-
International Journal of Molecular... Sep 2019A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated...
A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated pathways underlying the modulation of reactive microglia. In the present work, the TSPO activation was explored in an in vitro human primary microglia model (immortalized C20 cells) under inflammatory stimulus. Two different approaches were used with the aim to (i) pharmacologically amplify or (ii) silence, by the lentiviral short hairpin RNA, the TSPO physiological function. In the TSPO pharmacological stimulation model, the synthetic steroidogenic selective ligand XBD-173 attenuated the activation of microglia. Indeed, it reduces and increases the release of pro-inflammatory and anti-inflammatory cytokines, respectively. Such ligand-induced effects were abolished when C20 cells were treated with the steroidogenesis inhibitor aminoglutethimide. This suggests a role for neurosteroids in modulating the interleukin production. The highly steroidogenic ligand XBD-173 attenuated the neuroinflammatory response more effectively than the poorly steroidogenic ones, which suggests that the observed modulation on the cytokine release may be influenced by the levels of produced neurosteroids. In the TSPO silencing model, the reduction of TSPO caused a more inflamed phenotype with respect to scrambled cells. Similarly, during the inflammatory response, the TSPO silencing increased and reduced the release of pro-inflammatory and anti-inflammatory cytokines, respectively. In conclusion, the obtained results are in favor of a homeostatic role for TSPO in the context of dynamic balance between anti-inflammatory and pro-inflammatory mediators in the human microglia-mediated inflammatory response. Interestingly, our preliminary results propose that the TSPO expression could be stimulated by NF-κB during activation of the inflammatory response.
Topics: Aminoglutethimide; Anti-Inflammatory Agents; Aromatase Inhibitors; Base Sequence; Cell Line; Cell Survival; Cytokines; Gene Expression; Humans; Inflammation Mediators; Microglia; NF-kappa B; Phenotype; Purines; RNA Interference; Receptors, GABA
PubMed: 31510070
DOI: 10.3390/ijms20184467 -
Biomaterials Dec 2018The intrinsic characteristics of the tumor microenvironment (TME), including acidic pH and overexpression of hydrolytic enzymes, offer an exciting opportunity for the...
The intrinsic characteristics of the tumor microenvironment (TME), including acidic pH and overexpression of hydrolytic enzymes, offer an exciting opportunity for the rational design of TME-drug delivery systems (DDS). We developed and characterized a pH-responsive biodegradable poly-L-glutamic acid (PGA)-based combination conjugate family with the aim of optimizing anticancer effects. We obtained combination conjugates bearing Doxorubicin (Dox) and aminoglutethimide (AGM) with two Dox loadings and two different hydrazone pH-sensitive linkers that promote the specific release of Dox from the polymeric backbone within the TME. Low Dox loading coupled with a short hydrazone linker yielded optimal effects on primary tumor growth, lung metastasis (∼90% reduction), and toxicological profile in a preclinical metastatic triple-negative breast cancer (TNBC) murine model. The use of transcriptomic analysis helped us to identify the molecular mechanisms responsible for such results including a differential immunomodulation and cell death pathways among the conjugates. This data highlights the advantages of targeting the TME, the therapeutic value of polymer-based combination approaches, and the utility of -omics-based analysis to accelerate anticancer DDS.
Topics: Aminoglutethimide; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Doxorubicin; Drug Carriers; Drug Liberation; Female; Heterografts; Humans; Hydrogen-Ion Concentration; Mice, Inbred BALB C; Polyglutamic Acid; Triple Negative Breast Neoplasms; Tumor Microenvironment
PubMed: 30278346
DOI: 10.1016/j.biomaterials.2018.09.023 -
Molecular Human Reproduction Jan 2018Does 27-hydroxycholesterol (27OH) actively facilitate the progression of luteolysis?
STUDY QUESTION
Does 27-hydroxycholesterol (27OH) actively facilitate the progression of luteolysis?
SUMMARY ANSWER
There is increased mRNA expression of the enzyme that produces 27OH during luteolysis in vivo in rhesus macaques and sheep, and 27OH reduces progesterone secretion from human luteinized granulosa cells.
WHAT IS KNOWN ALREADY
There is an increase in mRNA expression of liver x receptor (LXR) and a decrease in sterol regulatory element binding protein 2 (SREBP2) target genes during spontaneous luteolysis in primates, which could result in reduced cholesterol availability for steroidogenesis. Concentrations of 27OH are also increased in primate corpora lutea (CL) during luteolysis, and 27OH is a dual LXR agonist and SREBP2 inhibitor.
STUDY DESIGN SIZE, DURATION
This was an in vitro study using primary human luteinized granulosa cells in a control versus treatment(s) design. Analyses of CL from sheep undergoing induced or spontaneous luteolysis were also performed, along with database mining of microarray data from rhesus macaque CL.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Primary luteinizing granulosa cells were obtained from 37 women aged 24-44 who were undergoing oocyte donation or IVF for male factor or idiopathic infertility, and cells were further luteinized in vitro using human chorionic gonadotropin. Three approaches to test the effect of 27OH produced via CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1) on luteinized granulosa cells were used: (i) direct 27OH supplementation, (ii) induction of endogenous CYP27A1 activity via pharmacologic inhibition of steroidogenesis, and (iii) siRNA-mediated knockdown to directly inhibit CYP27A1 as well as cholesterol transport into the mitochondria via the steroidogenic acute regulatory protein (STAR). Endpoints included: progesterone (P4) secretion into culture media determined by enzyme immunoassay, cholesterol efflux and uptake assays using fluorescent lipid analogs, and mRNA expression determined via semi-quantitative real-time PCR (QPCR). An additional experiment involved QPCR analysis of 40 CL collected from ewes undergoing induced or spontaneous luteolysis, as well as database mining of microarray data generated from 16 rhesus macaque CL collected during spontaneous luteolysis and 13 macaque CL collected during a luteinizing hormone ablation and replacement protocol.
MAIN RESULTS AND THE ROLE OF CHANCE
The mRNA expression of CYP27A1 was significantly increased during luteolysis in rhesus macaques and sheep in vivo, and CYP27A1 transcription was suppressed by luteinizing hormone and hCG. There was a significant decrease in hCG-stimulated P4 secretion from human luteinized granulosa cells caused by 27OH treatment, and a significant increase in basal and hCG-stimulated P4 synthesis when endogenous 27OH production was inhibited via CYP27A1 knockdown, indicating that 27OH inhibits steroidogenesis. Pharmacologic inhibition of steroidogenesis by aminoglutethimide significantly induced LXR and inhibited SREBP2 target gene mRNA expression, indicating that increased oxysterol production occurs when steroidogenesis is suppressed. Inhibiting cholesterol delivery into the mitochondria via knockdown of STAR resulted in reduced SREBP2 target gene mRNA expression, indicating that STAR function is necessary to maintain SREBP2-mediated transcription. The effects of 27OH treatment on markers of LXR and SREBP2 activity were moderate, and knockdown of CYP27A1 did not prevent aminoglutethimide-induced changes in LXR and SREBP2 target gene mRNA expression. These observations indicate that 27OH inhibits P4 secretion partially via mechanisms separate from its role as an LXR agonist and SREBP2 inhibitor, and also demonstrate that other oxysterols are involved in modulating LXR and SREBP2-mediated transcription when steroidogenesis is suppressed.
LARGE SCALE DATA
None.
LIMITATIONS REASONS FOR CAUTION
Luteinized granulosa cells may differ from luteal cells, and the effect on luteal function in vivo was not directly tested. The mechanisms that cause the initial rise in CYP27A1 mRNA expression during luteolysis are also not clear.
WIDER IMPLICATIONS OF THE FINDINGS
The factors causing luteolysis in primates have not yet been determined. This study provides functional evidence of a novel mechanism via increased 27OH synthesis during luteolysis, which subsequently represses progesterone secretion. Increased 27OH may also facilitate the progression of luteolysis in domestic animal species.
STUDY FUNDING AND COMPETING INTEREST(S)
The authors have nothing to disclose. Support was provided by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH), award number R00HD067678 to R.L.B.
Topics: Adult; Aminoglutethimide; Cells, Cultured; Cholestanetriol 26-Monooxygenase; Cholesterol; Chorionic Gonadotropin; Female; Humans; Hydroxycholesterols; Immunoenzyme Techniques; Luteinizing Hormone; Luteolysis; Progesterone; RNA, Small Interfering; Real-Time Polymerase Chain Reaction; Sterol Regulatory Element Binding Protein 2
PubMed: 29177442
DOI: 10.1093/molehr/gax061 -
International Journal of Molecular... Nov 2017Two new ergostane-type sterols; (22)-5,6α-epoxyergosta-8,14,22-triene-3β,7β-diol () and 5α,6α-epoxyergost-8(14)-ene-3β,7α-diol () were isolated from the fruiting...
Two new ergostane-type sterols; (22)-5,6α-epoxyergosta-8,14,22-triene-3β,7β-diol () and 5α,6α-epoxyergost-8(14)-ene-3β,7α-diol () were isolated from the fruiting bodies of king trumpet mushroom (), along with eight known compounds (-). All isolated compounds were evaluated for their inhibitory effects on aromatase. Among them, and exhibited comparable aromatase inhibitory activities to aminoglutethimide.
Topics: Agaricales; Aromatase; Aromatase Inhibitors; Biological Products; Enzyme Activation; Ergosterol; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Molecular Structure
PubMed: 29160820
DOI: 10.3390/ijms18112479 -
Medicine Jul 2017Steroid profiling was introduced to determine the endogenous steroid misuse in sports. Thus, screening for the exogenous use of these prohibited substances can be... (Randomized Controlled Trial)
Randomized Controlled Trial
Steroid profiling was introduced to determine the endogenous steroid misuse in sports. Thus, screening for the exogenous use of these prohibited substances can be established by monitoring a range of endogenous steroids, which constitute the steroid profile and evaluate their concentrations and ratios against reference values. The steroid profiling is currently based on population statistics. As large interindividual variations exist, athlete biological passport (ABP) analysis is ongoing. This study aimed to identify new biomarker(s) for aromatase inhibitor detection in sports using statistical analysis and adapt the model into ABP analysis.Forty-one Chinese nonathlete volunteers (21 males and 20 females) were administered 3 nonsteroidal aromatase inhibitors (aminoglutethimide, letrozole, and anastrozole) independently. Statistical analysis was performed on 16 steroid profile parameters.After administration, the concentrations of endogenous androgen biomarkers including testosterone (T), epitestosterone, androsterone (AN), etiocholanolone (ETIO), 5α-diol, 5β-diol, and dehydroepiandrosterone were increased, while the level of estrogen was decreased. These biomarkers returned to the baselines levels within 1 month. In females, the concentrations of endogenous biomarkers were affected by nonsteroidal aromatase inhibitors, without a common trend. Three new endogenous biomarkers (AN/estrone, ETIO/estrone, and T/estrone) elevated significantly after treatment. The 3 new models were more sensitive than the World Anti-Doping Agency ratio biomarkers. They were also effective in exponentially weighted moving average chart analysis.Verification experiment demonstrated that the biomarker T/estrone was valid in judging the steroidal aromatase inhibitor abuse. The screening of these new endogenous biomarkers can provide additional parameters to support ABP monitoring and specific information regarding the administered steroids.
Topics: Aminoglutethimide; Anastrozole; Aromatase Inhibitors; Biomarkers, Pharmacological; Doping in Sports; Female; Hormones; Humans; Letrozole; Male; Models, Biological; Nitriles; Single-Blind Method; Steroids; Substance-Related Disorders; Triazoles; Young Adult
PubMed: 28700478
DOI: 10.1097/MD.0000000000007411 -
Anesthesiology Sep 2017Liver X receptors, including α and β isoforms, are ligand-activated transcription factors. Whether liver X receptor α plays a role in neuropathic pain is unknown.
BACKGROUND
Liver X receptors, including α and β isoforms, are ligand-activated transcription factors. Whether liver X receptor α plays a role in neuropathic pain is unknown.
METHODS
A spared nerve injury model was established in adult male rats and mice. Von Frey tests were performed to evaluate the neuropathic pain behavior; Western blot and immunohistochemistry were performed to understand the underlying mechanisms.
RESULTS
Intrathecal injection of a specific liver X receptor agonist T0901317 or GW3965 could either prevent the development of mechanical allodynia or alleviate the established mechanical allodynia, both in rats and wild-type mice. GW3965 could inhibit the activation of glial cells and the expression of tumor necrosis factor-α (mean ± SD: 196 ± 48 vs. 119 ± 57; n = 6; P < 0.01) and interleukin 1β (mean ± SD: 215 ± 69 vs. 158 ± 74; n = 6; P < 0.01) and increase the expression of interleukin 10 in the spinal dorsal horn. All of the above effects of GW3965 could be abolished by liver X receptor α mutation. Moreover, more glial cells were activated, and more interleukin 1β was released in the spinal dorsal horn in liver X receptor α knockout mice than in wild-type mice after spared nerve injury. Aminoglutethimide, a neurosteroid synthesis inhibitor, blocked the inhibitory effect of T0901317 on mechanical allodynia, on the activation of glial cells, and on the expression of cytokines.
CONCLUSIONS
Activation of liver X receptor α inhibits mechanical allodynia by inhibiting the activation of glial cells and rebalancing cytokines in the spinal dorsal horn via neurosteroids.
Topics: Animals; Blotting, Western; Cytokines; Disease Models, Animal; Hyperalgesia; Immunohistochemistry; Inflammation; Interleukin-1beta; Liver X Receptors; Male; Mice; Mice, Knockout; Neuralgia; Neuroglia; Rats; Rats, Sprague-Dawley; Spinal Cord Dorsal Horn
PubMed: 28617705
DOI: 10.1097/ALN.0000000000001718 -
EBioMedicine Dec 2016Artemisinin (ARS) and its derivatives, which are clinically used antimalarial agents, have shown antitumor activities. Their therapeutic potencies, however, are limited...
UNLABELLED
Artemisinin (ARS) and its derivatives, which are clinically used antimalarial agents, have shown antitumor activities. Their therapeutic potencies, however, are limited by their low solubility and poor bioavailability. Here, through a pharmacophore hybridization strategy, we synthesized ARS-drug conjugates, in which the marketed chemotherapeutic agents chlorambucil, melphalan, flutamide, aminoglutethimide, and doxifluridine, were separately bonded to Dihydroartemisinin (DHA) through various linkages. Of these, the artemisinin-melphalan conjugate, ARS4, exhibited most toxicity to human ovarian cancer cells but had low cytotoxicity to normal cells. ARS4 inhibited the growth and proliferation of ovarian cancer cells and resulted in S-phase arrest, apoptosis, and inhibition of migration; these effects were stronger than those of its parent drugs, DHA and melphalan. Furthermore, ARS4 modulated the expression of proteins involved in cell cycle progression, apoptosis, and the epithelial-mesenchymal transition (EMT). Moreover, in mice, ARS4 inhibited growth and intraperitoneal dissemination and metastasis of ovarian cancer cells without observable toxic effects. Our results provide a basis for development of the compound as a chemotherapeutic agent.
RESEARCH IN CONTEXT
Artemisinin compounds have recently received attention as anticancer agents because of their clinical safety profiles and broad efficacy. However, their therapeutic potencies are limited by low solubility and poor bioavailability. Here, we report that ARS4, an artemisinin-melphalan conjugate, possesses marked in-vitro and in-vivo antitumor activity against ovarian cancer, the effects of which are stronger than those for its parent drugs, Dihydroartemisinin and melphalan. In mice, ARS4 inhibits localized growth of ovarian cancer cells and intraperitoneal dissemination and metastasis without appreciable host toxicity. Thus, for patients with ovarian cancer, ARS4 is a promising chemotherapeutic agent.
Topics: Animals; Antineoplastic Agents; Apoptosis; Artemisinins; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Drug Combinations; Drug Evaluation, Preclinical; Epithelial-Mesenchymal Transition; Female; Humans; Mice; Neoplasm Metastasis; Neoplasm Staging; Ovarian Neoplasms; Structure-Activity Relationship; Xenograft Model Antitumor Assays
PubMed: 27939426
DOI: 10.1016/j.ebiom.2016.11.026 -
Chemical & Pharmaceutical Bulletin 2016A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit enzymatic activity against aromatase (IC50=16.5 µg/mL). From the extract,...
A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit enzymatic activity against aromatase (IC50=16.5 µg/mL). From the extract, two new geranylated coumarins, mammeasins C (1) and D (2), were isolated together with seven coumarins: 8-hydroxy-5-methyl-7-(3,7-dimethyl-octa-2,6-dienyl)-9-(2-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (9), 8-hydroxy-5-methyl-7-(3,7-dimethyl-octa-2,6-dienyl)-9-(3-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (10), mammeas A/AA (14), A/AB (15), A/AA cyclo D (18), E/BA (23), and E/BC cyclo D (25). The structures of 1 and 2 were elucidated on the basis of spectroscopic evidence. Among the isolates including 17 previously reported coumarins, 1 (IC50=2.7 µM), 2 (3.6 µM), and mammea B/AB cyclo D (21, 3.1 µM) showed relatively strong inhibitory activities comparable to the activity of the synthetic nonsteroidal aromatase inhibitor aminoglutethimide (2.0 µM).
Topics: Aromatase; Aromatase Inhibitors; Coumarins; Dose-Response Relationship, Drug; Flowers; Humans; Mammea; Molecular Structure; Recombinant Proteins; Structure-Activity Relationship
PubMed: 27373643
DOI: 10.1248/cpb.c16-00218 -
International Journal of Molecular... Jun 2016The steroidogenic 18 kDa translocator protein (TSPO) is an emerging, attractive therapeutic tool for several pathological conditions of the nervous system. Here, 13 high...
The steroidogenic 18 kDa translocator protein (TSPO) is an emerging, attractive therapeutic tool for several pathological conditions of the nervous system. Here, 13 high affinity TSPO ligands belonging to our previously described N,N-dialkyl-2-phenylindol-3-ylglyoxylamide (PIGA) class were evaluated for their potential ability to affect the cellular Oxidative Metabolism Activity/Proliferation index, which is used as a measure of astrocyte well-being. The most active PIGA ligands were also assessed for steroidogenic activity in terms of pregnenolone production, and the values were related to the metabolic index in rat and human models. The results showed a positive correlation between the increase in the Oxidative Metabolism Activity/Proliferation index and the pharmacologically induced stimulation of steroidogenesis. The specific involvement of steroid molecules in mediating the metabolic effects of the PIGA ligands was demonstrated using aminoglutethimide, a specific inhibitor of the first step of steroid biosynthesis. The most promising steroidogenic PIGA ligands were the 2-naphthyl derivatives that showed a long residence time to the target, in agreement with our previous data. In conclusion, TSPO ligand-induced neurosteroidogenesis was involved in astrocyte well-being.
Topics: Astrocytes; Cell Line; Cell Proliferation; Humans; Indoles; Neurogenesis; Oxidation-Reduction; Pregnenolone
PubMed: 27367681
DOI: 10.3390/ijms17071028