In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.Activation of androgen receptor (AR) is crucial for prostate cancer growth. Remarkably, also castration-resistant prostate cancer (CRPC) is dependent on functional AR, and several mechanisms have been proposed to explain the addiction. Known causes of CRPC include gene amplification and overexpression as well as point mutations of AR. We report here the pharmacological profile of ODM-201, a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both  Prostate cancer (PC) is the most common cancer and among the three leading causes of cancer deaths in men in the United States349CRPC typically arises through mechanisms involving AR, as shown by studies demonstrating the role of autocrine synthesis of androgens and AR protein overexpression in CRPC111214151617181922232522in vitro and in vivo than other second-generation antiandrogens. Unlike other antiandrogens, ODM-201 exhibits negligible brain penetrance and does not increase serum testosterone levels in mice. These findings have been translated to a promising antitumor activity in CRPC patients, as shown by significant PSA reductions. The PSA response (50% or greater decrease) was present at 86% of chemotherapy-naïve patients at 700 mg twice daily (the highest dose in phase II dose expansion) in the ARADES study In the present study, we describe the discovery and preclinical development of ODM-201, a novel, nonsteroidal, orally active AR inhibitor that inhibits potently androgen binding to AR and androgen-induced nuclear translocation of AR in AR overexpressing cells. Using cells transiently transfected with expression vectors encoding the corresponding mutant AR and an androgen-responsive luciferase reporter gene construct, we show that ODM-201 retains antagonistic properties against clinically relevant AR mutations conferring resistance to antiandrogen therapies. ODM-201 is a full antagonist for the mutant AR(F876L) described to play a role in enzalutamide and ARN-509 resistance, and for mutants AR(W741L) and AR(T877A) shown to mediate resistance to bicalutamide and hydroxyflutamide. ODM-201 shows a more potent antitumor activity in nonclinical models of CRPC, AR-overexpressing VCaP cells, ODM-201 is a synthetic compound discovered by screening campaign using an AR transactivation assay in AR-HEK293 cells and by subsequent medicinal chemistry optimization. ODM-201 and its pharmacologically active main metabolite ORM-15341 are novel and structurally distinct from any known antiandrogens including the second-generation antiandrogens enzalutamide and ARN-509. 50 values of 26 and 38 nM as shown by transactivation assays in AR-HEK293 cells stably expressing full-length hAR and an androgen-responsive luciferase reporter gene construct. Enzalutamide and ARN-509 have IC50 values of 219 and 200 nM, respectively  values of ODM-201 and ORM-15341 were 11 and 8 nM, respectively, being clearly lower than those of enzalutamide (86 nM) and ARN-509 (93 nM)  that werectively . Thus, OEmergence of mutations in AR has been suggested to drive resistance to antiandrogen therapies. The effects of antiandrogens on mutant AR(F876L), AR(W741L), and AR(T877A) were studied in transactivation assays in human U2-OS osteosarcoma cells transiently transfected with expression vectors encoding the corresponding mutant AR and an androgen-responsive luciferase reporter gene construct. The F876L substitution in AR switched enzalutamide and ARN-509 from antagonists to agonists, whereas bicalutamide was agonistic for AR(W741L) mutation , as prev22232426To investigate the effect of antiandrogens on the subcellular localization of AR, immunocytochemical labeling with an anti-AR antibody in AR overexpressing HS-HEK293 cells was used. As shown in 50 values being 230 and 170 nM for ODM-201 and ORM-15341 vs. 410 and 420 nM for enzalutamide and ARN-509  once (qd) or twice daily (bid), or with enzalutamide  for 37 days. The dose for enzalutamide was selected based on previously published in vivo studies0–24) for this dose of enzalutamide was 2.5 times higher than that for ODM-201 . Moreover, enzalutamide exhibited a long plasma half-life (18.3 hours) while the half-life of ODM-201 in mice was not optimal (1.6 hours) supporting once daily dosing for enzalutamide and higher dose and more frequent dosing for ODM-201. PK data for enzalutamide and ODM-201 are presented in To elucidate the ODM-201 showed a significant antitumor activity with both doses, 50 mg/kg twice daily being more efficacious compared to castrated, untreated mice (p < 0.001) or enzalutamide (p = 0.0245) , which aSerum testosterone levels can increase due to central nervous system stimulation of luteinizing hormone (LH) signaling by the antiandrogen therapy14C]ODM-201 and its metabolites into the brain was confirmed with a quantitative whole-body autoradiography (QWBA) in rat (data not shown).To further evaluate the androgen feedback loop at the hypothalamic-pituitary-gonadal axis, the ability of ODM-201, ORM-15341, enzalutamide, or ARN-509 to enter the brain was evaluated in a series of PK studies. Test compound concentrations (including the ORM-15341 metabolite of ODM-201) were determined in mouse plasma and brain homogenates after 7 days of oral dosing of ODM-201  or enzalutamide . A single dose of ARN-509 (10 mg/kg) was enough to demonstrate the high penetrance of ARN-509 to the brain, the brain/plasma ratio being 62%. Brain/plasma ratios for different concentrations of ODM-201 and ORM-15341 were very low, 1.9–3.9% and 1.9–2.8%, respectively, with no dose-response, whereas enzalutamide showed marked brain penetrance with the brain/plasma ratio of 27% . The verHere, we describe a novel AR inhibitor ODM-201 which efficiently blocks AR signaling by binding to AR with superior affinity and impairing the nuclear translocation of the overexpressed AR. ODM-201 and its pharmacologically active main metabolite ORM-15341 are structurally distinct from any known antiandrogens including the second-generation antiandrogens enzalutamide and ARN-509. Since the diversity of chemical scaffolds in clinically used antiandrogens has thus far been narrow, new structures are of interest as they might have properties that differ from current antiandrogens.in vitro data of Joseph et al., 2013 and Balbas et al., 2013, it is likely that the F876L mutation arises also in patients treated with enzalutamide. Further, ODM-201 and its main metabolite functioned as antagonists with the AR mutants AR(T877A) (found in the LNCaP PC cell line) and AR(W741L) conferring the activation of AR-mediated transcription by hydroxyflutamide and bicalutamide. Thus, ODM-201 functions as an antagonist to all well-known AR mutations appearing in response to antiandrogen therapies. Our data support further investigations of clinical responses to ODM-201 in e.g. enzalutamide-resistant CRPC patients.It is now understood that PC is androgen-sensitive not only during the early stages of the disease but successful treatment with novel AR-targeting therapies indicates that also CRPC remains androgen-sensitive810In vivo, ODM-201 showed a significant antitumor activity in a VCaP xenograft model generated to mimic CRPC. In this model, castration of the mice stabilizes tumor growth at first, but after a few weeks, tumors start to grow again (the castration-resistant stage), and at that time treatment with ODM-201 and enzalutamide is initiated. Importantly, bicalutamide  does not have any antitumor activity in this model (data not shown). ODM-201 at 50 mg/kg  showed a more potent antitumor activity than enzalutamide , although the systemic exposure for enzalutamide was higher than that for ODM-201.In VCaP cells containing endogenous AR gene amplification and overexpressing AR, ODM-201 and ORM-15341 inhibited cell proliferation more efficiently than enzalutamide or ARN-509. Most of the tumors in the castration-resistant state of the PC still depend on AR for growth and increased androgen levels have been linked to the progression of CRPC113334The tendency to induce seizures has been recognized as an obstacle in discovery and development of the second-generation antiandrogensin vitro and in vivo models of CRPC, ODM-201 is more efficacious than other tested antiandrogens and it does not stimulate androgen feedback loop at the hypothalamic-pituitary-gonadal axis. Taken together, these results indicate that ODM-201 exhibits unique properties that may offer advantages for the treatment of CRPC over the first- and second-generation antiandrogens. These nonclinical findings have been translated to significant antitumor activity and a good tolerability and safety profile observed in phase 1 and 2 clinical trials in men with metastatic CRPCIn summary, ODM-201 is a high-activity, next-generation AR inhibitor, which antagonizes AR mutants AR(F876L), AR(W741L), and AR(T877A) known to mediate resistance to first- and second-generation antiandrogens. ODM-201 also functions as an antagonist in AR overexpressing cells and impairs nuclear translocation of the receptor. In nonclinical data not shown) and AR-overexpressing LNCaP cellsU2-OS cells were obtained from Dr. Piia Aarnisalo  and HEK293 cells from American Type Cell Culture . HEK293 cells stably expressing wtAR (AR-HEK293 cells) were created by transfecting HEK293 cells with an expression vector encoding full-length hAR (pSG5-hAR) and an androgen-responsive reporter gene construct (pcDNA3.1/GRE2-TK-Luc). A cell clone of AR-HEK293 having over 5-fold overexpression of AR  of DMEM and F12 Nutrient mixture. All media were supplemented with 10% fetal bovine serum (FBS), 100 UI/mL penicillin, 100 μg/mL streptomycin, and 2 mM  or 4 mM  GlutaMAX. For maintaining the selection, medium was supplemented with geneticin  or with hygromycin and geneticin . For 3H]mibolerone from Perkin Elmer, and DHT from Sigma. Bicalutamide was extracted from pellets (AstraZeneca), and ODM-201 and ORM-15341 were synthetized by Orion Pharma.Testosterone was purchased from Fluka, mibolerone and [3H]mibolerone for isolated rat ARs was determined in a saturation binding experiment as previously described3H]mibolerone were incubated with increasing concentrations of test compounds overnight. After the incubation, bound and free steroids were separated by treatment with 100 μL of dextran-coated charcoal suspension. Bound radioactivity was determined by counting 100 μL of supernatant fraction in 200 μL of scintillation fluid  using a microbeta counter . All procedures were carried out at 0–4 °C.AR binding affinities of test compounds were studied in cytosolic lysates obtained from ventral prostates of castrated rats  by a competition binding assay as previously described2, cells were lysed and luciferase activity was measured with a Centro LB 960 microplate luminometer (Berthold Technologies) using a luciferase assay system (Promega Corporation) according to manufacturer’s instructions.Functional activity and potency of antiandrogens to hAR were determined in AR-HEK293 cells. The cells were treated with test compounds and 0.45 nM testosterone in steroid-free assay medium supplemented with 2 nM GlutaMAX and 25 mM HEPES. After 24 hours at 37 °C with 5% COTM2000 (Invitrogen). The construction of the mutant AR expression vectors was done as previously described® (Gibco). Cells were treated with increasing concentrations of the test compounds in the absence or presence of a reference agonist inducing a submaximal reporter gene activation  in steroid-free assay medium and incubated for 24 hours. Luciferase activity was measured as described above.Human U2-OS osteosarcoma cells were transiently transfected with an androgen-responsive reporter gene construct (pGV5-basic-GRE-hiv-luc) and expression vectors encoding AR mutants AR(F876L), AR(T877A), or AR(W741L)  using Lipofectamin® 488 . Cells were washed, DNA was labeled with DAPI , and images were analyzed with a NucTrans. V3 assay algorithm (Thermo). For confocal imaging, polyclonal AR antibody  was used as a primary and Alexa Fluor® 546 anti-rabbit IgG  as a secondary antibody. Coverslips were mounted with Vectashield containing DAPI (Vector Laboratories). AR overexpressing LN-AR-C cells were treated with 3 μM of test compounds together with 0.3 nM testosterone for 4 hours, immunolabeled with the AR-antibody  and a secondary antibody  and imaged with the HCS reader.AR overexpressing HS-HEK293 cells immunolabeled with an AR-antibody were imaged either with a high-content screening (HCS) reader  or with a confocal microscope . HS-HEK293 cells in steroid-free assay medium were plated on poly-D-lysine coated microplates (BD) (HCS reader) or on coverslips . After a 48-hour incubation, the cells were treated with 0.3 (HCS reader) or 1 μM  of test compounds together with 0.3 nM testosterone for 5 hours. After fixation with 3.7% PFA, the cells were washed with phosphate-buffered saline (PBS), permealized with 0.1% Triton X-100 (Sigma), and treated with 3% BSA in PBS to block unspecific staining. For HCS reader, cells were incubated with polyclonal AR antibody conjugated with Alexa FluorVCaP cells were treated with a submaximal concentration of mibolerone (0.1 nM) and increasing concentrations of test compounds in steroid-free assay medium supplemented with 4 mM GlutaMAX. After a 4-day incubation with the compounds, cell viability was measured using a WST-1 cell proliferation assay (Roche), according to manufacturer’s instructions. To rule out non-AR –mediated toxicity, AR-negative PC cells (DU-145) and lung cancer cells (H1581) were treated with an increasing concentration of ODM-201, and cell viability was measured as described above.All animal studies were conducted in accordance with EU legislation2 × L/2 (mm3), where W is the shorter and L the longer diameter of the tumor. When the average tumor volume reached ~200 mm3, mice were castrated or SHAM-operated under Avertin anesthesia. Oral treatments with two doses of ODM-201 , enzalutamide , or vehicle were initiated upon tumor regrowth (when average tumor volumes were ~400 mm3) and were continued for 37 days. For all in vivo studies, Macrocol® (Merck) + propylene glycol +5% glucose  was used as a vehicle.BALB/c nude male mice  were subcutaneously injected with 2 million VCaP cells in 100 μL of RPMI-1640 medium and Matrigel (BD) (1:1). Tumor growth was monitored twice weekly by caliper measurements. The volume of the tumor was calculated according to the formula WOne million VCaP cells in 20 μL of RPMI-1640 medium were orthotopically injected into the dorsal prostate lobes of athymic nude male mice . Tumor growth was followed by measuring serum PSA . Oral treatments were initiated 4 weeks post-inoculation, when mean serum PSA values were approximately 5.5 ug/L. ODM-201 , enzalutamide , or vehicle were administered for 3 weeks. At autopsy, blood samples were collected by heart puncture, clotted overnight at +4 °C, and centrifuged. For serum testosterone determination, 25-μL aliquots were extracted twice with 2 mL of diethyl ether and evaporated under nitrogen to dryness. The residues were reconstituted in PBS and measured using a standard radioimmunoassay as described previously2EDTA tubes by cardiac puncture under CO2 anesthesia and plasma was separated by centrifugation. Brain samples (without olfactory bulbs and medulla oblongata) from each group and time point were pooled and homogenized before the analysis. Concentrations of ODM-201 and ORM-15341 in mouse plasma and brain were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method at Charles River, UK, with the lower limit of quantification (LLOQ) being in plasma 1.00 ng/mL for both ODM-201 and ORM-15341 and in brain 4.00 ng/g for ODM-201 and 10.00 ng/g for ORM-15341. Enzalutamide and ARN-509 concentrations were determined by LC-MS/MS method at Orion Pharma . Plasma and brain concentration vs. time were evaluated by noncompartmental analysis using WinNonlin® Professional v. 5.2 software (Pharsight Corporation). Brain/plasma ratios were calculated based on AUC0–24 values for plasma and brain.In the PK studies analyzing the penetration of test compounds to the brain, nude male mice  were orally dosed for 7 days with 25, 50, or 100 mg/kg of ODM-201 twice daily (n = 5) or with 20 mg/kg enzalutamide once daily (n = 4), or with a single oral dose of ARN-509 (10 mg/kg) (n = 3). Control mice received vehicle. Blood samples were collected into Kin vitro data were analyzed with GraphPad Prism 5 software (version 5.02) to obtain Ki and IC50 values. For the VCaP xenograft experiment, mean tumor volumes were calculated for each treatment group. A repeated measure ANOVA (RMANOVA) was used as a statistical method to analyze tumor volume changes over the treatment time. With regard to the serum testosterone levels, the differences between groups were analyzed using Wilcoxon rank sum test.If not otherwise stated, How to cite this article: Moilanen, A.-M. et al. Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies. Sci. Rep.5, 12007; doi: 10.1038/srep12007 (2015).


Background. Osteopontin (OPN) has been proved to be associated with allergic airway inflammation. However, the roles of OPN and its regulation in childhood allergic rhinitis (AR) are poorly understood. Objective. This study aims to evaluate the expression of OPN and miR-181a in children with AR and their association with Th1/Th2 immune response. Methods. Children who suffered from AR were included along with control subjects. Serum was collected to examine the level of OPN and Th1/Th2 cytokines by enzyme-linked immunosorbent assay (ELISA) and the level of miR-181a by quantitative polymerase chain reaction (qPCR). Results. Children with AR had significantly higher serum levels of OPN and lower serum levels of miR-181a than healthy controls. Furthermore, serum levels of OPN were positively correlated with Th2 cytokine and negatively correlated with Th1 cytokine. On the contrary, miR-181a level had a negative correlation with IL-4/IL-5 and positive correlation with IFN-γ/IL-12. More importantly, there was also significant negative correlation between OPN and miR-181a. Conclusion. The OPN protein and miR-181a levels may serve as predictors of disease severity in childhood AR and appear to be promising targets for modulating AR.  Allergic rhinitis (AR) is a common chronic respiratory disease of the upper airway which is characteristically presented with rhinorrhea, sneezing, and congestion . The oveOsteopontin (OPN) is an extracellular matrix protein and cytokine that could be produced by the airway epithelial cells and inflammatory cells around airways. It is a multifunctional protein in allergy and asthma, an immune modulator that has been recognized as a key regulator in immunoglobulin (Ig) E-mediated and Th2-skewed allergic response . Also, OMicroRNAs (miRNAs) are small non-protein-coding RNAs that function as posttranscriptional regulators of target genes expression by suppressing translation or through mRNA degradation . MicroarTwenty-five children with AR were recruited at the Department of Otolaryngology, Guangzhou Women and Children's Medical Center, from January 2015 to October 2015. The diagnosis of AR was made on history, clinical examination, skin prick test, and specific IgE measurement according to the Allergic Rhinitis and Its Impact on Asthma (ARIA) guideline (2010) . Twenty Venous blood samples were obtained into Vacuette tubes between 6 am and 8 am after an overnight fast. After centrifuging at 1000 g for 15 min at 4°C, serum samples were separated and stored at −80°C until assay.−ΔΔCt method and normalized to RNA U6 controls.For miR-181a, total RNA from each serum sample was reverse transcribed into cDNA using the TaqMan microRNA reverse transcription kit and TaqMan microRNA assays (Invitrogen), as per manufacturer's instructions. The results were calculated with the 2γ, IL-5, IL-12, and IL-4 were measured in duplicate using ELISA kits  according to the manufacturer's instructions. The detection limits of the assays were as follows: OPN (312.5 pg/mL), IFN-γ (12.5 pg/mL), IL-4 (1.56 pg/mL), IL-5 (7.8 pg/mL), and IL-12 (2.5 pg/mL). Eosinophil cationic protein (ECP) was detected by Unicap system.The serum levels of OPN, IFN-t-test. A P < 0.05 was set as a significant criterion.The data are presented as mean ± SD. The differences between groups were determined by Student's We enrolled 25 children with AR and 20 normal controls for the study. Anthropometric parameters of the AR children and controls were listed in r = −0.58, P < 0.05; r = −0.59, P < 0.05).Concomitant OPN and miR-181a levels in serum were evaluated. As shown in γ/IL-12 . The results also revealed a significant inverse correlation between serum miR-181a level and TNSS .To identify if OPN and miR-181a have relationship with the severity of pediatric AR patients, the TNSS of the children was assessed. As shown in There is limited literature regarding the roles of OPN and miRNAs in airway allergic inflammation and most relates to asthma and adult population. In this study, we demonstrated that the serum level of OPN was elevated and miR-181a was decreased in AR children compared to that in normal controls. OPN level was positively correlated with Th2 cytokine IL-4 and total nasal symptom score, whereas miR-181a level had a negative correlation with them. Our key finding was that the levels of OPN and miR-181a had an inverse correlation in the serum of children with AR.OPN is expressed in a variety of cells, including bronchial epithelial cells, airway and vascular smooth muscle cells, myofibroblasts, and inflammatory cells like T-lymphocytes and mast cells . The eleThe biological functions of OPN can be profoundly influenced by posttranslational modifications . AccumulA number of studies have recognized the important roles of OPN in Th1-associated immunological diseases such as Crohn's disease  and tubeγ. The findings are supported by previous observations demonstrating that serum OPN concentration was correlated with blood serum ECP and IL-5 levels which were also typical Th2 cytokines in adult AR [γ. Based on the above analysis, it seems plausible that miRNA-181a acted on OPN to modulate the expression of pro- and anti-inflammatory cytokines in the setting of childhood AR.Our present study in pediatric AR demonstrated a positive correlation between OPN and IL-4, a representative cytokine of Th2-mediated allergic responses, and negative correlation with Th1 cytokine IFN-adult AR . Furtheradult AR . MeanwhiWe emphasize that the altered systemic OPN and miR-181a are related to the modulation of Th2-mediated allergic immune response. Therefore, it is reasonable to ask whether both of them contribute to the severity of AR in clinical scenario. In the present study, serum OPN showed a significant positive relationship and miR-181a had an inverse correlation with total nasal symptom scores in pediatric AR patients. These findings suggest that OPN plays a crucial role in driving the progression of childhood AR and miR-181a is more likely to have opposing effect. Nonetheless, the question of whether decreased miR-181a mediated posttranscriptional silencing is a potential mechanism for the upregulation of OPN needs further investigation.In summary, children with AR showed a marked increase in serum level of OPN but a decrease in miR-181a level. Our findings may be beneficial for designing a potential strategy for the optimal prevention and management of AR children.


The current study investigates the role of hypoxia and autophagy in combined cediranib/quinacrine efficacy. EF5 immunostaining revealed a prevalence of hypoxia in mouse intracranial 4C8 glioma, consistent with high-grade glioma. MTS cell viability assays using 4C8 glioma cells revealed that hypoxia potentiated the efficacy of combined cediranib/quinacrine: cell viability reductions induced by 1 µM cediranib +2.5 µM quinacrine were 78±7% (hypoxia) vs. 31±3% (normoxia), p<0.05. Apoptosis was markedly increased for cediranib/quinacrine/hypoxia versus all other groups. Autophagic vacuole biomarker LC3-II increased robustly in response to cediranib, quinacrine, or hypoxia. Combined cediranib/quinacrine increased LC3-II further, with the largest increases occurring with combined cediranib/quinacrine/hypoxia. Early stage autophagy inhibitor 3-MA prevented LC3-II accumulation with combined cediranib/quinacrine/hypoxia and substantially attenuated the associated reduction in cell viability. Combined efficacy of cediranib with bafilomycin A1, another late-stage autophagy inhibitor, was additive but lacked substantial potentiation by hypoxia. Substantially lower LC3-II accumulation was observed with bafilomycin A1 in comparison to quinacrine. Cediranib and quinacrine each strongly inhibited Akt phosphoryation, while bafilomycin A1 had no effect. Our results provide compelling evidence that autophagic vacuole accumulation plays a causal role in the anti-glioma cytotoxic efficacy of combined cediranib/quinacrine. Such accumulation is likely related to stimulation of autophagosome induction by hypoxia, which is prevalent in the glioma tumor microenvironment, as well as Akt signaling inhibition from both cediranib and quinacrine. Quinacrine's unique ability to inhibit both Akt and autophagic vacuole degradation may enhance its ability to drive cytotoxic autophagic vacuole accumulation. These findings provide a rationale for a clinical evaluation of combined cediranib/quinacrine therapy for malignant glioma.We have previously reported that the  Malignant gliomas are the most frequently occurring primary malignant brain tumors in adults. Glioblastoma multiforme (GBM), the most common malignant glioma, represents their most severe manifestation with an average survival of 15 months, despite improvements in diagnosis and treatment We have previously reported that cediranib, a RTK inhibitor targeting VEGF and PDGF receptor signaling was largely unable to provide an effective therapeutic benefit in an intracranial mouse glioma model Mouse studies were conducted with the approval of the Oregon Health and Science University Institutional Animal Care and Use Committee (protocol #IS00001409) and under the supervision of the OHSU department of Comparative Medicine.c-neu2. Female C57BL/6× DBA/2 F1 hybrid mice (B6D2F1) were purchased from Charles River Laboratories . Brain tumors were induced by the intracerebral injection of 0.5–1×106 4C8 cells, suspended in DMEM/F12 (5–10 µl) using a stereotaxic frame as previously described The 4C8 mouse glioma cells were provided by Dr. G. Yancey Gillespie, University of Alabama at Birmingham.. The 4C8 cells were originally obtained from Dr. Charissa Dyer, at the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, and are derived from a clone of a MOCH-1 tumor that arose spontaneously in the brain of a B6D2F1 mouse transgenic for myelin basic protein promoter–driven For the assessment of hypoxic regions within the tumors, mice were injected intravenously with the 2-nitroimidazole hypoxia marker EF5 , 3 hours prior to sacrifice. Mouse brains were removed, placed in a cryomold with OCT on dry ice, and stored at −80°C. Sections (10 µm) were mounted on slides in a cryostat, fixed in 4% paraformaldehyde for 60 min, rinsed, and blocked overnight at 4°C. Sections were rinsed and incubated with Alexa 488 conjugated EF5 antibody (provided by Dr Cameron J. Koch) for 6 hours at 4°C while covered with aluminum foil. Sections were subsequently rinsed and mounted with Prolong Gold Anti-fade reagent with DAPI (Life Technologies).Cells were plated in 96-well plates at a density of 3000 cells/well and were exposed to a range of treatment conditions. To assess cell viability in response to treatment, a mixture of 2 ml of MTS solution  and 100 µl of phenazine methosulfate solution  was added to cells in 96 well plates (20 µL/well) and left for 1 hour at 37°C. A negative control of MTS/PMS stock solution and medium alone was also included. The absorbance was measured using ELX800 Micro Plate Reader  at 490 nm.4 cells per well in 6-well plates and treated with vehicle or drugs. After the indicated time of treatment, cells were lysed by brief sonication in whole-cell lysis buffer , and cellular proteins were collected in the supernatant fraction after centrifugation at 14,000 g for 10 minutes. Proteins were reduced by heating at 100°C for 5 minutes in 50 mmol/L DTT with 0.05 bromphenol blue, electrophoresed in 4–20% Tris-HCl gels, and transferred onto nitrocellulose using the NuPage electrophoresis system . The membranes were probed with antibodies to LC3 and cleaved caspase 3  and actin . Membranes were treated with appropriate secondary antibody, exposed to enhanced chemiluminescence solutions  and visualized using the Molecular Imager Gel Documentation System.Cells were plated at density of 4.8×10EMD Millipore, Billerica, MA) to stably express the RFP-LC3 fusion protein. Cells were then treated with combined cediranib/quinacrine at the concentrations indicated in the figure legends, for 24 hours. RFP-LC3 expressing cells were then visualized using an EVOS fl fluorescence microscope (AMG).Cells were tranduced with lentiviral particles  was employed to test for statistical significance.Values are expressed as mean ± SEM. Student's t test  in terms of tumor oxygenation During the process of autophagy, autophagosome formation involves conjugation of the cytosolic form of LC3 (LC3-I) to phosphatidylethanolamine to form LC3-II, which becomes recruited to the autophagosome membranes. LC3-II has been proposed to have important functions in the sequestration of ubiquitinated substrates targeted for degradation through its interaction with p62/SQSTM1 We next sought to determine whether the autophagic vacuole accumulation in response to combination treatment under hypoxic conditions is not only associated with, but also plays a causal role in the anti-glioma effects. To accomplish this, we exposed 4C8 cells to combination treatment under hypoxic conditions with and without the addition of early stage autophagy inhibitor, 3-Methyladenine (3-MA). 3-MA blocks autophagosome formation via the inhibition of type III Phosphatidylinositol 3-kinases (PI-3K) A key control point of autophagy is mTOR . Inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been shown to potently induce autophagy in tumors + ATPase (V-ATPase) enzyme, which drives acidification in lysosomes, thereby inhibiting the autophagic vacuole degradation process. Because quinacrine's Akt inhibitory capability has the potential to increase its therapeutic efficacy, we compared it to bafilomycin A1, a commonly employed late stage autophagy inhibitor. Bafilomycin A1 is a specific inhibitor of the HRTK signaling plays a critical role in tumorigenic processes, driving proliferation, survival, and angiogenesis through modulation of key downstream pathways such as PI3K/Akt/mTOR, Ras/Raf/MEK/MAPK, and others in vivo effects that we reported previously in intracranial 4C8 glioma with combined cediranib/quinacrine treatment relates at least partly to an induction of autophagic cell death. The findings also support the concept that the in vivo efficacy with the combined agents could substantially derive from their direct effects on glioma cells. However, because cediranib's molecular targets are also potently anti-angiogenic, a similar efficacy against endothelial cells cannot be excluded, consistent with the marked tumor devascularization that was observed. Various reports have indicated a susceptibility of proliferating endothelial cells to autophagic cell death and/or autophagy inhibition. Employing Western blot and fluorescent microscopy to assess both the levels and spatial distribution of autophagic vacuole biomarker LC3, our study determined that, consistent with our previous findings, treatment with cediranib, quinacrine, or hypoxia each induced autophagic vacuole accumulation in glioma cells. in vivo observations of combined cediranib/quinacrine efficacy in 4C8 glioma. in vivo setting, via cediranib-induced reductions of blood flow within the tumor core region. in vivo success with combined cediranib/quinacrine treatment in that model, is consistent with this premise.Consistent with the known stimulation of autophagy by hypoxia, our study has clearly identified an important role for hypoxia in enhancing the synergistic efficacy of the cediranib/quinacrine combination in 4C8 glioma. Importantly, both the reduction of cell viability and the induction of apoptosis induced by the therapeutic combination was markedly potentiated by hypoxia  and was In our study, we determined that autophagic pathway stimulation not only occurs with hypoxia, but also as a direct result of cediranib treatment. Such an effect is likely to occur via inhibition of one or more of cediranib's RTK targets, which can activate various important downstream signaling pathways, such as PI3K/Akt/mTOR, inhibition of which would stimulate autophagic flux through downregulation of mTOR, the primary autophagy control point. + ATPase. Importantly, our data clearly demonstrates that unlike quinacrine, it does not inhibit Akt phosphorylation (While quinacrine's lysosomotropic action is well documented and required for its anti-malarial capability, it has pleiotropic biological effects. rylation . NotablyIn conclusion, our results provide compelling evidence that the unique anti-glioma cytotoxic efficacy of combined cediranib/quinacrine is causally linked to modulations in the autophagic pathway by the two agents, and by hypoxia. Our study provides a basis for an examination of this combination therapy in future clinical trials.


Study of the first steps of ligand recognition and the molecular basis of ligand binding to the orthosteric site is essential for understanding the pharmacological properties of the receptor. In this work we investigated the characteristic features of the agonist association–dissociation process to and from the different conformational forms of β2-AR by use of advanced molecular modeling techniques. The investigation was focused on estimating the free energy profiles (FEPs) corresponding to the process of a full agonist -fenoterol) and an inverse agonist (carazolol) binding and unbinding to and from β2-AR. The two different conformational forms of β2-AR, i.e. active β2-AR–PDB: 3P0G and inactive β2-AR–PDB: 2RH1 were included in this stage of the study. We revealed several significant qualitative differences between FEPs characteristic of both conformational forms. Both FEPs suggest the existence of three transient binding sites in the extracellular domain of β2-AR. Comparison of the residues surrounding these transient binding sites in both β2-AR states revealed the importance of the aromatic residues F194, H932.64, H2966.58, and H178 (extracellular part of β2-AR) in the early stages of the binding process. In addition, slightly different exit and entry paths are preferred by the ligand molecule in the extracellular part of β2-AR, depending on the conformation of the receptor.The βThe online version of this article (doi:10.1007/s00249-015-1010-4) contains supplementary material, which is available to authorized users. It is crucial for physiological regulation of cardiovascular and pulmonary functions. β2-AR, similar to other GPCRs, consists of seven-transmembrane α-helices (TMs I–VII) connected by three extracellular (ECLs I–III) and three intracellular (ICLs I–III) loops, with an extracellular N-terminus and an intracellular C-terminus  are a family of seven-transmembrane receptors that, upon activation by extracellular signals, couple with trimeric G proteins or β-arrestins to transduce signals from the cellular environment into the cell. GPCRs are activated by a variety of species ranging from photons to small-molecular-weight molecules and peptides. The β2-AR and ligands, and the mechanism of receptor activation have been intensively studied   to the different conformations of β2-AR, stabilized either by the agonist [3H]-methoxyfenoterol or by the antagonist [3H]CGP-12177  than for the conformation stabilized by the antagonist (inactive state). The process of antagonist displacement can affect both the orthosteric and other binding sites of β2-AR. Because [3H]CGP-12177, used as the radioligand marker, binds at two sites of the receptor -methoxyfenoterol) revealed significant differences between the thermodynamic characteristics of binding of the fenoterol stereoisomers to different conformations of β2-AR  and showed that structurally similar compounds (stereoisomers of fenoterol), the full agonists of β2-AR, bind to the inactive and active conformational states of β2-AR with different affinities and thermodynamic data -carazolol (PDB: 2RH1)  . The 3,5-dihydroxyphenyl group of fenoterol   only of the active state of β2-AR. The fenoterol molecule does not interact simultaneously with S204, S203, S207 (TM V), and N3127.39 (TM VII) of the inactive conformation of β2-AR. Movement of TM V toward TM VI on agonist activation enables interaction between the agonist (fenoterol) and serines of TM V and N3127.39  the two different high-resolution X-ray structures of β PDB: 2RH (Cherezoeinstein ), which einstein , inverseeinstein , and anteinstein . The proresidues of the second extracellular loop (ECL II) ;3.29 (TM III); orT1107.32, Y3087.35 or W3137.40).residues of TMVII  and ECL II  residues, is important in ligand binding  forms a hydrogen bond with an acceptor atom (oxygen) in the p-position of the fenoterol molecule   PDB: 2RH1) -fenoterol), Fig. 2-AR by taking into account that β2-AR can adopt distinct conformational forms (i.e. active and inactive states). The resulting FEPs were subjected to analysis revealing the main types of the ligand–receptor interaction responsible for the selected, characteristic regions of the given FEP. Next, we compared the data with results obtained for dissociation of an inverse agonist, (S)-carazolol  molecule complexed with the inactive conformational state of β2-AR in its inactive and active states was modeled on the basis of the crystal structure of human β2–AR–T4 lysozyme fusion protein (PDB: 2RH1)  -carazolol and BI-167107 co-crystallized with β2-AR in its inactive and active conformations, respectively. . The initial position of the carazolol molecule corresponded to the crystal structure of β2-AR co-crystallized with carazolol (PDB: 2RH1).β2–AR models obtained  with bound ligands were inserted into an equilibrated palmitoyl–oleoyl–phosphatidylcholine (POPC) cell-membrane model by use of the InflateGro procedure (http://www.csb.bit.uni-bonn.de/inflategro.html) and solvated with ~16,300 simple point charge (SPC) -fenoterol) was inserted into the binding cavity of each of the considered proteins.The two βR,R′)-fenoterol-β2-AR complexes was performed, lasting up to 100 ns. The final frames of these MD runs were used for subsequent ligand-pulling simulations .Energy minimization was conducted by applying 2000 steps of the steepest descent algorithm followed by 2000 steps of the l-bfgs algorithm. The four-step MD simulation then was performed with position restraints on the selected atoms (e.g. protein backbone). Finally, unconstrained MD simulation of each type of . The temperature was maintained close to its reference value (310 K) by applying the V-rescale thermostat -fenoterol–Ac_β2-AR and -fenoterol–In_β2-AR complexes by applying the US procedure. The distance between the centers of masses of ligand and protein (z) was accepted as the coordinate describing the binding–unbinding process. During the first step (pulling simulations) the force was applied to the ligand center of mass to obtain the constant velocity (0.0025 nm/ps) of ligand dissociation. The vector of the force was chosen in such a way as to ensure that the ligand dissociation path will be (approximately) in accordance with the most likely path  identified by González et al.  and ~5 nm (ligand outside the binding cavity). This resulted in 31 independent US simulations for each system studied. During the US runs, each lasting 40 ns, the distance between the ligand and the protein was restrained to its initial value by using the so-called umbrella potential with the force constant at 5000 kJ/mol/nm2. The FEP curves were calculated by using the WHAM  procedure  corresponding to association–dissociation of the ligand to and from the binding cavity were calculated for the , because this coordinate can describe both association and dissociation. In other words, the calculated profiles do not contain the dynamic (or kinetic) information and all the “non-equilibrium features” of the initial trajectory used to generate the US frames are assumed to be lost during 40-ns sampling.z) are depicted in Fig. Plots of FEP as a function of protein–ligand distance  cause the increase of the free energy. It is supposed that after dissociation of the ligand from the binding cavity, pulling it a sufficiently large distance further from the binding channel would result in a decrease of the free energy (which would then represent only ligand–solvent interactions). Although for the maximum value of the reaction coordinate the ligand is outside the binding cavity (i.e. there is no direct ligand–receptor contact) it can still interact strongly  with the receptor, which affects the FEP values. Moving the ligand further from β2-AR will result in screening of the unfavorable interactions and reduction of the FEP. Consequently, the actual difference between the FEP global minimum and the characteristic FEP region for the ligand not interacting with β2-AR will be smaller and closer to the actual binding free energy.Differences between the global minimum (corresponding to the ligand located in the binding cavity) and the “plateau” region  for considered FEPs both were larger than those expected on the basis of experimental data . In both cases the global minima can be associated with the ligand–receptor complexes studied during the previous step of the investigation (i.e. during the unconstrained MD simulations). This observation confirms the correctness of the initial structures obtained from the docking studies. First, let us notice that the minimum values of the accepted coordinate (z) differ for complexes involving Ac_β2-AR and In_β2-AR. This is caused both by the different conformational forms of Ac_β2-AR and In_β2-AR, which affect the optimum ligand–protein distances (expressed relative to their centers of masses) and the favorable position of the ligand, which, for Ac_β2-AR, is slightly shifted toward the extracellular part of the receptor; despite this shift, for both cases the spatial orientations of the ligand molecules in the binding cavity are very similar to each other. In both cases the 3,5-dihydroxyphenyl group is directed toward TM V and TM VI and the amine group of the ligand interacts with D1133.32 (TM III) whereas the p-hydroxyphenyl group is directed toward TM VII or ECL II.Furthermore, because of the approximate course of the FEP curves, it is hard to define the exact “plateau”. The “oscillating” character of FEPs observed for larger values of the coordinate is most likely caused by interaction of the ligand with more flexible parts of the protein, e.g. the N-terminus region and extracellular loops; the timescale of this movement may be too large to enable a perfectly smooth curve to be obtained during 40 ns of sampling.  than that observed for the In_β2-AR–fenoterol system (10 kJ/mol). This seems to confirm experimental studies indicating that the agonist molecule binds more strongly to β2-AR in its active conformation than to β2-AR in the inactive conformation -fenoterol for the active conformer of β2-AR is 86 times higher than that for the inactive conformer  , W2866.48 (In_β2-AR), and D192, K3057.32 (both conformations of the receptor).hydrogen bonds involving, e.g., N3122-AR is connected with hydrogen bonds created by the protonated amine and β-OH groups of the ligand and N3127.39. These interactions are present when the ligand is bound to active β2-AR but absent from the fenoterol–In_β2-AR complex. The above-mentioned hydrogen bonds break when the ligand leaves the favorable position in the binding cavity; this is accompanied by a shift on the FEP plot . This process causes the larger free energy change and the higher energy barrier between the global and first local minimum observed for the Ac_β2-AR conformer in comparison with the In_β2-AR conformer.On the basis of the results obtained, we speculate that the most important difference between interaction of fenoterol with the active and inactive conformations of β2-AR complex and compare the results with those reported elsewhere  . Figure R,R)-fenoterol molecule in the binding cavities of Ac_β2-AR and In_β2-AR; both cases correspond to the global minima on the corresponding FEP plot. The main type of attractive interaction, common to both systems, is the ionic bridge between the protonated amine group of the ligand and carboxyl group of D1133.32. Interestingly, this interaction occurs only at the global minimum and is disrupted during further steps of the ligand-dissociation process , respectively. The remaining interactions are mainly of HB-type and include:In the most favorable interaction pattern in the fenoterol–Ac_βHB between the 3,5-dihydroxyphenyl group of the ligand and both T195 and F193 (carbonyl oxygen atoms of the protein backbone);p-hydroxyphenyl group (ligand) and the side chain of D192 (ECL II) or K305 (TM VII) or H932.64 (TM II); andHB between the β-hydroxyl group (β-OH) of the ligand and the side chain of N2936.55 (TM VI) or N3127.39.HB between the β-OH–D113 hydrogen bonding is also possible but infrequent. Moreover, interaction of the ligand with D113 causes disruption of HB between β-OH and N2936.55. Lack of stable HB with the serines of TM V was observed. The distance between one of the hydroxyl groups of the 3,5-dihydroxyphenyl and S2035.42 is ~4–5 Å, and the contribution of the water molecule is essential for water-mediated HB with S203.2-AR complex the network of HB in the structure corresponding to the FEP global minimum is substantially different than that observed for the ligand–Ac_β2-AR complex. Interestingly, the ligand molecule bends relative to its main chain and the 3,5-dihydroxyphenyl group becomes closer to TM VI. The 3,5-dihydroxyphenyl group of the ligand is located between aromatic rings of W2866.48 and F2896.51 and can create very stable HB with W286 6.48 (the carbonyl oxygen atom of the protein backbone). Moreover, the 3,5-dihydroxyphenyl group interacts via Π–Π stacking with the aromatic rings of W2866.48, F2896.51, F2906.52, F193, Y3167.43, and Y3087.35 . Finally, the β-OH group participates in very stable HB with D1133.32 (carbonyl oxygen atom of the protein backbone); at the same time the carboxyl group of D113 forms an ionic bridge with the ligand amine group.For the ligand–In_β2-AR and Ac_β2-AR) preferential ligand–protein interactions have their source in the strong ionic bridge created between the central part of the ligand (–NH2+ group) and the carboxyl group of D1133.32, accompanied by different networks of hydrogen bonds involving all the polar parts of the ligand molecule  is reduced comparing with the ligand–In_β2-AR complex. This is only a qualitative estimate  but it harmonizes well with one of the hypotheses explaining the diverse results of the stereoselectivity-related simulations described above  and N2936.55, which has been found to be responsible for stereoselective binding to β2-AR  US simulation. This issue will be discussed in detail in forthcoming papers.Moreover, we observed that fenoterol interacts directly  with W2862-AR reveals the existence of multiple retention points represented by the local minima on the FEP plots. The most significant of these are located at approximately z = 2.5 nm (Ac_β2-AR) and z = 2.15 nm (In_β2-AR)  of the FEP is determined mainly by ligand–receptor HB. Table For the ligand–Ac_β3.32 and the lack of any direct ligand–protein interactions engaging the ligand amine group (except the extremely scarce HB with F193) in general; andthe lack of an ionic bridge between the protonated amine group of fenoterol and D113p-hydroxyphenyl group which can interact with D192 and H178 (ECL II), but not simultaneously.the slightly changed position of the z increments varying from 2 to 2.5 nm, corresponding to the transition from the main to the secondary minimum of free energy.The remaining (HB-type) interactions are comparable for both the global minimum and first local minimum, leading to the conclusion that disruption of the ligand–D113 salt bridge is responsible for the large increase on the FEP plot for z = 2.78 nm, as a result of four ligand–receptor interactions:Furthermore, the additional  local minimum on the FEP curve is visible at approximately p-hydroxyphenyl group (ligand) and the side chain of D300 and Q299  with H178 located on the ECL II);strong HB between the 6.55, Y3087.35, and F193 ;HB of the dihydroxyphenyl group of fenoterol with N293β-OH group and N312 is lost, resulting in rotation of the β-OH and directing it towards the extracellular part of β2-AR; at the second local minimum the β-OH group interacts via HB with D192 (frequently) and F193  but not simultaneously; andduring exit from the first local minimum, HB between the ligand the amine group (ligand) acts as an HB donor to D192.2+– group exposed to the solvent environment. On the basis of observation of the MD trajectories representing ligand transfer from the first to the second local minimum of FEP, we suggest a novel effect of F194 (ECL II) and histidines: H932.64, H2966.58, and H178. These histidines, surrounding the F194 side chain, can create a network of Π–Π interactions with each other  is disrupted, which results in the shift of ECL II and the N-end and, at the same time, increases the space available for the ligand molecule. This enables the β-OH and NH2+ groups to participate in stable interactions with D192 and F193 (ECL II)  and G16 (N-end) was observed. The latter type of interaction is especially interesting because of the β2-AR polymorphism at the 16th residue; it will be studied in more detail in forthcoming studies.The course of the FEP plot reveals the existence of the third local minimum for the ligand complex with Ac_β2-AR. All are very stable, however. The first and the second local minima of the free energy are located very close to each other, at approximately z = 1.7 nm and z = 2.1 nm, respectively. They are separated by a relatively small free energy barrier (~9 kJ/mol). The orientations of the fenoterol molecule corresponding to these two minima are quite similar. The slight differences are mainly because of orientation of the p-hydroxyphenyl group. At the first local minimum, the p-hydroxyphenyl group is closer to ECL III whereas at the second local minimum it is shifted toward ECL II  with N2936.55, which has previously been identified as responsible for stereoselective binding to the receptor .Similarly to Ac_ββ-OH, and amine groups of the ligand are also maintained for the configurations representing the second local minimum of the free energy , Q1975.36 (TM V), or N301 (ECL III). In contrast with the fenoterol–Ac_β2-AR complex, stable interactions with serines on TM V were observed for both the first and second minima of FEP. It is supposed that these attractive interactions partially compensate for the energetically unfavorable disruption of the D113-ligand salt bridge and are the reason for the relatively small (16–19 kJ/mol) FEP difference between the global and the first two local minima characteristic of In_β2-AR. For Ac_β2-AR, in which interactions with serines are absent, the corresponding difference is much larger (45 kJ/mol).Most of the mentioned interactions involving the 3,5-dihydroxyphenyl, z = 2.8 nm) corresponds to the ligand shifted closer to the extracellular part of the receptor. The fenoterol molecule loses its direct interactions with the TM V (S204), TM VI (N293), and TM VII (Y308) residues. The amine group of the ligand is located between ECL II and ECL III and does not participate in HB with β2-AR. Overall, the ligand molecule interacts only with residues on the extracellular loops. The 3,5-dihydroxyphenyl group participates in HB simultaneously with T195 (ECL II) and H2966.58. The β-OH group forms two hydrogen bonds, with H2966.58 and N301 (ECL III). The p-hydroxyphenyl group of fenoterol can interact with the T177 residue (ECL II) only. The stability of these HBs is weakened by the large mobility of the extracellular part of β2-AR.The third local minimum  and only two local minima . The ligand position in the global minimum is very similar to that observed in the crystal structure of the carazolol–β2-AR complex (PDB: 2RH1); the average RMSD was 0.21 Å .In contrast with the fenoterol–β2-AR complex are created between the amine group of the ligand and D1133.32, N3127.39, and Y3167.43. The other interactions include HB between:The most favorable interactions in the carazolol–In_β5.42, S2045.43) and the aromatic groups of ligand; andserines on the TM V (S2033.32 (Table the hydroxyl group (ligand) and D1132-AR complexes, disruption of HB between the hydroxyl group of carazolol and D113 seems to be of fundamental importance for dissociation from the global minimum. Slight rotation of the ligand β-OH group causes disruption of the β-OH–D113 HB and creation of new HB between β-OH and N3127.39. Moreover, the ligand becomes more distant from the serines on TM V. The distances between the nitrogen atom of the aromatic ring (ligand) and S2035.42 and S2045.43 increases to 4–6 Å  and TM VII (N312), compared with fenoterol, seem to be the result of the different chemical structures of the ligands. In the carazolol molecule the distance between the aromatic and amine groups is larger by two C–C bonds than for the fenoterol molecule; this enables the carazolol molecule to maintain stable interactions with N3127.39 without disrupting the network of interactions around the aromatic group. A significant energy barrier located between the first and second local minima is associated with disruption of HB between the ligand and the D1133.32, N3127.39, and Y3167.43 residues  dissociation processes. For both the fenoterol–β2-AR and carazolol–β2-AR complexes, the residues involved in direct contact with the ligand are similar to those reported by González et al. , can lead to slightly different results because of accuracy-related issues. The use of steered MD and Jarzynski equality assumes the ligand dissociation process (as accepted by the pulling direction) whereas use of time-independent umbrella potentials results in ligand association and dissociation processes described by the same profile (reversibility is assumed). The sampling times (~30 ns for each US window) are much larger than the time of ligand pulling (~3 ns) described by González et al. , allowinThe types of the ligands, which have two different pharmacological characteristics (agonist and inverse agonist); moreover, the dimensions of their molecules differ substantially  which did not contain the uncrystallized region of the N-terminus which, according to our US simulations, can be of crucial importance in the initial stages of the ligand-binding process. The proximity of the positively charged N-teminus region interacting with the cationic ligand can explain the larger free energy barrier observed for z > 3 nm compared with the equivalent results obtained for carazolol. The artificial N-terminus of the incomplete crystal structure of β2-AR would be located in the vicinity of the extracellular loops and obstructs entry to the core and/or binding site of β2-AR, i.e. further from the preferred ligand dissociation path. The fully reconstructed N-terminus region has much conformational flexibility which has a larger effect on the ligand–β2-AR interaction pattern.Details of the composition of the systems. The FEP profiles reported by González et al.  were obtThe differences between the FEP profiles calculated for fenoterol and carazolol could result from three factors:2-AR and In_β2-AR conformational forms include:To summarize, the most significant differences between the ligand binding–unbinding modes characteristic of the Ac_βthe ligand positions in the global and local minima of the free energy;the quantitatively different course of the FEP plots associated with the binding–unbinding processes; and2-AR and In_β2-AR (described below).the favorable entry and exit routes of the ligand to and from the binding cavity of Ac_β2-AR complex is closer to the extracellular part of the receptor -fenoterol and carazolol, were closer to TM V in comparison with Ac_β2-AR. Fenoterol interacts with S2035.42 and S2045.43 only when the system state corresponds to the first and second local minima of free energy (for In_β2-AR).As expected on the basis of different ligand–receptor interaction patterns (described above), slightly different dissociation paths of the ligand from Ac_βrgy Fig. . During 2-AR) the protonated amine group of fenoterol interacts with T195 (ECL II), being much closer to this residue than in the fenoterol–Ac_β2-AR complex  creates the additional space for the ligand extracted from Ac_β2-AR     , 5.43 (S204 in β2-AR), 5.46 (S207 in β2-AR)        -fenoterol) and inverse agonist (carazolol) association–dissociation process to and from the β2-adrenergic receptor (β2-AR). For fenoterol, the calculations were performed separately for the two conformational forms of β2-AR, i.e. the inverse agonist-bound  and the agonist-bound  to elucidate potential similarities and differences.Enhanced sampling MD simulations have been performed to calculate the free energy profiles associated with the full agonist  free energy barriers. The difference between the global minima and “plateaus” reach 80 kJ/mol for complexes containing fenoterol and 50 kJ/mol for carazolol. Note that these values do not necessarily correspond to the free energy of binding, as explained in the section “2-AR, with more metastable states and hindrance points experienced by the ligand during its binding and unbinding. Analysis of the profiles enabled identification of the crucial type of interactions responsible for each characteristic region of the free energy profiles. For both forms of β2-AR leaving the primary global minimum of the free energy is connected with disruption of the most favorable, attractive ligand–receptor interaction, i.e. disruption of the salt bridge between D1133.32 and the positively charged amine group of the ligand. For the carazolol–β2-AR complex, leaving the global minimum is associated with disruption of HB between the hydroxyl group of the ligand and the carboxyl group of D113. Irrespective of the conformational form of β2-AR, most of the attractive fenoterol–receptor interactions in the secondary minima of the free energy  are of hydrogen-bonding-type. Further differences between the active and inactive states of β2-AR include the slightly different association–dissociation paths of the ligand in the extracellular parts of the receptor. Comparison of these results with those reported for the inverse agonist (carazolol) molecule binding to the inactive form of β2-AR led to speculation about the effect of the N-termini in the initial stages of ligand-binding process. Finally, we observed that the fenoterol molecule may interact directly with the W2866.48 residue when is bound by the inactive β2-AR conformer. This observation can lead to better understanding of the effect of the ligand on the initial steps of the β2-AR conformational rearrangements (“activation”) but requires further studies.For both conformational forms of βSupplementary material 1 (DOCX 3153 kb)Below is the link to the electronic supplementary material.


The number of older adults living with lower limb amputation (LLA) who require rehabilitation for improving their walking capacity and mobility is growing. Existing rehabilitation practices frequently fail to meet this demand. Nintendo Wii Fit may be a valuable tool to enable rehabilitation interventions. Based on pilot studies, we have developed “Wii.n.Walk”, an in-home telehealth Wii Fit intervention targeted to improve walking capacity in older adults with LLA.The objective of this study is to determine whether the Wii.n.Walk intervention enhances walking capacity compared to an attention control group.This project is a multi-site , parallel, evaluator-blind randomized controlled trial. Participants include community-dwelling older adults over the age of 50 years with unilateral transtibial or transfemoral amputation. Participants will be stratified by site and block randomized in triplets to either the Wii.n.Walk intervention or an attention control group employing the Wii Big Brain cognitive software. This trial will include both supervised and unsupervised phases. During the supervised phase, both groups will receive 40-minute sessions of supervised group training three times per week for a duration of 4 weeks. Participants will complete the first week of the intervention in groups of three at their local rehabilitation center with a trainer. The remaining 3 weeks will take place at participants’ homes using remote supervision by the trainer using Apple iPad technology. At the end of 4 weeks, the supervised period will end and the unsupervised period will begin. Participants will retain the Wii console and be encouraged to continue using the program for an additional 4 weeks’ duration. The primary outcome measure will be the “Two-Minute Walk Test” to measure walking capacity. Outcome measures will be evaluated for all participants at baseline, after the end of both the supervised and unsupervised phases, and after 1-year follow up.Study staff have been hired and trained at both sites and recruitment is currently underway. No participants have been enrolled yet.Wii.n.Walk is a promising in-home telehealth intervention that may have useful applications for older adults with LLA who are discharged from rehabilitation or live in remote areas having limited or no access to existing rehabilitation programs.Clinicaltrial.gov NCT01942798; http://clinicaltrials.gov/ct2/show/NCT01942798 (Archived by WebCite at http://www.webcitation.org/6V0w8baKP). In 2003, it was estimated that more than 2 million individuals were living with lower limb amputation (LLA) in North America with an annual incidence of 150,000 , a grant from the Amputee Coalition of Canada, and the University of Alberta-Franklin Fund. BI is a Vanier Canada Graduate Scholar.Study staff have been hired and trained at both sites and recruitment is currently underway. No participants have been enrolled yet.The lack of continuity in rehabilitation treatment after discharge leads to decreased mobility, walking capacity, functional independence, and HRQOL. Wii.n.Walk presents a promising in-home telehealth intervention that may have useful applications for older adults with LLA who are discharged from rehabilitation, live in remote areas, or have limited or no access to existing rehabilitation programs. The application of a home treatment intervention reduces the client’s burden for travel and is particularly advantageous for clients with greater disability. The benefits extend beyond improved walking capacity and include increases in physical activity tolerance and better health and participation in important life activities that may ultimately ameliorate social and financial costs.This study has a number of limitations. RCTs in rehabilitation are subject to numerous threats to study validity . First,


Androgen receptor (AR) is expressed in normal murine and human kidneys of both genders, but its physiologic role is uncertain. Several studies showed loss of AR in renal cell carcinoma (RCC) in conjunction with increasing clinical stage and pathological grade, but others found that higher AR expression correlated with worse outcomes. Limited functional studies with renal cell lines suggested tumor-promoting activity of AR. In this study, we queried transcriptomic, proteomic, epigenetic and survival data from The Cancer Genome Atlas (TCGA) to evaluate AR expression and its association with overall survival in three subtypes of RCC . We found that although there was no significant difference in AR mRNA expression in ccRCC of males vs. females, AR protein expression in ccRCC was significantly higher in male compared to female patients. More importantly, higher expression of AR at both transcript and protein levels was associated with improved overall survival in both genders with ccRCC, but did not predict survival of either gender with pRCC or chRCC. Genes whose transcript levels were associated with AR mRNA levels significantly overlapped between ccRCC and pRCC, but not with chRCC, suggesting a similar transcriptional program mediated by AR in ccRCC and pRCC. Ingenuity pathway analysis also identified overlapping pathways and upstream regulators enriched in AR-associated genes in ccRCC and pRCC. Hypermethylation of CpG sites located in the promoter and first exon of AR was associated with loss of AR expression and poor overall survival. Our findings support a tumor suppressor role for AR in both genders that might be exploited to decrease the incidence or progression of ccRCC. Similarly, 79 out of 252 (31%) genes positively associated with AR in pRCC were found in the database. Fifty-six out of 120 (47%) genes positively associated with AR overlapping between ccRCC and pRCC were found in the database. Finally, 38 out of 170 (22%) genes positively associated with AR in chRCC were found in the database. These results suggest that AR plays an active role in regulating gene expression in all three subtypes of RCC and that it mediates a similar transcriptional program in ccRCC and pRCC.When genes associated with AR expression were queried against ARGD [Androgen responsive gene database , patients with higher expression of genes positively-associated with AR as well as lower expression of genes negatively-associated with AR displayed significantly longer survival . AR-assop<0.05). Forty-two and 35 pathways were enriched for pRCC and chRCC, respectively. The top 21 most significant pathways are listed in Pathways enriched in genes positively and negatively correlated with AR expression in each of the three RCC subtypes were identified using Ingenuity Pathway Analysis (IPA) . Sixty pUsing IPA, the top 50 upstream regulators of genes associated with AR expression in each of the three RCC subtypes were also identified . Thirty-To explore the subtype-specific role of AR in ccRCC and pRCC, we identified genes associated with AR expression only in ccRCC but not in pRCC or vice versa and pathways that were enriched in these genes. Seventy-five genes were found associated with AR in ccRCC but not in pRCC and 136 were associated with AR in pRCC but not in ccRCC . FifteenA possible mechanism for loss of AR expression in RCC is methylation. In a previous analysis of the relationship of gene expression and methylation of AR between ccRCC of stage I versus stage IV, a significant negative correlation (-0.19) was noted . In our Survival analysis of 208 cases stratified by median methylation level of AR demonstrated that higher methylation was associated with poor survival . When maOur study found that AR expression in ccRCC was prognostic and that high expression predicted better overall survival in both males and females. Others have reached similar conclusions using different cohorts ,10. Our AR expression was variable in cases with pRCC and chRCC, but did not associate with overall survival. It is noteworthy that genes whose expression correlated with AR expression in TCGA showed a high degree of overlap between ccRCC and pRCC, but differed considerably in chRCC. Different subtypes of RCC are believed to arise in different regions of the kidney, with ccRCC and pRCC originating from the proximal tubules while chRCC arises from distal tubules ,20. chRCIn an effort to gain insight into the function of AR in RCC, we identified genes whose expression most strongly correlated with that of AR. Many of the top AR-positively correlated genes in ccRCC and pRCC have functions consistent with potential regulation by AR. For example, LMTK2 (lemur tyrosine kinase 2) had the strongest positive correlation with AR expression among the genes positively correlated with AR in both ccRCC and pRCC. This gene encodes a novel membrane-anchored kinase with a role in endosomal membrane cycling, and an essential role in spermatogenesis. Interestingly, LMTK2 negatively regulates AR expression in prostate cancer cells and decreased expression of LMTK2 has been observed in prostate cancer , indicatLoss or gain of AR function has been implicated in cancers arising from other AR-expressing tissues. Previous studies have indicated that AR may have distinct and contrasting roles in different hormone-related tumors. For example, AR contributes to the pathobiology of breast cancer  suggestiWhether AR has tumor suppressor or promoter activity in RCC has clinical implications. If AR is in fact a tumor suppressor, then treatment of patients bearing AR-expressing RCC with AR antagonists could be deleterious and promote, rather than prevent, progression. Conversely, treatment with AR agonists could be efficacious. In a phase II study, 30 patients with metastatic RCC received high dose testosterone proprionate plus provera, and 3 showed partial response . Given tPathways enriched in genes associated with AR expression identified by IPA analysis may provide some clues in the mechanisms of AR signaling in RCC. For example, the top common pathway affected by AR signaling in both ccRCC and pRCC is NGF signaling, which has been shown to be overexpressed in metastatic ccRCC compared to primary tumors . The secAR and its associated gene expression are prognostic in ccRCC with higher expression predicting longer survival. AR mediates a similar transcriptional program in ccRCC and pRCC that is different from chRCC. The tumor suppressive role of AR suggests that therapies that promote retention of AR signaling may be beneficial to ccRCC patients.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


These effects were attenuated by inhibition of AKT, AR and MMP9. AR overexpression upregulated MMP9 protein levels, whereas this effect was counteracted by AR siRNA. Inhibition of AKT by siRNA or an inhibitor (MK-2206 2HC) decreased AR protein expression in both stably transfected and parental SGC-7901 cells. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that AR bound to the AR-binding sites of the MMP9 promoter. In summary, AR overexpression induced by AKT phosphorylation upregulated MMP9 by binding to its promoter region to promote gastric carcinogenesis. The AKT/AR/MMP9 pathway plays an important role in GC metastasis and may be a novel therapeutic target for GC treatment.Androgen receptor (AR) plays an important role in many kinds of cancers. However, the molecular mechanisms of AR in gastric cancer (GC) are poorly characterized. Here, we investigated the role of AR in GC cell migration, invasion and metastatic potential. Our data showed that AR expression was positively correlated with lymph node metastasis and late TNM stages. These findings were accompanied by activation of AKT and upregulation of matrix metalloproteinase 9 (MMP9). AR overexpression induced increases in GC cell migration, invasion and proliferation  Gastric cancer (GC), hepatocellular carcinoma (HCC), and pancreatic cancer are all male-predominant cancers -3. In viGC is the fourth most common cancer worldwide and more common in developing countries, and the incidence of GC is higher in males than in females with a ratio of 2:1 [in vitro and in vivo studies has shown that AR not only mediates the effects of androgen but also functions as an oncoprotein by interacting with other molecules implicating the proliferation and metastasis of cancer cells [AR, also known as NR3C4 , belongs to a family of nuclear receptors that act as transcription factors. Many studies have described the functions of AR in both androgen-dependent and -independent manners . For exaer cells , 13.Moreover, AR is functionally associated with a broad range of oncogenic signaling pathways. Ha et al found that AR levels are regulated by the AKT . ActivatRecent data have demonstrated that AR is closely related to the development of various types of human cancer by elevating cell migration and invasion in response to various stimuli including inflammatory factors . It is ain vitro and in vivo. Finally, we characterized the molecular mechanism by which AR mediates the metastatic potential of GC cells.Here, we investigated AR expression, its relationship to clinicopathological features and P-AKT/MMP9 expression in GC tissues by immunohistochemistry (IHC). Additionally, the effect of AR on the invasion and metastasis of GC cells was examined To explore the role of AR in GC, we performed IHC to analyze the protein expression of 40 GC tissue samples. We identified 15 and 25 GC tissue samples that were positive and negative for AR, respectively. It has been previously reported that dysregulated AR is an independent unfavorable prognostic factor in GC with a positivity rate of 20% , the autTo elucidate the effect of AR on the malignant phenotypes of GC cells, WB was used to examine the endogenous AR expression of 7 adherent parental gastric cell lines. Our data showed that AR expression level in SGC-7901 cells was lower than CRL-5822 but higher than other cell lines Fig. , which aAs shown in Fig. http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 and http://jaspar.genereg.net/), and found two putative sites with high scores (−139 to −148 nt and −1208 to −1223 nt)  assays were performed. We analyzed the MMP9 promoter region for potential AR-binding sites using online tools (nt) Fig. . Then, fnt) Fig. . Then thnt) Fig. . Taken tTo examine the effect of AR downregulation on GC cell migration, invasion, and proliferation, we first assessed the effects of AR, AKT and MMP9 inhibition by siRNA or inhibitors on cell motility in wound healing assays. As shown in Fig. in vivo. SGC-7901/AR or control cells were injected subcutaneously or peritonealy into nude mice, and subcutaneous tumor formation was monitored by growth curves ves Fig. . The avein vivo, P-AKT, AR, and MMP9 were detected by IHC  Fig. . To asseIHC Fig. . The datIHC Fig. . These dAR expression has been documented in liver, colon and brain tumors -26. In GThe present study revealed that there was no difference in the AR expression of tumors in male and female subjects. This result reflects the fact that activation of the AR pathway can be either ligand dependent or mediated by ligand-independent mechanisms. Castration-resistant prostate cancer may develop as a consequence of the dysregulated AR signaling that results from AR amplifications, altered interactions of co-regulatory molecules during transcription as well as non-steroidal activation by growth factors, cytokines, and neurosecretory products .Considering the male predominance of GC and the lack of information available on AR expression in the stomach, detailed mechanistic studies need to be conducted to reveal the mechanism of AR functions in normal stomach and GC. Accumulated evidences implicate AR was involved in the process of carcinogenesis. AR promotes prostate cancer progression by direct suppression of the tumor suppressor gene CCN3/NOV, and it enhances ligand-independent prostate cancer progression through c-Myc upregulation , 30. Thein vivo experiments. Recent in vitro studies focusing on the molecular interactions of hormone receptors with oncoproteins have been confirmed s in animal models [We demonstrated that AR overexpression promoted GC cell migration, invasion and proliferation, and our results were confirmed by l models , 32. Howl models . Howeverl models . These dl models .Our results indicated that an increase in AKT activity correlated with increased AR protein expression. To confirm this, we assessed the effect of AKT inhibition on GC cells and data showed that AKT suppression resulted in downregulation of AR protein expression and decreases in GC cell migration, invasion, and proliferation. This is the first report to demonstrate that AR induces GC cell migration and invasion via activation of AKT. However, the exact mechanisms by which AR signaling enhanced gastric carcinogenesis remain largely unknown. One potential target by which AR increases the invasion and migration of cancer cells is MMPs. It is well established that secretion of MMPs with the capacity for ECM degradation is a feature of metastatic cancer cells . MMP2 anIn summary, we have shown for the first time that AR is functionally involved in the regulation of metastasis in GC. This molecular mechanism involves AR-mediated up-regulation of MMP9 via activation of AKT. Our study strongly suggests that the P-AKT/AR/MMP9 pathway may be a potential target for therapeutic strategies to enhance the survival of GC patients.2 at 37°C. Exponentially growing cells were used for experiments.Tissues were resected for GC treatment at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine  between 2012 and 2014. Sections were prepared from 10% formalin-fixed, paraffin-embedded GC tissues. All cases of GC were histologically diagnosed according to the Japanese Classification of Gastric Carcinoma and Lauren's classification , 41. HumFor IHC, all staining steps were performed at room temperature and the samples were washed with PBS between steps. Sections were de-waxed before incubation with primary antibodies (rabbit anti-P-AKT/AR/MMP2/MMP9)  for 1 h. A biotinylated swine anti-rabbit  secondary antibody was applied for an additional hour following the removal of primary antibodies. Staining was developed with an avidin-biotinylated horseradish peroxidase complex (Dako) and 3, 3′-diaminobenzidine (Dako) following the manufacturer's instructions. Expression of P-AKT/AR/MMP2/MMP9 was detected in the cytoplasm, nuclei or intercellular areas. Positivity of more than 15% of the carcinoma cells was considered as overexpression of the proteins. For WB, tissue samples were lysed using T-PER Tissue Protein Extraction Reagent (Pierce) in the presence of a protease inhibitor cocktail (Pierce). For cultured cells, trypsinized cells were washed with PBS and then subjected to sonication in 50mM Tris-HCl buffer (pH 8.0) containing 1% glycerol, 1mM EDTA, 0.5mM PMSF, and 2mM benzamidine followed by centrifugation. Equal amounts of protein (100μg) were electrophoresed on 12% reducing SDS-polyacrylamide gels. The proteins were electrotransferred to Immobilon-P membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline  containing 0.1% Tween-20 for 2h, and subsequently incubated with rabbit antibodies at 1μg/ml. Antigen-antibody complexes were visualized using enhanced chemiluminescence. In most cases, blots were re-probed with an antibody against glyceraldehyde-3-phosphate dehydrogenase to assess protein loading. Band intensities were quantitated using a Tanon 2500 imaging system (TANON).5 cells/cm2, cultured for 24 h, and then transfected with vectors using 4μg DNA and Lipofectamine (Invitrogen). After 24h, every 48h thereafter for 4 weeks, the culture medium was replaced with fresh medium containing 800μg/ml G418. Pools of 16 clones were isolated as stable transfectants with an AR expression vector or an empty vector.Wild-type AR (WT-AR) in a pcDNA3.1 (+) plasmid was kindly provided by Dr. Masahiro Takeyoshi  and Prof. Li-Chun Xu . The pcDNA3.1 (+) mammalian expression vector was purchased from NOVOBIO. GC cells were plated at a density of 2 × 106) using a Lip2000 transfection reagent kit according to the manufacturer's instructions. For plasmid transfections, plasmid DNA and Lipofectamine were diluted separately in serum-free medium and then incubated at room temperature for 5 min. The diluted DNA and Lipofectamine were then mixed and incubated at room temperature for 20 min. Aliquots of the transfection mixture were then added to the cells. For inhibitor treatments, we used an AKT Inhibitor (MK-2206 2HC) (Selleck) at 15nM.SiRNA used in our study mainly target the gene coding regions, which allowed it to knockdown both the endogenous and exogenous AR protein expression effectively and efficiently. 750pmol scrambled siRNA oligos or SiRNAs that specifically targeted human AKT, AR, or MMP9  were transfected into GC cells .3 cells/well) were seeded into 96-well plates and incubated for 5 days. Cell proliferation was assessed by a water-soluble tetrazolium salt assay using Cell Counting Kit-8  according to the manufacturer's instructions. All experiments were performed in triplicate.At 24h post-transfection, cells (2 × 105 cells/well) were added to the upper chambers and allowed to invade for 24h at 37°C in a CO2 incubator. Cells remaining attached to the upper surface of the filters were carefully removed with cotton swabs. Invaded cells were stained with 0.1% crystal violet for 10 min at room temperature and examined by light microscopy.We employed the Boyden chamber technique . Briefly, 8μm pore size filters were coated with 100μl of 1mg/ml matrigel (dissolved in serum-free RPMI-1640) (migration assays omit matrigel). A total of 600μl RPMI-1640 containing 10% FBS was added to the lower chambers. Single cell suspensions (1 × 10The human MMP9 gene promoter was amplified by PCR and cloned into the multi-cloning site of a pGL3 reporter plasmid (Ambion) containing the WT firefly luciferase reporter gene. To introduce mutations into the seed sequences of the two AR-binding sites in the promoter and generate MMP9 MT1 and MT2 reporters, plasmids were constructed by overlap extension PCR. Cells seeded into 24-well plates were co-transfected with different plasmids . Firefly and Renilla luciferase activities were measured at 24h post-transfection by the Dual Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.To evaluate the interaction between AR and MMP9 promoter region, ChIP assays were performed using a kit (Millipore) according to the manufacturer's instructions. Cells were fixed with formaldehyde for protein/DNA crosslinking and then lysed. The DNA was sheared by sonication  and then added to a well coated with an anti-AR antibody. Washes were performed to remove unbound material, and then AR-bound DNA was released by protein digestion with proteinase K. The DNA was purified through a column. PCR was performed using primers designed to target the MMP9 promoter region spanning the site of the interaction with AR. Primers were as follows: MMP9-F: GCAAAGTGTTCATTG GTTAGTGAACTTTAGAACTTCAA; MMP9-R: TTTTTTGCCCCCCACCCCCCGAGAAACAGGGAT TTAAC. Genomic DNA and IgG were used as controls.6 or 2 × 106 in 150μL) were injected subcutaneously or into the peritoneal cavity of 4-week-old male nude mice . Mice were checked weekly, and tumor nodules were measured with a caliper. Tumor volumes were evaluated using the following formula: volume = (width + length) / 2 × width × length × 0.5236. Mice were sacrificed after 5 weeks. Tumor growth curves and promotion rates were calculated. All tumors were excised, weighed, harvested and embedded in paraffin. A mouse anti-human Ki-67 antigen monoclonal antibody  was used to determine nuclear expression of Ki-67 by IHC.Stably transfected cells with the AR or control plasmid (1 × 10All animal experiments were conducted according to the Chinese guidelines for animal experimentation and approved by the Institutional Animal Care Committee of Shanghai Jiao-Tong University School of Medicine, Ruijin Hospital. All patients provided written informed consent before enrollment, and the Ethics Committee of Shanghai Jiao-Tong University School of Medicine, Ruijin Hospital approved the study protocol. The study was carried out according to the provisions of the Helsinki Declaration of 1975.Statistical analyses were performed with SPSS Version 19.0 using the Student's t-test or one-way analysis of variance, chi-squared test, Fisher's exact test or Mann–Whitney U-test. Differences were considered statistically significant in two-tailed tests with p-values of <0.05.


Curcuma longa. It has been suggested that ar-turmerone inhibits microglia activation, a property that may be useful in treating neurodegenerative disease. Furthermore, the effects of ar-turmerone on neural stem cells (NSCs) remain to be investigated.Aromatic (ar-) turmerone is a major bioactive compound of the herb In vivo, naïve rats were treated with a single intracerebroventricular (i.c.v.) injection of ar-turmerone. Proliferative activity of endogenous NSCs was assessed in vivo, by using noninvasive positron emission tomography (PET) imaging and the tracer [18F]-fluoro-L-thymidine ([18F]FLT), as well as ex vivo.We exposed primary fetal rat NSCs to various concentrations of ar-turmerone. Thereafter, cell proliferation and differentiation potential were assessed. In vitro, ar-turmerone increased dose-dependently the number of cultured NSCs, because of an increase in NSC proliferation (P < 0.01). Proliferation data were supported by qPCR-data for Ki-67 mRNA. In vitro as well as in vivo, ar-turmerone promoted neuronal differentiation of NSCs. In vivo, after i.c.v. injection of ar-turmerone, proliferating NSCs were mobilized from the subventricular zone (SVZ) and the hippocampus of adult rats, as demonstrated by both [18F]FLT-PET and histology (P < 0.05).in vitro and in vivo data suggest that ar-turmerone induces NSC proliferation. Ar-turmerone thus constitutes a promising candidate to support regeneration in neurologic disease.Both  Curcuma longa. Although many studies have demonstrated curcumin to possess antiinflammatory and neuroprotective properties , to datiewed by  and inhiiewed by , have be -fluoro-L-thymidine ([18F]FLT) was synthesized as described previously [18F]FLT (1.0 to 2.2 mCi/rat), and emission data were acquired for 60 minutes. PET data were reconstructed in two time frames of 1,800 seconds. The last frame  was used for image analysis.FLT that enables imaging and measuring of proliferation, thereby allowing noninvasive detection and quantification of endogenous NSC mobilization in the adult rat brain in vivo[18F]FLT-PET, we here found ar-turmerone to mobilize NSCs from both neurogenic niches, the SVZ and the dentate gyrus of the hippocampus, in vivo. Thus, this study provides further evidence for NSC activation by ar-turmerone, spanning from cell-culture findings to in vivo imaging.Noninvasive iotracer FFLT that in vivo. By usinin vitro and in vivo. Ar-turmerone increased the number of NSCs both in cell culture and in the adult rat brain in vivo. This increase resulted from enhanced NSC proliferation and led to promoted neurogenesis during differentiation. In vivo, ar-turmerone mobilized endogenous NSCs from both neurogenic niches, the SVZ and the hippocampus. We propose that ar-turmerone constitutes a promising future drug candidate to support regeneration in neurologic disorders.In this study, we investigated the effects of ar-turmerone on NSCs


The model was used to determine the effect of different AVB and AS configurations on flow proportion and pressure of the aortic valve and the AVB. Results showed that the AVB leads to a significant reduction in transvalvular pressure gradient. The percentage of flow through the AVB can range from 55.47% to 69.43% following AVB with a severe AS. LV stroke work was also significantly reduced following the AVB surgery and reached a value of around 1.2 J for several AS severities. Findings of this study suggest: 1) the AVB leads to a significant reduction in transvalvular pressure gradients; 2) flow distribution between the AS and the AVB is significantly affected by the conduit valve size; 3) the AVB leads to a significant reduction in LV stroke work; and 4) hemodynamic performance variations can be estimated using the model.Aortic valve bypass (AVB) has been shown to be a viable solution for patients with severe aortic stenosis (AS). Under this circumstance, the left ventricle (LV) has a double outlet. The objective was to develop a mathematical model capable of evaluating the hemodynamic performance following the AVB surgery. A mathematical model that captures the interaction between LV, AS, arterial system, and AVB was developed. This model uses a limited number of parameters that all can be non-invasively measured using patient data. The model was validated using  Aortic stenosis (AS) is the most common valvular disease in the elderly population. Untreated symptomatic AS is associated with a poor prognosis and significant morbidity. Aortic valve replacement (AVR) is currently the standard of care for reducing the left ventricular overload and improving the quality of life of patients . HoweverIn AVB, also called apico-aortic bypass, the AS and the aorta are never manipulated and a second outflow tract for blood flow during systolic phase is created by inserting a valved conduit at the level of the left ventricle apex. This leads to a double outlet ventricle . The outvs. the bypass conduit. In addition, previous studies ..2 and 0.2); 2) severe AS with EOA of 0.7 cm2. As expected, the presence of a severe AS induces very large transvalvular pressure gradients: TPGmax = 99 mmHg; TPGmean = 52 mmHg. The results for an EOA of 0.7 cm2 are displayed because this EOA corresponds to the median value reported in the study of Lund et al. [2. Here the results for an AVB with a conduit valve size of 19 mm and two different conduit sizes (18 mm and 26 mm) are displayed. The AVB induced a significant reduction in LV peak pressure and TPGs that can be reduced down from 99 mmHg .2 (vs.1.19 J).2 . Now wit2 . A large2 to 0.75 cm2). LV stroke work values corresponding to the AVB for each AS severity were averaged over all configurations of conduit valve size and conduit size tested in this study. For all AS severities, the AVB contributed to a significant reduction in the LV stroke work. The LV stroke work values reached following the AVB surgery are not significantly affected by the selected conduit valve size/conduit size configurations for the AVB and reached a value around 1.2 J. This value remains however higher than normal LV stroke work values (1.03 J).We have introduced a lumped-parameter method which captures the interaction between left ventricle, AS, arterial system, and AVB. This method only needs few non-invasively measured quantities described as follows: 1) total stroke volume; 2) EOA of the aortic valve and aortic cross sectional area (A); (3) EOA of the conduit valve and conduit area. All the above mentioned parameters can easily be measured in patients non-invasively ,22. The AVB is not a new technique, the first reference to AVB was in 1910  and in 1TAVI seems to be a very attractive option for such inoperable patients. However, TAVI may be complicated if performed in patients with calcified aorta , ostial encroachment or bicuspid valve. In a recent study, Garcia et al.  showed tIn this study, we showed that the AVB leads to a significant reduction in TPG. It should be mentioned however that the reduction in the TPG can mainly be attributed to the significant reduction in the flow rate crossing the aortic valve following the AVB surgery.in vivo and in silico studies [This flow distribution is a major determinant of the residual transvalvular pressure gradient through the native aortic valve following AVB. To determine this ratio, the model developed in this study needs only non-invasive parameters that are routinely measured in daily clinical practice and the EOA of the bioprosthetic valve in the conduit (listed in Pibarot and Dumesnil ). The re studies ,10,26.vs. 1.10±0.05 J, p<0.001). However, the differences appear not to be clinically significant.Another significant result found in this study is that the AVB significantly reduces the LV stroke work. The expected post-operative LV stroke work is appeared to be quite independent from the selected AVB configurations. Interestingly, it has been hypothesized that the AVB might provide better overall hemodynamic performance since the net effective orifice area should be the summation of the effective orifice area of the stenotic aortic valve plus the effective orifice area of the bioprosthetic valve included in the conduit . Our resin vivo and in vivo studies are still required to determine further the validity of the equation mainly for large conduit valve sizes.Since no formulation has been developed to express the net instantaneous pressure gradient through a conduit valve yet, we elected to use the same formulation as the one used for modeling the aortic valve. In order to assess the validity of this formulation, data resulting from our mathematical simulations were compared with in vivo findings , resultvs. AVB) can be predicted mathematically and its main determinant is conduit valve size. Finally, AVB leads a significant reduction in LV stroke work whatever is the configuration of conduit valve size and conduit size. Finally, the positive hemodynamic results obtained in this study following AVB may suggest a need for a randomized trial comparing TAVI and AVB.AVB is a viable solution for patients with AS and contraindications to both AVR and TAVI. AVB leads to a double outlet LV. In this study, we have shown by using mathematical modelling that AVB leads to a significant reduction in pressure gradient across the AS and LV stroke work. Furthermore, the results show that the flow distribution (through AS