a bifunctional enzyme known as 3ß-hydroxysteroid dehydrogenase/C4-decarboxylase (3ßHSD/D), which removes an oxidized methyl (carboxylic) group at C4 while simultaneously catalyzing the 3ß-hydroxyl→3-keto oxidation. Its loss-of-function mutations cause ergosterol-dependent growth in yeast and congenital hemidysplasia with ichthyosiform erythroderma and limb defect (CHILD) syndrome in humans. Although plant 3ßHSD/D enzymes were well studied enzymatically, their developmental functions remain unknown. Here we employed a CRISPR/Cas9-based genome-editing approach to generate knockout mutants for two Arabidopsis 3ßHSD/D genes, HSD1 and HSD2, and discovered the male gametophytic lethality for the hsd1 hsd2 double mutation. Pollen-specific expression of HSD2 in the heterozygous hsd1 hsd2/+ mutant not only rescued the pollen lethality but also revealed the critical roles of the two HSD genes in embryogenesis. Our study thus demonstrated the essential functions of the two Arabidopsis 3ßHSD/D genes in male gametogenesis and embryogenesis.

Ketonization of Ginsenoside C-K by Novel Recombinant 3-ß-Hydroxysteroid Dehydrogenases and Effect on Human Fibroblast Cells.

BACKGROUND AND OBJECTIVE: The ginsenoside compound K (C-K) (which is a de-glycosylated derivative of major ginsenosides) is effective in the treatment of cancer, diabetes, inflammation, allergy, angiogenesis, aging, and has neuroprotective, and hepatoprotective than other minor ginsenosides. Thus, a lot of studies have been focused on the conversion of major ginsenosides to minor ginsenosides using glycoside hydrolases but there is no study yet published for the bioconversion of minor ginsenosides into another high pharmacological active compound. Therefore, the objective of this study to identify a new gene (besides the glycoside hydrolases) for the conversion of minor ginsenosides C-K into another highly pharmacological active compound. METHODS AND RESULTS: Lactobacillus brevis which was isolated from Kimchi has showed the ginsenoside C-K altering capabilities. From this strain, a novel potent decarboxylation gene, named HSDLb1, was isolated and expressed in Escherichia coli BL21 (DE3) using the pMAL-c5X vector system. Recombinant HSDLb1 was also characterized. The HSDLb1 consists of 774 bp (258 amino acids residues) with a predicted molecular mass of 28.64 kDa. The optimum enzyme activity was recorded at pH 6.0-8.0 and temperature 30 °C. Recombinant HSDLb1 effectively transformed the ginsenoside C-K to 12-ß-hydroxydammar-3-one-20(S)-O-ß-D-glucopyranoside (3-oxo-C-K). The experimental data proved that recombinant HSDLb1 strongly ketonized the hydroxyl (-O-H) group at C-3 of C-K via the following pathway: C-K → 3-oxo-C-K. In vitro study, 3-oxo-C-K showed higher solubility than C-K, and no cytotoxicity to fibroblast cells. In addition, 3-oxo-C-K induced the inhibitory activity of ultraviolet A (UVA) against matrix metalloproteinase-1 (MMP-1) and promoted procollagen type I synthesis. Based on these expectations, we hypothesized that 3-oxo-C-K can be used in cosmetic products to block UV radiations and anti-ageing agent. Furthermore, we expect that 3-oxo-C-K will show higher efficacy than C-K for the treatment of cancer, ageing and other related diseases, for which more studies are needed.

The Non Catalytic Protein ERG28 has a Functional Role in Cholesterol Synthesis and is Coregulated Transcriptionally.

The enzymatic pathway of cholesterol biosynthesis has been well characterized. However, there remain several potential interacting proteins that may play ancillary roles in the regulation of cholesterol production. Here, we identified ERG28 (chromosome 14 open reading frame 1 [C14orf1]), a homologue of the yeast protein Erg28p, as a player in mammalian cholesterol synthesis. ERG28 is conserved from yeast to humans but has been largely overlooked in mammals. Using quantitative RT-PCR, luciferase assays, and publicly available chromatin immunoprecipitation sequencing data, we found that transcription of this gene is driven by the transcription factor SREBP-2, akin to most cholesterol synthesis enzymes, as well as identifying sterol-responsive elements and cofactor binding sites in its proximal promoter. Based on a split luciferase system, ERG28 interacted with itself and two enzymes of cholesterol synthesis (NSDHL and SC4MOL). Huh7 ERG28-KO cell lines were generated, revealing reduced total cholesterol levels in sterol-depleted environments. In addition, radiolabeled metabolic flux assays showed a 60-75% reduction in the rate of cholesterol synthesis in the KO versus wild-type cells, which could be rescued by expression of ectopic ERG28. Unexpectedly, KO of ERG28 also impaired the activation of SREBP-2 under sterol-replete conditions, by a yet-to-be defined mechanism. These results indicate that ERG28 is clearly involved in cholesterol synthesis, although the precise role this noncatalytic protein plays in this complex metabolic pathway remains to be fully elucidated. A deeper understanding of ERG28, and other ancillary proteins of cholesterol synthesis, may help inform therapeutic strategies for diseases associated with aberrant cholesterol metabolism.

Integrative Analysis of Transcriptomic and Metabolomic Data for Identification of Pathways Related to Matrine-Induced Hepatotoxicity.

Matrine (MT) is a major bioactive compound extracted from Sophorae tonkinensis. However, the clinical application of MT is relatively restricted due to its potentially toxic effects, especially hepatotoxicity. Although MT-induced liver injury has been reported, little is known about the underlying molecular mechanisms. In this study, transcriptomics and metabolomics were applied to investigate the hepatotoxicity of MT in mice. The results indicated that liver injury occurred when the administration of MT (30 or 60 mg/kg, i.g) lasted for 2 weeks, including dramatically increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), etc. The metabolomic results revealed that steroid biosynthesis, purine metabolism, glutathione metabolism, and pyruvate metabolism were involved in the occurrence and development of MT-induced hepatotoxicity. Further, the transcriptomic data indicated that the downregulation of NSDHL with CYP51, FDFT1, and DHCR7, involved in steroid biosynthesis, resulted in a lower level of cholic acid. Besides, Gstps and Nat8f1 were related to the disorder of glutathione metabolism, and HMGCS1 could be treated as the marker gene of the development of MT-induced hepatotoxicity. In addition, other metabolites, such as taurine, flavin mononucleotide (FMN), and inosine monophosphate (IMP), also made a contribution to the boosting of MT-induced hepatotoxicity. In this work, our results provide clues for the mechanism investigation of MT-induced hepatotoxicity, and several biomarkers (metabolites and genes) closely related to the liver injury caused by MT are also provided. Meanwhile, new insights into the understanding of the development of MT-induced hepatotoxicity or other monomer-induced hepatotoxicity were also provided.

Absence of some cytochrome P450 (CYP) and hydroxysteroid dehydrogenase (HSD) enzymes in hagfishes.

Corticosteroids are synthesized from cholesterol by steroidogenic enzyme catalysts belonging to two main families: the cytochrome p450s (CYPs) and hydroxysteroid dehydrogenases (HSDs). The action of these steroidogenic enzymes allows the genesis of the terminal active corticosteroids 11-deoxycortisol (S), 1ɑ-hydroxycorticosterone (1α-OH-B), or cortisol in different fish species. However, for Cyclostomes like hagfishes, the terminal corticosteroid is still undefined. In this study, we examined the presence or absence of CYPs and HSDs as traits in fishes to gain insight about the primary corticosteroid synthesis pathways of the hagfishes. We used published cytochrome c oxidase I (COXI) amino acid sequences to construct a phylogeny of fishes and then mapped the CYPs and HSDs as morphological traits onto the tree to predict the ancestral character states through ancestral character reconstruction (ACR). There is a clear phylogenetic signal for CYP (i.e., CYP11a1, 17, 21, and 11b) and HSD (i.e., 11-ßHSD and 3ß-HSD) derivatives of interest throughout the more derived fishes. Using trait-based ACR, we also found that hagfishes possess genes for 3ß-HSD, CYP11a1, CYP17, and CYP21. Importantly, the presence of CYP21 implies that hagfish can synthesize 11-deoxycorticosterone (11-DOC) and S. Previous research demonstrated that despite hagfish having CYP21, neither 11-DOC nor S could be detected in hagfish. This discrepancy between the presence of steroidogenic enzymes and products brings into question the expression and/or function of CYP21 in hagfishes.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

Inflammatory linear verrucous epidermal nevus (ILVEN) encompasses a spectrum of inflammatory mosaic disorders.

BACKGROUND: Inflammatory linear verrucous epidermal nevus (ILVEN) is a rare skin disease characterized by pruritic erythematous scaly plaques distributed along the lines of Blaschko. Two cases of ILVEN with CARD14 mutations and one case with a GJA1 mutation have been previously reported. OBJECTIVE: To elucidate the genetic cause of a cohort of patients diagnosed based on clinical and histopathological evaluation with ILVEN. METHODS: We recruited patients diagnosed with ILVEN based on clinical and histopathological criteria. Exome sequencing of affected skin with or without blood/saliva was performed and germline and somatic pathogenic variants were identified. RESULTS: Five patients were enrolled. All had skin lesions from birth or early childhood. Two patients developed psoriasis vulgaris after the diagnosis of ILVEN. The first had a germline heterozygous CARD14 mutation and a post-zygotic hotspot mutation in KRT10. The histopathologic evaluation did not show epidermolytic hyperkeratosis. The second had a post-zygotic hotspot mutation in HRAS. Her ILVEN became itchy once psoriasis developed. One patient was re-diagnosed with linear porokeratosis based on a germline mutation in PMVK and a post-zygotic second-hit mutation. Two patients were re-diagnosed with congenital hemidysplasia with ichthyosiform nevus and limb defect nevus based on germline NSDHL mutations. CONCLUSION: ILVEN is a clinical descriptor for a heterogenous group of mosaic inflammatory disorders. Genetic analysis has the potential to more precisely categorize ILVEN and permits pathogenesis-directed therapies in some cases.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Physiopathological role of the enzymatic complex 5α-reductase and 3α/ß-hydroxysteroid oxidoreductase in the generation of progesterone and testosterone neuroactive metabolites.

The enzymatic complex 5α-reductase (5α-R) and 3α/3ß-hydroxysteroid oxidoreductase (HSOR) is expressed in the nervous system, where it transforms progesterone (PROG) and testosterone (T) into neuroactive metabolites. These metabolites regulate myelination, brain maturation, neurotransmission, reproductive behavior and the stress response. The expression of 5α-R and 3α-HSOR and the levels of PROG and T reduced metabolites show regional and sex differences in the nervous system and are affected by changing physiological conditions as well as by neurodegenerative and psychiatric disorders. A decrease in their nervous tissue levels may negatively impact the course and outcome of some pathological events. However, in other pathological conditions their increased levels may have a negative impact. Thus, the use of synthetic analogues of these steroids or 5α-R modulation have been proposed as therapeutic approaches for several nervous system pathologies. However, further research is needed to fully understand the consequences of these manipulations, in particular with 5α-R inhibitors.

NSDHL promotes triple-negative breast cancer metastasis through the TGFß signaling pathway and cholesterol biosynthesis.

PURPOSE: Metastasis is the main cause of breast cancer mortality. Recent studies have proved that lipid metabolic reprogramming plays critical roles in breast cancer carcinogenesis and metastasis. We aim to identify critical lipid metabolism genes in breast cancer metastasis. METHODS: We designed and cloned a CRISPR pooled library containing lipid metabolic gene guide RNAs and performed a genetic screen in vivo. Transwell assay and animal experiments were used to evaluate cell metastatic ability in vitro or in vivo, respectively. We performed immunohistochemistry with breast cancer tissue microarray to study the clinical significance of NSDHL. FINDINGS: We identified a cholesterol metabolic enzyme, NSDHL, as a potential metastatic driver in triple-negative breast cancer. NSDHL was highly expressed in breast cancer tissues and predicted a poor prognosis. NSDHL knockdown significantly suppressed cell proliferation and migration. Mechanistically, NSDHL activated the TGFß signaling pathway by inhibiting the endosomal degradation of TGFßR2. In addition, blocking the upstream metabolism of NSDHL with ketoconazole rescued cancer metastasis and TGFßR2 degradation. However, the inactivation of NSDHL (Y151X) did not rescue the migration ability and the TGFßR2 protein expression. CONCLUSION: Taken together, our findings established that NSDHL serves as a metastatic driver, and its function depends on its enzyme activity in cholesterol biosynthesis and is mediated by the NSDHL-TGFßR2 signal pathway. Our study indicated that NSDHL and steroid biosynthesis may serve as new drug targets for patients with advanced breast cancer.

Oxysterol gradient generation by lymphoid stromal cells guides activated B cell movement during humoral responses.

7α,25-dihydroxycholesterol (7α,25-OHC) is a ligand for the G protein-coupled receptor EBI2; however, the cellular sources of this oxysterol are undefined. 7α,25-OHC is synthesized from cholesterol by the stepwise actions of two enzymes, CH25H and CYP7B1, and is metabolized to a 3-oxo derivative by HSD3B7. We showed that all three enzymes control EBI2 ligand concentration in lymphoid tissues. Lymphoid stromal cells were the main CH25H- and CYP7B1-expressing cells required for positioning of B cells, and they also mediated 7α,25-OHC inactivation. CH25H and CYP7B1 were abundant at the follicle perimeter, whereas CH25H expression by follicular dendritic cells was repressed. CYP7B1, CH25H, and HSD3B7 deficiencies each resulted in defective T cell-dependent plasma cell responses. These findings establish that CYP7B1 and HSD3B7, as well as CH25H, have essential roles in controlling oxysterol production in lymphoid tissues, and they suggest that differential enzyme expression in stromal cell subsets establishes 7α,25-OHC gradients required for B cell responses.

Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer.

Prostate cancer resistance to castration occurs because tumours acquire the metabolic capability of converting precursor steroids to 5α-dihydrotestosterone (DHT), promoting signalling by the androgen receptor and the development of castration-resistant prostate cancer. Essential for resistance, DHT synthesis from adrenal precursor steroids or possibly from de novo synthesis from cholesterol commonly requires enzymatic reactions by 3ß-hydroxysteroid dehydrogenase (3ßHSD), steroid-5α-reductase (SRD5A) and 17ß-hydroxysteroid dehydrogenase (17ßHSD) isoenzymes. Abiraterone, a steroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitor, blocks this synthetic process and prolongs survival. We hypothesized that abiraterone is converted by an enzyme to the more active Δ(4)-abiraterone (D4A), which blocks multiple steroidogenic enzymes and antagonizes the androgen receptor, providing an additional explanation for abiraterone's clinical activity. Here we show that abiraterone is converted to D4A in mice and patients with prostate cancer. D4A inhibits CYP17A1, 3ßHSD and SRD5A, which are required for DHT synthesis. Furthermore, competitive androgen receptor antagonism by D4A is comparable to the potent antagonist enzalutamide. D4A also has more potent anti-tumour activity against xenograft tumours than abiraterone. Our findings suggest an additional explanation-conversion to a more active agent-for abiraterone's survival extension. We propose that direct treatment with D4A would be more clinically effective than abiraterone treatment.

The brain 3ß-HSD up-regulation in response to deteriorating effects of background emotional stress: an animal model of multiple sclerosis.

The brain 3ß-hydroxysteroid dehydrogenase (3ß-HSD), is the enzyme that catalyzes the biosynthesis of a neuroprotective factor, progesterone. The regulation of 3ß-HSD in response to stress exposure in the cuprizone-induced model of Multiple Sclerosis was investigated and the reaction related to the demyelination extremity. 32 female Wistar rats divided into four groups (i.e., control group (Cont), non-stress cuprizone treated (N-CPZ), physical stress- cuprizone treated (P-CPZ) and emotional stress- cuprizone treated (E-CPZ). A witness foot-shock model used to induce background stress for 5 days. An elevated-plus maze applied to validate the stress induction. Followed by 6 weeks of cuprizone treatment, the Y-maze test performed to confirm brain demyelination. 3ß-HSD gene expression as an indicator of progesterone synthesis examined. At the behavioral level, both stressed groups reflected more impaired spatial memory compared to the N-CPZ group (p < 0.01), with more severe results in the E-CPZ group (p < 0.01). The results of mRNA expression of 3ß-HSD illustrated significant elevation in all cuprizone treated groups (p < 0.001) with a higher up-regulation (p < 0.001) in the E-CPZ group. Background stress -particularly emotional type- exacerbates the demyelination caused by cuprizone treatment. The brain up-regulates the 3ß-HSD gene expression as a protective response relative to the myelin degradation extent.

NAD(P)-dependent steroid dehydrogenase-like protein and neutral cholesterol ester hydrolase 1 serve as novel markers for early detection of gastric cancer identified using quantitative proteomics.

BACKGROUND: Gastric cancer (GC) is the third most common cause of cancer deaths worldwide. In the present study, we aimed to identify novel GC biomarkers by integrating isobaric tags of relative and absolute quantitation (iTRAQ) for aberrantly expressed proteins in GC patients. METHODS: Using stable isotope tags, we labeled an initial discovery group comprising four paired gastric cancer and adjacent gastric tissue samples, and subjected them to LC-ESI-MS/MS. We used a validation set comprising 129 paired gastric cancer and adjacent gastric tissues from patients and benign healthy controls to validate the candidate targets. RESULTS: We identified two proteins, NAD(P)-dependent steroid dehydrogenase-like (NSDHL) and neutral cholesterol ester hydrolase 1 (NCEH1), that were significantly overexpressed in GC tissues. The sensitivity and specificity of NSDHL were 80.6% and 74.4%, respectively, in GC compared with a sensitivity of 25.6% in adjacent tissues and 24% in benign healthy controls. The area under the ROC curve (AUC) for NSDHL was 0.810 for GC detection. Overexpression of NSDHL in GC was significantly correlated with local tumor invasion. The sensitivity and specificity of NCEH1 were 77.5% and 73.6%, respectively, in GC compared with a sensitivity of 26.4% in adjacent tissues and 20% in benign controls. The AUC for NSDHL was 0.792. Overexpression of NCEH1 was significantly associated with tumor histological classification and local invasion. Moreover, a combined analysis of NSDHL and NCEH1 achieved a sensitivity and specificity of 85.7% and 83%, respectively, and the AUC was 0.872. The combined analysis of NSDHL and NCEH1 was significantly correlated with histological grade and TNM Ⅱ-Ⅳ staging. CONCLUSIONS: iTRAQ-labeled quantitative proteomics represents a powerful method to identify novel cancer biomarkers. The present study identified NSDHL and NCEH1 as useful biomarkers for screening, diagnosis, and prognosis of patients with gastric cancer.

Possible roles of gonadotropin-releasing hormone (GnRH) and melatonin in the control of gonadal development of clam Ruditapes philippinarum.

Gonadotropin-releasing Hormone (GnRH) is a key reproductive endocrine regulator, and melatonin is considered as a potent candidate in the regulation of photoperiod-related reproductive endocrinology. Nevertheless, their function during gonadal development of molluscs has not been uncovered yet. In the present study, RNAi of GnRH and melatonin injection were conducted on marine bivalve manila clam Ruditapes philippinarum. Tissue section showed that gonadal development was significantly inhibited in male clams injected with GnRH dsRNA for 21 days. For GnRH RNAi treatment group, the expression levels of steroid synthetic enzyme genes 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), cytochrome P450 (CYP3A) and melatonin receptor homolog (MTNR) gene were significantly down-regulated in female clams while significantly up-regulated in male clams. In melatonin injection group, the expression of GnRH was significantly inhibited and the expression of 3ß-HSD, 17ß-HSD, CYP3A and MTNR genes also increased which was in line with the GnRH dsRNA injection group in male clams. These results suggest that melatonin may affect GnRH expression and both have effects on gonadal development of bivalves. This study provides evidence for understanding the effects of melatonin and GnRH on reproductive endocrinology and gonadal development in bivalve molluscs.